家兔卵子体外受精研究

从供体母兔输卵管冲取成熟的卵母细胞,与体外获能的附睾精子或射出精子体外受精。三种获能方法分别是 His-DM 法、肝素法和附睾精子 DM 预培养法,获能精子与卵母细胞体外受精后的2-细胞胚胎发育率分别是36.37%(24/66)、12.57%(13/102)和35.33%(53/150)。体外受精后体外发育到2-至4-细胞期胚胎经移植给7只同期发情受体母兔,结果有2头怀孕并产仔9只,胚移产仔率为28.57%(2/7),移入胚胎成仔率为11.39%(9/79)。     

提高牛体外性控胚胎效率关键技术研究

为了提高牛体外受精效果,试验从性控冷冻精液体外获能方法及抗氧化剂的添加、体外成熟24h后卵母细胞是否部分脱除卵丘细胞3个方面,研究了影响牛性控精液体外受精效率的关键技术。结果表明,一次离心体外获能法体外受精卵裂率[(68.3±3.7)%]、囊胚率[(30.9±2.3)%]与精子上浮获能法体外受精卵裂率[(67.8±2.6)%]、囊胚率[(29.7±3.7)%]无显著差异(P>0.05),但显著高于二次离心体外获能法体外受精卵裂率[(56.1±4.1)%]、囊胚率[(21.6±4.6)%](P<0.05);获能液和体外受精液添加1.0%抗坏血酸组体外受精卵裂率[(70.2±3.2)%]、囊胚率[(35.0±4.7)%]显著高于不添加组体外受精卵裂率[(60.0±4.5)%]、囊胚率[(28.3±3.6)%](P<0.05);在体外受精前,成熟卵母细胞周围的卵丘细胞脱除情况为部分脱除组体外受精卵裂率[(70.1±4.6)%]、囊胚率[(35.5±3.7)%]显著高于不脱除组体外受精卵裂率[(52.9±4.1)%]、囊胚率[(26.1±3.6)%](P<0.05)。由此可见,性控精液体外受精时,获能方法宜采用一次离心体外获能法或上浮获能法,且精子获能液和体外受精液中宜添加抗坏血酸;体外受精前,成熟卵母细胞部分脱除卵丘细胞对于提高性控精液体外受精胚胎发育率具有重要意义。     

树鼩卵母细胞的体外成熟培养及体外受精

利用树鼩卵母细胞的成熟培养、体外受精等方法,生产树鼩的试管婴儿,期望通过此路径来实现生产转基因树鼩动物模型。使用促性腺激素进行树鼩超数排卵、TCM199完全培基对卵母细胞进行体外成熟培养、卵母细胞与体外获能的附睾精子进行体外受精、受精卵发育至桑椹胚、囊胚的实验。结果表明,卵母细胞成熟率A级76.7%、B级55.01%、C级17.39%。受精率52%,受精卵采用体细胞共培养与非共培养的分裂率分别为37.14%和9.6%。桑椹胚/囊胚发育率分别为13.57%和0,(P<0.05)。树鼩卵母细胞的成熟培养、体外受精实验方法可行,受精卵在体外能发育到囊胚阶段。     

猪卵子体外受精方法研究

本试验分析研究了：（1）猪卵子体外成熟所需要的培养时间；（2）促性腺激素和猪卵泡液对猪卵子体外成熟的作用；（3）不同体外受精次数对猪卵子体受精率的影响；（4）不同精子获能辅助剂对猪精子体外获能的作用；（5）不同种类猪精液的体外受精能力。研究结果表明，猪卵子体外成熟所需培养时间是32－36小时，在体外成熟培养液中加入0.25IU/ml PMSG（或FSH）和10％猪卵泡液可以促进猪卵子体外成熟，在精子体外获能培养液中加入0.05mg/ml肝素，在体外受精培养液中加入2mg/ml咖啡因可以促进猪精子体外获能并可提高体外受精率，体外成熟后的猪卵子进行2次受精可以获得较高的体外受精率，本试验用新鲜射出精子，冷冻射出精子，新附睾精子，冷冻附睾精子对体外成熟猪卵子进行体外受精，受精后卵裂率（2－4细胞期）分别是24.26％、23.40%,22.65%和24.24%。     

微流路精子分离器体外受精方法对奶牛体外受精效果的影响

为应用牛性控精液进行体外受精(IVF)获得大量廉价的性控胚胎,本实验利用微流控精子分离器(MFSS)分离有活力的精子,将体外成熟的卵母细胞放置在MFSS的C室进行体IVF,与常用的微滴-IVF法进行对比,检测2种受精方法的受精率和囊胚发育率。结果表明:MFSS-IVF的单精子入卵率和囊胚率(分别为64.38%、40.12%)均显著高于微滴-IVF法(29.02%、24.55%)(P0.05),受精率和卵裂率差异不显著(P0.05)。由以上得出,MFSS-IVF方法可获得较高的单精子入卵率和囊胚发育率,利用MFSS使用奶牛性控精液进行体外受精生产胚胎是可行的。     

