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1.
牛体外成熟卵母细胞冷冻保存的研究 总被引：10，自引：1，他引：9
本实验探讨了用６各不同方法冷冻保存的牛体外成熟卵母细胞体受精后的发育潜力及其冷冻损伤，尽管用不同方法冷冻的牛卵母细胞多数（８６．１％）形态正常，但体外受精后的受精率（３０．０％）和卵裂率（７．４％）明显低于对照组（６９．０％和５２．９％）。卵裂后继续发育的能力严重受损，在６种冷冻方法中，１．６Ｍ丙二醇分步平衡、程序冷冻效果最好，受精率和卵裂率分别达３６．４％和１３．５％，冷冻后卵母细胞发育潜力下降 相似文献
2.
将不同直径牛卵泡内卵母细胞体外成熟培养22h后，对其体外受精后的卵裂率，胚胎发育速度和胚胎染色体异常发生率进行了研究。结果表明，体外受精后48h，直径3－6mm卵泡内卵母细胞外受精后的卵裂率明显高于直径1－2mm和7－10mm卵泡内卵母细胞体外受精后的卵裂率（P＜0．05）。体外受精后48h，直径3－6mm包泡内卵母细胞体外受精后的胚胎的发育速度明显快于直径1－2mm和7－10mm卵泡组胚胎（P＜0．05）。通过对不同卵裂期胚胎的染色体标本分析表明，直径1－2mm卵泡内卵母细胞体外受精后的胚胎染色体异常发生率明显高于直径3－6mm卵泡组（P＜0．05），但与直径7－10mm卵泡组之间无显著性差异。 相似文献
3.
试验选用5-6周龄雌性昆明小鼠的成熟卵母细胞，在不同前处理液（10％EG或10％EG＋10％DMSO）中平衡5min，然后在冷冻溶液（EFS30、EFS40、EDFS30或EDFS40）中平衡30s后进行OPS法和SSV法玻璃化冷冻保存。试验结果表明：（1）小鼠成熟卵母细胞的OPS法冷冻保存，用EFS冷冻的卵母细胞解冻后形态正常率为75．8％，激活后卵裂率为47．8％；在EDFS30中冷冻保存，解冻后形态正常率为87．2％，卵裂率为62．0％；在EDFS40中冷冻保存，解冻后形态正常率为86．6％，卵裂率为57．4％，与对照组（99．0％和80．5％）相比，差异极显著（P〈0．01）。（2）小鼠成熟卵母细胞的SSV法冷冻保存，用EFS冷冻的卵母细胞解冻后形态正常率为82．7％，卵裂率为47．1％；在EDFS30中冷冻保存，解冻后形态正常率为91．5％，卵裂率为57．5％；在EDFS40中冷冻保存，解冻后形态正常率最高为85．1％，卵裂率为55．9％，与对照组（99．0％和80．5％）相比，差异极显著（P〈0．01）。（3）OPS法和SSV法冷冻小鼠成熟卵母细胞解冻后形态正常率为86．5％和91．6％，卵裂率为53．8％和55．6％．与对照绢相比。差异极显著（P〈0．01）。 相似文献
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牛性机能发育阶段与卵泡、黄体因素对卵母细胞体外成熟及体外受精的影响 总被引：1，自引：0，他引：1
利用屠宰母牛卵巢．对来自不同性机能发育阶段母牛的卵巢卵母细胞．不同状态卵巢卵泡的卵母细胞的体外成熟（ＩＶＭ）、体外受精（ＩＶＦ）进行了系列研究。结果表明．来自初情期、性成熟母牛卵巢的卵母细胞受精后的卵裂率（ ７４． ７％和 ８１． ５％）、囊胚率（ ２３．０％和 ２６． ８％）显著高于来自初情期前母牛卵巢卵母细胞的卵裂率（１８．８％）和囊胚率（１．８％）,Ｐ＜０．０５;有黄体卵巢的卵母细胞体外受精后的发育能力显著低于无黄体卵巢卵母细胞（卵裂率５８．０％：７９． ９％,囊胚率１３．％：２７．１％,Ｐ＜０．０５）;优势卵泡的有无对卵母细胞的卵裂率和囊胚率无明显影响。 相似文献
6.
对MII期水牛卵母细胞进行人工诱导激活可以帮助人们间接判断体外成熟卵母细胞质量的优劣。并且，卵母细胞的充分激活也是提高核移植效率的关键因素之一。试验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小时或30小时的囊胚发育率（19．0％或17．7％）明显高于体外成熟21小时或24小时的囊胚发育率（12．3％或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（13．0％vs21．7％）。 相似文献
7.
