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1.
澳洲波尔山羊胚胎3种冷冻方法对其胚胎移植效果的影响 总被引：2，自引：0，他引：2
在25℃室温下,采用细管法(一步法、二步法)和OPS法,以不同浓度的EFS、EDFS为玻璃化冷冻液,对澳洲波尔山羊致密桑椹胚和囊胚进行玻璃化冷冻保存。同时利用1.5mol/LEG为抗冻保护剂对胚胎进行常规法冷冻保存。分别将上述3种方法冷冻解冻后的胚胎移植于同期发情后6~7d的云南黑山羊受体。结果表明,细管法胚胎玻璃化冷冻保存效果均以EFS40组为佳,解冻后胚胎移植产羔率分别为40.54%(15/37;一步法)和51.35%(19/37;二步法)。与新鲜胚胎移植产羔率(52.50%,21/40)和常规法冷冻保存的胚胎移植产羔率(45.16%,14/31)相比无显著性差异(P>0.05)。另外,用EDFS30玻璃化溶液,OPS法冷冻解冻后的胚胎移植产羔率高达51.43%(18/35),为整个玻璃化冷冻试验的最佳值。玻璃化冷冻方法简便、迅速,无论是细管法还是OPS法均获得了比较理想的胚胎移植效果。 相似文献
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自1985年胚胎玻璃化冷冻(v itrification)保存技术发明以来,玻璃化法先后在小鼠、兔、绵羊、牛、猪、山羊等动物胚胎上获得成功,近年来有关哺乳动物胚胎玻璃化冷冻保存的研究主要集中在冷冻和解冻方法上,相继发明了一些新的玻璃化冷冻方法:冷环玻璃化法(cryo loop)和开放式细管法(open pu lled straw,OPS),并且对解冻后细管内直接脱除防冻剂进行了广泛深入的研究,使得冷冻胚胎移植更易于在生产上推广应用。现将胚胎玻璃化冷冻的原理、冷冻保护剂、冷冻方法、解冻后保护剂脱除方法的最新研究进展作一综述。 相似文献
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不同冷冻和解冻方法对小鼠桑椹胚发育的影响 总被引：1，自引：0，他引：1
本试验以2种程序化冷冻液和2种玻璃化冷冻液对昆明白系小鼠的桑椹胚进行细管法冷冻保存,比较程序化冷冻-管外解冻和玻璃化冷冻-管内解冻对胚胎体内、外发育的影响。胚胎体外培养结果表明:玻璃化冷冻组及程序化冷冻组胚胎发育率(95.3%￣95.8%,98.9%)无显著(P>0.05)差异。将程序化冷冻、EFS30玻璃化冷冻以及新鲜的胚胎各168枚移植给假孕受体鼠,妊娠受体产活仔率各组间相比(50.8%,58.3%,54.9%)无显著性(P>0.05)差异。结果证明,玻璃化冷冻保存的胚胎管内解冻效果好,为生产中家畜的胚胎移植提供了理论和技术参考。 相似文献
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小鼠2-细胞胚胎细管法和OPS法玻璃化冷冻保存技术的研究 总被引：8，自引：0，他引：8
本试验在室温 (2 0℃和 2 5℃ )条件下 ,利用不同浓度的玻璃化溶液 (EFS和EDFS) ,对小鼠 2 细胞胚胎进行细管法和OPS法玻璃化冷冻保存。在 2 0℃室温条件下 ,用EFS4 0平衡 1min细管一步法冷冻 ,解冻后囊胚发育率仅为35 .0 % ,和新鲜 2 细胞体外培养的对照组 (6 5 .0 % )的差异极显著 (P <0 .0 1)。当 2 细胞胚胎在 10 %EG +10 %D溶液中预处理 5min ,再移入EDFS中平衡 30s二步法冷冻保存 ,解冻后囊胚发育率达 4 7.8%～ 4 8.8% ;当室温升至2 5℃时 ,二步法冷冻保存后 2 细胞的囊胚发育率达到 5 2 .2 % ,与对照组无显著差异 (P >0 .0 5 )。改用OPS二步法EFS30冷冻组保存后的 2 细胞胚胎的囊胚发育率高达 6 2 .2 % ,为试验中的最佳组。用最佳细管法和OPS法冷冻组解冻后培养至囊胚移植给受体母鼠均获得产仔 相似文献
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近17年来,采用建立起来的牛非手术采卵及冷冻方法,已能将牛桑葚胚和囊胚冷冻,并获得较高的存活率。现在试验中和商业上牛胚胎冷冻大多数是采取控制降温和解冻的程序。解冻后存活率按形态评定可达90％～100％,非手术移植的妊娠率为60％。绵羊、山羊和马的桑葚胚和囊胚用控制降温和解冻程序也能有效的冷冻。简化的控制降温和解冻程序的一步法用于牛胚胎,妊娠率可达50％。此法免去了解冻后较繁琐的脱除冷冻保护剂和胚胎评定过程。玻璃化法和快速冷冻法是较先进的方法,可大大缩短胚胎冷冻时间,而且无需复杂的冷冻设备。不过,迄今家畜胚胎冷冻的结果还是有待提高的,尽管牛、绵羊、山羊桑葚胚和囊胚用玻璃化法冷冻已经成功,移植后产了仔。另外,卵母细胞、早期胚胎、显微手术(切割、克隆、活组织检查)后的胚胎、猪胚胎冷冻成功率还很低。因此,为使胚胎移植技术能在生产中应用和开展胚胎显微操作、体外受精、克隆和基因转移之类生物技术的试验及应用,很有必要加强研究建立起更为有效的胚胎冷冻解冻方法。 相似文献
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试验首次采用OPS法玻璃化冷冻小鼠GV期卵母细胞(不带卵丘细胞,下同),同时尝试用EDFS30对小鼠卵巢进行细管法玻璃化冷冻,以研究GV期卵母细胞冷冻后的发育潜力。首先,利用MEM培养和MEM-腔前卵泡培养新鲜GV期卵母细胞,并把较好的培养方式用于冷冻后培养试验。2种培养方式培养24h后新鲜GV期卵母细胞成熟率无显著性差异;OPS法冷冻的GV期卵母细胞解冻后成熟率及体外受精后卵裂率与对照组差异不显著(P>0.05)。细管法冷冻卵巢组织的GV期卵母细胞成熟率极显著低于对照组(P<0.01),其受精后未获得受精卵。结果表明:OPS法可有效地冷冻保存小鼠GV期卵母细胞,而细管法冷冻小鼠卵巢对GV期卵母细胞损伤较大。 相似文献
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Conventional freezing of in vitro-produced and biopsied bovine blastocysts in the presence of a low concentration of glycerol and sucrose 总被引：1，自引：0，他引：1