CTC法结合体外受精比较不同方法对绵羊精子体外获能的影响

为比较不同获能方法对绵羊精子的获能效果，新鲜绵羊精液分别经肝素上游法(swim-up)、钙离子载体(IA，A23187)诱导法、Percoll离心法和Percoll离心后肝素孵育法进行精子获能的诱导后与体外成熟的绵羊卵母细胞进行体外受精，并利用金霉素荧光染色（chlortetracycline，CTC）法结合各自相应的体外受精结果对其获能效果进行检验。结果显示上游法处理后精子的获能比例和受精后的卵裂率均高于其它组, 说明肝素上游法可以较好的诱导绵羊精子获能。     

用淘汰奶牛卵巢生产体外受精奶牛胚胎的研究

本文研究了淘汰奶牛卵巢采集方法、卵丘细胞和颗粒细胞对卵母细胞体外受精后发育的影响.结果表明奶牛屠宰后取其卵巢,用剖解法可比抽吸法得到更多的可用卵母细胞(8.5:6.2枚/卵巢,P＜0.05).经成熟培养和体外受精后,无卵丘细胞包围的裸露卵母细胞卵裂率为40.2%,但仅28.2%停留在8～16细胞期,最高发育阶段为16细胞期;而有3层以上卵丘细胞紧密包围的卵丘-卵母细胞复合体,其卵裂率(60.2%:53.2%,P＜0.05)和囊胚发育率(31.5%:19.6%,P＜0.05)均高于卵丘细胞包被不全的卵母细胞.在培养液中添加颗粒细胞,提高囊胚细胞数(96.2±5.2:70.4±4.6,P＜0.05),但没有促进牛受精卵的卵裂率.     

肉羊体外胚胎高效生产技术的优化研究

为了探讨放线菌酮(CHX)对卵母细胞预成熟的影响及囊胚滋养层细胞囊泡(TVS)、维生素C对肉羊体外胚胎质量的影响,试验采用从屠宰厂(场)收集的肉羊卵巢,抽取卵巢表面2~8 mm的卵泡卵母细胞进行体外成熟、体外受精和早期胚胎体外培养。结果表明:经过5%CHX预成熟的卵母细胞卵裂率、桑葚胚率、囊胚率显著高于对照组(P0.05);在体外受精及早期胚胎培养液(SOFaa)中添加1%维生素C组与TVS共培养组的卵裂率和囊胚发育率均高于其他试验组(P0.05);体外胚胎与TVS共移植受胎率显著高于对照组(P0.05)。说明核质同期成熟处理及抗氧化剂可以用于高效生产体外胚胎。     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育比较

比较了水牛卵母细胞体外成熟时间对孤雌激活胚发育能力的影响,以及不同化学激活方法对水牛卵母细胞孤雌激活效果的影响,并在相同条件下,对孤雌激活胚与体外受精胚的发育能力进行了比较.结果表明,水牛卵母细胞体外成熟27 h或30 h的囊胚发育率(19.0%、17.7%)明显高于体外成熟21 h或24h的囊胚发育率(12.3%、13.8%);Ion联合6-DMAP激活水牛卵母细胞的效果优于其他几组激活方法;在相同条件下,孤雌激活胚与体外受精胚的发育能力存在着差异,其中卵裂率差异不显著,但孤雌激活胚的囊胚发育率显著高于体外受精胚.     

雪龙黑牛试管胚胎产业化生产培养方法的建立

为充分利用雪龙兼松屠宰场废弃的优秀母牛卵巢,加速雪龙黑牛培育进程,满足高档牛肉市场需求,本文对胚胎生产体外受精3个关键技术环节进行了试验研究,共做了15批次试验,包括卵母细胞体外成熟培养的试验研究,卵母细胞体外受精试验研究以及受精卵体外培养试验研究。结果显示,卵母细胞的成熟率达98.6%,卵裂率为86.5%,囊胚发育率为36.8%。初步建立了一整套雪龙黑牛试管胚胎产业化生产培养方法和技术。     

牛卵母细胞体外受精技术研究

本研究从3方面进行了牛卵母细胞的体外受精试验,即用不同离心法、不同温度上浮法、不同精子浓度等方法对COCs进行体外受精,测定其体外受精率。结果表明,两种离心法中,以上清液二次离心法的精子获能效果最好,受精率最高,该法首次采用低速离心技术,突破了离心法的传统方法,离心效果明显提高;不同温度条件下的精子上浮效果以CO2培养箱恒温（38.5℃）上浮法最好,受精率最高;对于选择受精时获能精子的浓度,宜控制在1.0×106～1.5×106个/ml,不仅能提高受精率,还可避免或降低多精受精现象。     