本试验通过牛卵母细胞玻璃化冷冻过程中采用载体、冷冻时卵母细胞所带颗粒细胞层数和其成熟培养时间这3个方面对牛卵母细胞的玻璃化冷冻进行了探讨。(1)不同载体的对比:玻璃OPS管和塑料OPS管冷冻效果较好。玻璃OPS管冷冻卵母细胞,解冻后形态正常率、体外受精后卵裂率和囊胚率分别为91.2%、41.3%和14.52%。塑料OPS管冷冻卵母细胞,解冻后形态正常率、体外受精后卵裂率和囊胚率分别为89.09%、40.90%和14.54%。(2)保留2￣3层颗粒细胞的卵母细胞玻璃化冷冻的冷冻效果较好,其冷冻解冻后形态正常率、体外受精后卵裂率和囊胚率分别为90.79%、40.13%和15.13%。(3)体外成熟培养22h的卵母细胞冷冻效果较好,其冷冻解冻后形态正常率、体外受精后卵裂率和囊胚率分别为94.14%、41.80%和15.23%。 相似文献
8.
本试验主要比较了离子霉素、电脉冲两种方法激活牛、羊体外成熟卵母细胞的效率。两种激活方法中。牛胚胎卵裂率无显著差异（90．61％对94．40％，P〉0．05），而离子霉素激活胚胎的囊胚发育率极显著高于电激活方法（12．3％对2．4％，P〈0．01）。两种方法对羊胚胎的研究中，羊胚胎卵裂率无显著差异（72．4％对77．4％，P〉0．05）。但是离子霉素激活胚胎的囊胚发育率显著高于电激活方法（3．67％对10．40％，P〈0．05）。本试验中还比较了用化学激活法（离子霉素）激活牛体外成熟卵母细胞后，用SOFaa体系培养，换液与不换液对孤雌激活胚胎体外发育的影响。结果表明：在第4天不换液的胚胎卵裂率和囊胚率极显著高于换液的胚胎（11．64％对3．49％。P〈0．01）。 相似文献
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牛胚胎体外生产技术的简化研究 总被引：2，自引：0，他引：2
利用屠宰牛卵巢分离的卵母细胞，以卵裂率和囊胚发育率为标准，对牛胚胎体外生产过程进行了简化试验。结果显示：不加卵丘细胞的高密度卵母细胞体外成熟（１００～２００枚／平皿）可代替加卵丘细胞的标准密度（５０枚／平皿）培养，其卵裂率（５７．６％比６２．５％）和囊胚发育率（２３．７％比２９．１％）没有显著差异（Ｐ＞０．０５），体外授精时间可从成熟培养后２２ｈ延长至２７ｈ，卵裂率和囊胚发育率均没有显著变化，卵母细胞体外成熟后可以不经洗涤直接移入受精滴，原成熟培养皿内残留卵丘细胞再培养４８ｈ形成的单层细胞，可代替标准方法制作的单层小滴用于体外受精胚胎的共同培养，供胚胎发育培养的单层细胞使用多次不影响囊胚发育率，经简化的ＩＶＦ技术获得的胚胎非手术移入情期同步受体牛子宫角２～２．５个月后直检有５８．３％（７／１２）妊娠。我们认为，传统的牛胚胎体外生产技术经过简化可以提高生产效率，不会减少囊胚发育率和妊娠率，值得推广应用。 相似文献
11.
In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection
Matsukawa K Akagi S Adachi N Sato F Hasegawa T Takahashi S 《The Journal of reproduction and development》2007,53(4):877-885
In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing. 相似文献
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采用PBS作为冷冻基础液,分别用甘油和二甲基亚砜(DMSO)作为冷冻保护液,在程序化冷冻保存和玻璃化冷冻保存条件下,研究小鼠生发泡期(GV期)卵母细胞的抗冻能力。结果表明,2种冷冻方法对小鼠GV期卵母细胞解冻后形态正常率和存活率无显著影响(P>0.05)。冷冻保护剂种类对小鼠GV期卵母细胞解冻后形态正常率无显著影响(P>0.05);但对存活率有显著影响,玻璃化冷冻采用二甲基亚砜作为冷冻保护液效果极显著优于甘油(P<0.01)。以冷冻效果较好的二甲基亚砜作为冷冻保护液,采用玻璃化冷冻不同发育阶段(GV期和MⅡ期)的小鼠卵母细胞,解冻后形态正常率无显著差异(P>0.05),但存活率GV期要显著优于MⅡ期卵母细胞(P<0.05)。 相似文献
14.
①用EFS30、EFS40、EDFS30、EDFS40四种玻璃化冷冻液对MⅡ期水牛卵母细胞进行毒性试验,结果表明:试验组卵母细胞形态正常率与对照组均无显著性差异(P>0.05);对卵母细胞孤雌激活后EDFS30、EDFS40组的卵裂率与对照组(75.28%)及EFS30、EFS40组差异显著(P<0.05);利用4种冷冻保护剂采用OPS法冷冻保存MⅡ期水牛卵母细胞,其中以EDFS40作为冷冻液时,卵母细胞冷冻解冻后孤雌激活卵裂率最高,达31.60%;以EDFS40作为冷冻液,比较了GMP法和OPS法的冷冻效果,结果表明GMP法冷冻效果好于OPS法。②采用不同预处理时间和平衡时间使用细管法常规冷冻G V期卵母细胞,结果表明预处理5 min、平衡15min组的形态正常率和极体排出率相对较高,分别为72.73%、27.27%。 相似文献
15.
Chian RC Kuwayama M Tan L Tan J Kato O Nagai T 《The Journal of reproduction and development》2004,50(6):685-696
Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology (ART). Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have attempted to improve the survival rates of oocytes by a vitrification technique using bovine models. In vitro matured oocytes with or without cumulus cells were vitrified with either 15.0% (v/v) ethylene glycol (EG) + 15% (v/v) dimethylsulfoxide (DMSO) + 0.5 M sucrose or 15% (v/v) EG + 15% (v/v) 1,2-propanediol (PROH) + 0.5 M sucrose, using 'Cryotop' or 'thin plastic sticker', respectively. The oocyte survival rates after vitrifying-warming, and the capacity for fertilization and embryonic development were examined in vitro. The rate of embryonic development to blastocyst was significantly higher (P<0.05) in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose than in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose (7.4% +/- 4.1 vs. 1.7% +/- 3.0, respectively). Oocytes vitrified without cumulus cells had a higher survival rate after thawing and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. Prolonged pre-incubation time after thawing adversely affected the rates of embryonic cleavage and development. These results indicate that in vitro matured bovine oocytes can be vitrified successfully with the mixture of the cryoprotectants, EG + PROH, the absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and vitrified and warmed oocytes do not require pre-incubation before in vitro fertilization. 相似文献
16.
The fertilization and cleavage of bovine oocytes matured by intra- or extra-follicular methods were investigated. Oocytes were fertilized in vitro or in the rabbit oviduct and cleavage was assessed after in vitro culture of in vitro fertilized oocytes and after in vivo culture (rabbit oviducts) of xenogenously fertilized oocytes. The effect of fertilization with fresh-diluted or frozen-thawed semen were also examined. The intra-follicular method did not increase the nuclear maturation rate as compared with the extra-follicular method (57.9 and 52.7%, respectively). However, the proportions of in vitro fertilized eggs (54.8%) and of cleaved eggs (two to eight cells; 34.6%) in the rabbit oviduct for 48 h after xenogenous fertilization were higher (P less than .025) in the intra-follicular oocytes than those of the extra-follicular oocytes (37.1 and 21.3%, respectively). It was also found that the use of fresh-diluted semen resulted in more cleaved eggs from the rabbit oviduct than the use of frozen-thawed semen (43.4 and 23.3% in the intra-follicular oocytes, P less than .025; 31.0 and 7.8% in the extra-follicular oocytes, P less than .05), while the appearance of cleaved eggs following in vitro fertilization was extremely low (0 to 6.6%). The present results demonstrated that the intra-follicular culture method of bovine oocytes provided a physiological environment for cytoplasmic maturation leading to higher fertilizability and development than the conventional in vitro culture of extra-follicular oocytes. 相似文献
17.
Kohaya N Fujiwara K Ito J Kashiwazaki N 《The Journal of reproduction and development》2011,57(6):675-680
Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility. 相似文献
18.
Weiping HUANG Masashi NAGANO Sung-Sik KANG Yojiro YANAGAWA Yoshiyuki TAKAHASHI 《The Journal of reproduction and development》2014,60(1):9-13
The objective of this study was to clarify the effects of prematurational culture
(pre-IVM) supplemented with 3-isobutyl-1-methylxanthine (IBMX) on nuclear and cytoplasmic
maturation of in vitro-grown bovine oocytes. In experiment 1, oocytes (95
μm in diameter) derived from early antral follicles (0.5–1 mm in diameter) were cultured
for 12 days for in vitro growth (IVG). IVG oocytes with a normal
appearance were subjected to examinations of diameter and chromatin structure in the
germinal vesicle (GV) before IVM. In addition, percentages of metaphase II (M II) were
examined after IVM. Regardless of pre-IVM, the mean diameters of IVG oocytes were about
115 μm. The proportions of GV3 (50.0%) and M II stages (80.1%) of IVG oocytes with pre-IVM
were higher than those without pre-IVM (28.0 and 49.4%, respectively). In experiment 2,
the fertilizability and developmental competence of IVG oocytes were examined. Regardless
of pre-IVM, the normal fertilization rates of IVG oocytes were similar (around 70%) but
were lower than that of in vivo-grown oocytes (88.0%). Cleavage and
blastocyst rates of IVG oocytes with pre-IVM (63.0 and 26.1%, respectively) were higher
than those without pre-IVM (45.8 and 12.7%, respectively). The blastocyst rate based on
cleaved IVG oocytes with pre-IVM (41.7%) was similar to that of in
vivo-grown oocytes (48.7%), although the cleavage rate of IVG oocytes with
pre-IVM was lower than that of in vivo-grown oocytes. In conclusion,
pre-IVM with IBMX improved the maturational and developmental competences of IVG oocytes,
probably due to promotion of their chromatin transition and synchronization of meiotic
progression. 相似文献
19.
Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm 
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下载免费PDF全文 Katsuyoshi Fujiwara Maki Kamoshita Tsubasa Kato Junya Ito Naomi Kashiwazaki 《Animal Science Journal》2017,88(1):180-184
The objective of this study was to evaluate fertility and full‐term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co‐cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full‐term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm. 相似文献