The purpose of this study was to develop a practical cryopreservation method for in vitro-produced (IVP) and sex-predetermined bovine blastocysts that will be applicable to direct transfer of the post-thaw embryos. Blastocysts were harvested 7 days after IVF and allocated to either an intact or biopsy group. The cryoprotective solution contained 0.7 M glycerol and 0, 0.05 or 0.1 M sucrose. Slow cooling at a rate of -0.5 C/min was terminated at -25, -30, or -35 C, and rapid cooling in liquid nitrogen was followed. After one-step thawing and dilution, the IVP blastocysts were cultured for 3 days to assess their survival. The post-thaw survival rate of intact blastocysts after termination of slow cooling at -30 C in 0.7 M glycerol plus 0.1 M sucrose (96.2%) was significantly higher than that at -25 C in 0.7 M glycerol alone (44.4%). The post-thaw survival rate of biopsied bovine blastocysts after termination of slow cooling at -25 C in 0.7 M glycerol alone (53.8%) tended to be lower than that at -25 C in 0.7 M glycerol plus 0.05 M sucrose (91.3%) or -30 C in 0.7 M glycerol plus 0.1 M sucrose (92.3%). Thus, addition of a small amount of sucrose to 0.7 M glycerol cryoprotective solution shortened the process of slow cooling for both the intact and biopsied bovine embryos. Judged from the survival levels in vitro after thawing and one-step dilution of embryos (>80%), this is an improved method of cryopreservation for subsequent direct transfer of IVP and biopsied bovine blastocysts. 相似文献
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[目的]为了增加精液数量,提高种公牛的利用率,降低精液能量消耗,补充适当营养和保护物质的活动延长精子的寿命,有利于精子的冷冻和保存。[方法]冷冻精液稀释液的筛选采用正交设计方法并使用L9（3^4）正交表安排试验,优选出冷冻保存效果最佳的稀释液配方,再用较好的稀释液进行比较试验,得出最理想的冷冻稀释液。对要进行试验的稀释液,试验时先将同一头BMY牛精液分成九等分,依次标记为1-9号,然后在精液中分别加入等量的对应编号的冷冻稀释液,进行稀释冷冻效果的比较。[结果]B糖不同水平之间的差异极显著（P〈0.01）,B1与B3的差异极显著,与B2的差异不显著,B2与B3的差异不明显;最佳的组合是A2B1C1。添加不同种氨基酸和水平的稀释液BMY牛精液冻精解冻效果均无明显差异（P〉0.05）,添加不同氨基酸和水平的稀释液稀释BMY牛原精的效果亦无明显差异。 相似文献
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Hiroshi Tajimi Tsugumi Yamazaki Syoko Oike Tatsuyuki Yoshida Konosuke Okada Masashige Kuwayama Hitoshi Ushijima 《Animal Science Journal》2018,89(8):1194-1200
This study was conducted to examine the utility of vitrification for bovine embryos with low‐quality grade, and simple cryoprotectants dilution method for practitioners. In Experiment 1, survival of frozen embryos was compared with that of vitrified embryos using minimum volume cooling (MVC). Then, vitrified embryos were used to confirm the optimum sucrose concentration in Experiment 2. The survival rates of embryos that had been vitrified following diluted cryoprotectants with the one‐step in‐straw method were compared with those of fresh control embryos in Experiment 3. Frozen‐thawed or vitrified‐warmed blastocysts were cultured with TCM‐199 supplemented with 100 μmol/L beta‐mercaptoethanol +5% fetal bovine serum at 38.5°C in an atmosphere of 5% CO2 in air, their survival after 24 hr were compared. The development to term of fair quality in vivo embryos after vitrification was examined in Experiment 4. Results show that survival rates of frozen‐thawed embryos were lower (p < .05) than that of vitrified‐warmed ones. When vitrified embryos were warmed in 0.3 mol/L sucrose in straws, their survival rate was 100%. The total cell numbers of vitrified‐warmed embryos were comparable to those of fresh control embryos. The six calves from 13 vitrified embryos were delivered in Experiment 4. These results indicate that MVC vitrification following one‐step cryoprotectants dilution is utilized to preserve low‐quality bovine embryos. 相似文献
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Naomi NAKAGATA Toru TAKEO Kiyoko FUKUMOTO Yukie HARUGUCHI Tomoko KONDO Yumi TAKESHITA Yuko NAKAMUTA Tomoko UMENO Shuuji TSUCHIYAMA 《The Journal of reproduction and development》2014,60(2):168-171
Sperm cryopreservation has been widely adopted for maintenance of the genetically
engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes
worldwide. However, the recipients are not always able to obtain high fertilization rates
with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm
via various methods and performed in vitro
fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm
preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In
addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF
in the same manner. The fertilization rates of both the sperm cryopreserved
via the methods applied in some countries and the cryopreserved GEM
sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that
the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain
of frozen mouse sperm. 相似文献
15.
Erika HAYASHI Sayaka WAKAYAMA Daiyu ITO Ayumi HASEGAWA Keiji MOCHIDA Masatoshi OOGA Atsuo OGURA Teruhiko WAKAYAMA 《The Journal of reproduction and development》2022,68(2):118
Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at −80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at −80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at −80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed. 相似文献
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The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196 degrees C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitro culturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitro without freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitro culturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival. 相似文献
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稀释方法对绒山羊冷冻精液品质的影响 总被引：2，自引：2，他引：0
选取7只精液品质优良的雁门绒山羊种公羊,通过测定其冷冻精液解冻后的精子活率（0、4 h）、精子存活指数、顶体完整率及精清中透明质酸酶（HYD）、谷草转氨酶（GOT）、乳酸脱氢酶（LDH）酶活力,研究一步稀释法和二步稀释法对冷冻精液品质的影响。结果表明,二步稀释法解冻后的精子存活指数显著（P＜0.05）高于一步稀释法,其解冻后精清中透明质酸酶、谷草转氨酶、乳酸脱氢酶的活力均显著（P＜0.05）低于一步稀释法。表明采用二步稀释法冷冻的绒山羊精液品质好于一步稀释法。 相似文献
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转基因兔胚胎玻璃化冷冻保存的研究 总被引：4，自引：0，他引：4
在２５℃条件下,将兔体外受精精子载体转基因兔桑椹胚置于含有４０％乙二醇、１８％Ｆｉｃｏｌ、０．３ｍｏｌ蔗糖的ｍＰＢＳ溶液（ＥＦＳ４０）中平衡２分钟,然后直接投入液氮,成功地进行了玻璃化冷冻保存。解冻后桑椹胚发育至囊胚和孵化囊胚的比例分别为６５．８１％和３９．２４％,与未经冷冻的鲜胚发育比例（７１．０５％和４３．４２％）相比,没有明显的差异。７８枚经玻璃化冷冻和解冻的桑椹胚移植给５只受体,其中２只妊娠,共产下８只活仔兔。 相似文献
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RE Green BFS Santos CC Sicherle FC Landim-Alvarenga SD Bicudo 《Reproduction in domestic animals》2009,44(3):406-410
The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions. 相似文献
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Transfer of frozen-thawed embryos in sheep 总被引：3，自引：0，他引：3
Embryos collected from ewes six days after oestrus were frozen in straws using ethylene-glycol as a cryoprotectant. The efficiency of the simplified freezing and thawing procedure was assessed after transfer, which resulted in an overall survival rate of 58.3 per cent. Forty-two lambs were born from 72 frozen embryos which had been transferred without any attempt to evaluate them after the thawing and sucrose dilution process. 相似文献