猪卵巢卵母细胞的体外成熟和体外受精

将从屠宰母猪卵巢采得的卵母细胞-卵丘细胞复合体(Oocyte-cumulus Cell Complex.OCC)在含PMSG的M199培养40～44小时,卵丘细胞大部分扩散(86.4％)。48.1％(142/295)的卵母细胞排出第一极体(PBI)。将体外成熟的卵母细胞与体外获能精子授精后30～70小时,80.5％(103/128)的卵母细胞受精并可在体外发育到2～8细胞甚至桑椹胚。本文还对裸卵母细胞的体外成熟和体外受精进行了研究,对体外受精卵的早期发育作了观察。实验结果表明:PMSG对诱导卵丘细胞扩散及卵母细胞的全面成熟有重要作用,在OCC中的卵母细胞成熟率高于裸卵母细胞体外授精后8～10小时将受精卵放入改良KRB液培养可使卵裂比例明显提高。     

牛卵母细胞体外受精和体外成熟技术

文章从卵母细胞的来源、精子体外获能、卵母细胞体外成熟、体外受精和体外受精早期胚胎培养五个方面综述了牛卵母细胞体外受精和体外成熟技术的研究进展.同时指出了影响精子体外获能的因素、影响卵母细胞成熟培养的因素、体外受精存在的问题及解决办法等.并且对影响该技术的主要因素进行了系统分析.     

牛卵母细胞体外成熟和体外受精技术的研究进展

论述牛卵母细胞体外成熟和体外受精的最新研究进展，包括卵丘卵母细胞复合体、卵母细胞的体内、体外成熟，以及体内、体外的异常成熟和卵母细胞的体外成熟方法，无蛋白质、无血清系统的限定性培养液的研究进展，卵母细胞体外成熟状态与标志的某些理论上的突破；体外受精中精子供体的选择、精子活力和正常形态的选择、冷冻解冻精液的体外获能、精子的体外受精力，以及体外的异常受精；还论述了牛卵母细胞体外成熟和体外受精技术在家畜育种和胚胎克隆等方面的应用前景     

Nuclear and cytoplasmic maturation of bovine oocytes cultured with dbc AMP, FSH and hCG

The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus.     

水牛附睾尾精子的体外受精

采用肝素诱导获能 ,比较了 TAL P液和 BO液处理水牛附睾尾精子进行体外受精和细管冷冻精液体外受精的效果。结果表明 ,用 BO液和 TAL P液分别处理水牛附睾尾精子 ,受精后的卵裂率分别为 4 6 .6 7%和 5 3.73% ,发育率分别为 2 1.6 7%和 2 6 .87% ,其受精效果差异不显著。综合 2种方法 ,水牛附睾尾精子的受精率为 5 7.14 % ,受精后的卵裂率为 5 0 .39% ;发育率相对于培养卵为 2 4 .4 1% ,相对于卵裂卵为 4 8.4 4 % ;与细管冷冻精液 (5 6 .0 0 % ,5 4 .31% ,2 6 .72 % ,4 9.2 1% )相比 ,差异不显著。形态学观察还表明 ,用保温干储的方法可获得活率好、存活时间长的附睾尾精子。试验结果说明水牛附睾尾精子用于体外受精可以得到与细管冷冻精液相当的效果。     

半胱胺对牛卵母细胞早期胚胎发育能力的影响

半胱胺（Cysteamine）是一种重要的巯基物,在体外培养体系中添加半胱胺能够增强谷胱甘肽（GSH）的合成和胚胎发育能力。本研究旨在探讨不同浓度半胱胺处理对牛卵母细胞体外成熟和早期胚胎发育能力的影响。结果表明,在牛卵母细胞体外成熟液中添加100μM的半胱胺,其囊胚率与对照组有显著差异;但是,与同时在成熟液和发育液中都添加100μM的半胱胺组差异不显著。研究结果也表明,在牛卵母细胞体外成熟培养液中添加半胱胺会提高牛卵母细胞的囊胚发育率,而且随着浓度的增加这种作用将达到饱和,其最佳作用浓度为100μM。     

牛成熟卵母细胞体外受精的研究进展

这篇论文论述了牛成熟卵母细胞体外受精的研究进展,主要从精子的制备方法,获能液的组成、精子获能的判定、受精方,法以及卵母细胞受精与否的评定5个方面加以阐述,并展望了体外受精的研究方向。     

Effect of cumulus cell removal and sperm pre‐incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre‐incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre‐incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre‐incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3–4 cell stage zygotes were obtained with fresh sperm pre‐incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.     

Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes

Routinely, swim‐up method is used to separate high‐quality sperm; however, long processing time and close cell‐to‐cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex^? and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus–oocyte complexes (COC s) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO ₂ incubator at 38.5°C and 5% CO ₂. Matured COC s were rinsed twice in fertilization TALP and placed in the pre‐warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex^?, glass wool filtration and swim‐up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15–20 min in CO ₂ incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co‐incubation with sets of 10–15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA , while in vitro fertilizing rates were compared by chi‐squared test using SPSS ‐20. Least significant difference (LSD ) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex^? filtration improved (p  <  .05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim‐up (control). In conclusion, cryopreserved Nili‐Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo.