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1.
不同培养体系对牛胚胎体外发育的影响 总被引：2，自引：0，他引：2
采用离子霉素和6-二甲氨基嘌呤(6-DMAP)对牛体外成熟卵母细胞进行联合激活,激活后采用不同的培养体系进行体外培养,观察不同的培养液对牛孤雌激活胚体外发育能力的影响。3种培养体系分别为:A(0~48 h:CR1aa+3 mg/mL BSA;48 h~7 d:CR1aa+10%FBS),B(连续7 d均为SOFaa+3 mg/mL BSA),C(0~5 d:SOFaa+3 mg/mL BSA;6~7 d:SOFaa+10%FBS)。结果表明:3种培养液对卵裂率没有显著性影响,分别为87.22%,94.33%和91.30%,但囊胚发育率存在显著性差异,以A效果最好,其囊胚发育率为25.56%;C次之,囊胚发育率为11.80%;B最低,囊胚发育率为3.55%。之后选择最佳的培养液进行体外受精实验,结果表明CR1aa可用做牛胚胎体外生产的培养液。 相似文献
2.
利用Leptin和ITS促进体外成熟和体外培养的牛卵母细胞的发育和质量,探讨提高胚胎体外生产的质量和数量的方法和技术。试验1：体外受精胚胎的培养液：添加BSA的KSOM中添加10mL/L浓度的ITS,结果使胚胎的桑椹胚率和囊胚率显著（P〈0.05）高于培养液中不加ITS的对照组（桑椹胚率：43.48%vs29.07%,囊胚率：22.83%vs11.63%）,卵裂率、正常分裂率和8细胞率与对照组差异不显著（P〉0.05）。试验2：在卵母细胞的体外成熟液中添加10μg/L具有生物学活性的重组鸡Leptin成熟肽融合蛋白,Leptin处理组和对照组卵母细胞经体外成熟、受精后转入加有10mL/LITS的KSOM培养液进行体外培养。试验组卵裂率和正常分裂率极显著（P〈0.01）高于对照组（88.96%vs66.81%和61.11%vs29.36%）,8细胞率显著（P〈0.05）高于对照组（84.84%vs69.57%）。Leptin处理的卵母细胞在受精后的桑椹胚率和囊胚率与对照组差异不显著,但最后的囊胚数量较对照组增加1倍多,分别为IVF卵母细胞总数的14.8%和6.4%（P〈0.01）。这说明,添加Leptin对牛卵母细胞体外成熟有促进作用,可显著提高卵母细胞受精后早期胚胎的卵裂率、正常分裂率和8细胞率;加入ITS则能提高桑椹胚率和囊胚率;而Leptin和ITS的按顺序结合使用,则能大大增加体外生产胚胎的桑椹胚和囊胚的数量,从而提高胚胎体外生产的效率。 相似文献
3.
猪卵母细胞的体外成熟影响因素的研究 总被引：4，自引：1，他引：4
本试验研究了猪卵母细胞的体外成熟体系，着重研究了季节因素、FCS、pFF、胰岛素和17β-E2对猪卵母细胞体外成熟的影响。结果表明：①3月~4月和10月~11月卵母细胞成熟率最高，与其它月份相比差异显著（P<0.05）；②卵泡期卵母细胞成熟率和卵裂率均显著高于黄体期；③添加10%FCS、10% pFF、20% FCS和10% FCS+10% pFF的卵裂率差异不显著（P>0.05），添加10% FCS或10% pFF的成熟率差异不显著，与添加20% FCS或10% FCS+10% pFF成熟率差异显著；④培养液中添加一定量的胰岛素和17β-E2有助于提高成熟率和卵裂率。 相似文献
4.
为了探讨5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对德保黑猪手工克隆（HMC）重构胚胎体外发育效果的影响，本研究分别从供体细胞和重构胚入手，比较了5个不同处理浓度（0、5、10、20和40 nmol/L）5-Aza-CdR处理HMC重构胚的体外发育效果，筛选最佳处理浓度；在最佳浓度下比较5个不同处理时间（0、24、48、72和96 h）对HMC重构胚的体外发育效果，筛选最佳处理时间；用4个不同浓度（0、0.25、0.5和1 μmol/L）5-Aza-CdR结合最佳浓度和最佳时间处理供体和重构胚，比较其体外发育潜能。结果显示，与空白对照组相比，5、10、20和40 nmol/L 5-Aza-CdR处理72 h对重构胚卵裂率均无显著差异（P>0.05），20 nmol/L 5-Aza-CdR处理能显著提高重构胚的囊胚率（P<0.05），10和20 nmol/L 5-Aza-CdR处理均能显著提高囊胚细胞数（P<0.05），其中以20 nmol/L 5-Aza-CdR效果最佳；与空白对照组相比，利用20 nmol/L 5-Aza-CdR处理HMC重构胚72 h能显著提高重构胚的囊胚率和囊胚细胞数（P<0.05），其余处理时间对重构胚卵裂率、囊胚率和囊胚细胞数均无显著影响（P>0.05）；在囊胚的最佳处理浓度（20 nmol/L）和最佳处理时间（72 h）下，结合供体的4个处理浓度（0、0.25、0.5和1 μmol/L），同时处理重构胚和供体，各处理组HMC重构胚的发育潜能均有提高，但效果均不显著（P>0.05），其中0.25~0.5 μmol/L 5-Aza-CdR处理效果较佳。综上表明，适宜浓度（0.25~0.5 μmol/L）的DNA甲基化酶抑制剂5-Aza-CdR处理供体细胞72 h并结合20 nmol/L 5-Aza-CdR处理重构胚72 h均能有效提高德保黑猪HMC重构胚胎的体外发育潜能，该结果可为今后研究德保黑猪HMC胚胎DNA甲基化调控机制提供参考。 相似文献
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6.
不同共培养细胞类型对绵羊孤雌生殖胚发育的影响 总被引：2，自引：0，他引：2
体外成熟的绵羊卵母细胞经孤雌激活4 h后,分别在5种体细胞单层(成熟前颗粒细胞、成熟后颗粒细胞、输卵管壶腹部上皮细胞、输卵管峡部上皮细胞、子宫内膜细胞)中进行共培养,比较其对绵羊孤雌生殖胚体外发育的影响.结果表明:成熟前后颗粒细胞对绵羊孤雌胚体外发育的卵裂率和囊胚率差异不显著(P>0.05);但输卵管壶腹部上皮细胞(27.5%)、子宫内膜细胞组囊胚率(27.7%)高于输卵管峡部上皮细胞和颗粒细胞(17.7%、20.3%),且差异显著(P<0.05);壶腹部上皮细胞与子宫内膜细胞在囊胚率上差异不显著(P>0.05).说明在绵羊孤雌生殖胚体外发育中,选择成熟前及成熟后的颗粒细胞都可达到相同的共培养效果;使用输卵管壶腹部上皮细胞、子宫内膜细胞进行共培养效果优于输卵管峡部上皮细胞和颗粒细胞,可以得到较高的囊胚率. 相似文献
7.
研究培养液中血清的添加时间和添加浓度及不同培养体系对牛孤雌胚胎体外发育潜力的影响.结果表明:牛成熟卵母细胞孤雌激活后的第3天添加10%的FBS对孤雌胚的体外发育效果良好.4 种培养体系在孤雌激活后的第3天添加10%的FBS,平均囊胚率分别可达46.25%(SOFaa)、43.58%(CRlaa)、40.25%(KSOMaa)、17.98%(TCM199);TCM199组的囊胚率显著低于其他3组(P＜0.05),SOFaa优于CRlaa和KSOMaa组,但差异不显著(P＞O.05). 相似文献
8.
采用不同型血清分别添加到TCM199和mTCM199培养液、RPMI1640和mRPMI1640培养液中，对兔原核期受精卵进行了体外序贯培养，并对各组间不同时期发育率进行了分析比较。结果显示：体外培养至第72h时，3个体外序贯培养体系间8-细胞胚率、桑椹胚率差异不显著（P〉0．05），当体外培养至囊胚时，100mL／L NBS＋TCM199（mTCM199）培养体系、100mL／L NBS＋RPMI1640（mRPMI1640）培养体系的囊胚率均显著低于100mL／L FBS＋RPMI1640（mRPMI1640）培养体系的囊胚率（三组的囊胚率依次是27．3％、35．9%、97．2%，P〈0．01）。但前两组之间差异不显著。结果表明，不同型血清对兔早期胚胎体外正常发育具有很大影响，序贯培养中添加国产NBS的培养液的培养效果明显低于添加进口FBS的培养液的培养效果。 相似文献
9.
研究培养液中血清的添加时间和添加浓度及不同培养体系对牛孤雌胚胎体外发育潜力的影响。结果表明：牛成熟卵母细胞孤雌激活后的第3天添加10％的FBS对孤雌胚的体外发育效果；良好。4种培养体系在孤雌激活后的第3天添加10％的FBS，平均囊胚率分别可达46．25％（SOFaar）、43．58％（CRlaa）、40．25％（KSOMaa）、17．98％（TCM199）；TCM199组的囊胚率显著低于其他3组（P〈0.05），SOFaa优于CRlaa和KSOMaa组，但差异不显著（P〉0．05）。 相似文献
10.
本试验探讨了3种不同分割液对奶牛桑葚胚和囊胚分割效果的影响。借助显微操作仪，将发育至第6～8天的体内常规生产的桑葚胚和囊胚进行分割，体外培养半胚，观察其发育情况，选择形态恢复好的半胚与一个囊胚滋养层细胞囊泡（trophoblastic vesicles，TRV）共移植。结果显示，在PBS+0.2 mol/L蔗糖与PBS+5%PVP中分割桑葚胚，其分割成功率显著高于PBS（P<0.05），分别为89.13%、86.73%和69.67%，而半胚的囊胚发育率及移植妊娠率三者均无显著差异（P>0.05）；在PBS+0.2 mol/L蔗糖与PBS+5%PVP中分割囊胚， 其分割成功率显著高于PBS（P<0.05），分别为94.52%、92.52%和70.52%，而半胚培养的囊胚发育率及移植妊娠率三者均无显著差异（P>0.05）；说明在PBS中分别添加0.2 mol/L的蔗糖和5%的PVP有利于提高奶牛桑葚胚和囊胚的分割成功率。 相似文献
11.
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
12.
Yotsushima K Sakaguchi M Shimizu M Okimura T Izaike Y 《The Journal of reproduction and development》2004,50(4):471-476
The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos. 相似文献
13.
Effect of serum-free co-culture and synchrony of recipients on development of cultured sheep embryos to fetuses 总被引：1，自引：0，他引：1
The percentage of sheep embryos that continued to develop after collection and immediate transfer on d 2 after estrus was similar when phosphate-buffered saline with 10% fetal calf serum (PBSFCS, 45%), physiological saline (50%), or tissue culture medium 199 supplemented with 10% fetal calf serum (M199FCS, 47%) was used to flush embryos from oviducts. Co-culture of sheep embryos for 3 d with oviductal cells tended (P = .1) to reduce the percentage of embryos that developed to fetuses after transfer compared with those embryos transferred immediately. Tissue culture medium 199 supplemented with .3% BSA (M199BSA) was an adequate substitute for M199FCS for culture of sheep oviductal cells if tissue culture wells were pretreated with fibronectin. Estradiol in concentrations from 10 to 1,000 pg/ml and progesterone at concentrations of 1 or 10 ng/ml in M199BSA failed to stimulate embryo development during 3 d of co-culture beyond that seen in co-culture with M199FCS or M199BSA without added steroid. Transfer of sheep embyros co-cultured for 3 d in M199BSA or M199FCS to recipients synchronized with donors resulted in about 19% of the embryos developing to fetuses, whereas transfer to recipients that were in estrus 24 h after donors resulted in 33% of embryos developing to fetuses. The significant (P less than .05) improvement for delayed recipients may reflect the relatively lesser developmental rate of co-cultured embryos compared with that of embryos in vivo. Embryo development into fetuses was similar after co-culture in M199FCS or M199BSA co-cultures; therefore, serum is not required for the co-culture of sheep embryos. 相似文献
14.
供体细胞培养处理方法对水牛核移植效果的影响 总被引：4，自引：1，他引：4
以经常规培养法 (DMEM 10 % FCS)、血清饥饿法 (DMEM 0 .5 % FCS培养 5～ 10 d)和 Apidicolin- APD结合血清饥饿法 (0 .1mg/ L APD培养 2 4 h,DMEM 0 .5 % FCS培养 1～ 18d)培养处理的水牛卵巢颗粒细胞和水牛成体耳部成纤维细胞作供核 ,分别采取带下注核法和胞质内注核法进行核移植。同一供核细胞各处理组间的核移植胚融合率 (以颗粒细胞作供核 )以及重组胚的囊胚发育率无明显差异 (P>0 .0 5 ) ,但经 APD 0 .5 % FCS培养处理供体细胞核移植后的分裂率显著高于其他组 (P<0 .0 5 )。用 7%乙醇处理的成体耳部成纤维细胞进行核移植 ,其重组胚的分裂率和囊胚发育率与对照组 (不含乙醇 )均无明显差异 (P>0 .0 5 )。结果表明 ,(1)血清饥饿处理水牛供体细胞对其核移植效果没有影响 ;(2 ) DNA合成抑制剂 APD结合血清饥饿培养处理水牛颗粒细胞和成体耳部成纤维细胞 ,可提高其核移植效果 ;(3)乙醇预激活处理水牛成体耳部成纤维细胞 ,对其核移植效果没有影响 相似文献
15.
采用离子霉素和6-DMAP对黄淮白山羊体外成熟卵母细胞进行联合激活,分别在不同的培养体系进行体外培养,观察孤雌胚胎的发育情况。培养体系分别为:无共培养条件下,M199(10?S)、CR1aa和HTF P1;颗粒细胞共培养条件下,CR1aa、HTF P1和SOFaa。结果发现,无共培养条件下,CR1aa和HTF P1组孤雌胚胎的卵裂率显著高于M199组(P<0.05),但3组均未有囊胚;共培养条件下,HTF P1组的卵裂率显著高于CR1aa和SOFaa组(P<0.05),而CR1aa组的囊胚率却显著高于其他2组(P<0.05)。综合试验结果说明,CR1aa联合颗粒细胞共培养能够获得较好的培养效果,适用于山羊孤雌胚胎的体外培养。 相似文献
16.
BV Sanches JHF Pontes AC Basso CR Ferreira F Perecin MM Seneda 《Reproduction in domestic animals》2013,48(1):e7-e9
In this work, we evaluated whether embryo development and pregnancy rates would be affected by culturing bovine Bos indicus embryos in Synthetic Oviductal Fluid with amino acids (SOFaa) or G1/G2 sequential medium under a low‐oxygen atmosphere. Using Ovum Pick Up, we obtained 1,538 oocytes, divided into G1/G2 (n = 783) and SOFaa (n = 755). No difference was observed for blastocyst development among the groups (27.8% ± 14.6 and 34.9% ± 20.0 for G1/G2 and SOFaa respectively, p > 0.05). Transferring the embryos (n = 450) from both groups to recipients resulted in similar pregnancy rates for the G1/G2 (38.4% n = 78/203) compared to the SOFaa (39.7% n = 98/247). Our findings confirm that Bos indicus embryos cultured in SOFaa and G1/G2 under low‐oxygen atmosphere have similar in vitro (blastocyst rate) and in vivo (pregnancy rate) developmental capacity. However, embryos cultured in G1/G2 medium have higher cleavage than those cultured in SOFaa medium. 相似文献
17.
Co-culture of ovine ova with oviductal cells in medium 199 总被引：7，自引：0，他引：7
Three experiments were conducted to test the suitability of medium 199 supplemented with 10% fetal calf serum (M199FCS) as a medium for co-culture of one-cell sheep ova with sheep oviductal cells. In Exp. 1, ova were co-cultured for 5 d in 5 ml of M199FCS or in Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS). Co-culture did not increase the number of cleavages at the end of 5 d of culture, but M199FCS supported more cleavages than did F10FCS (P = .016). In Exp. 2, ova were cultured for 1 to 3 d in M199FCS alone or on oviductal, uterine or kidney cell monolayers from ewes 2 d postestrus and transferred to recipients from which they were recovered at 8 d postestrus. Co-culture with oviductal cells improved (P less than .001) the cleavage index of recovered embryos compared with culture in medium alone or co-culture with other cell types. In Exp. 3, monolayers of oviductal cells from ewes 2 d postestrus and from luteal-phase ewes were cultured as in Exp. 2. No difference was observed between the two sources of oviductal cells for their ability to support in vitro development of one-cell sheep eggs for 1 or 2 d. These studies suggest that M199FCS may be a good medium to use in an oviductal cell co-culture system for one-cell sheep ova. Results further suggest that specific secretions of oviductal cells may be important for early embryo development in vivo. 相似文献
18.
Varga S Diez C Fernández L Alvarez J Katchicualula A Hidalgo CO Tamargo C Carbajo M 《Acta veterinaria Hungarica》2011,59(1):129-139
The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed. 相似文献
19.
猪植入前胚胎体外培养条件的优化 总被引：2，自引：1，他引：1
探讨了更换胚胎培养液及添加FBS、高渗透压和不同浓度VE对猪卵母细胞体外受精(IVF)和孤雌激活(PA)胚胎体外发育的影响,进一步优化了猪植入前胚胎体外培养体系。试验一：在第2天、第4天更换新的培养液(换液组),在换液基础上第4天更换为添加10%FBS的培养液(FBS组)。试验二：胚胎分别在0.05 mol/L蔗糖(蔗糖组)和138 mmol/L氯化钠(氯化钠组)的PZM-3(300～320 mOsmol)中培养2 d后移至PZM-3(288 mOsmol)中培养5 d。试验三：在培养液中分别添加50、100和200 μmol/L VE。对照组均在PZM-3(288 mOsmol)中培养7 d。结果表明：试验一,IVF和PA胚胎FBS组囊胚率显著高于对照组和换液组(P<0.05);试验二,IVF胚胎氯化钠组卵裂率、囊胚率均显著高于对照组与蔗糖组(P<0.05);试验三,IVF胚胎添加100 μmol/L VE组囊胚率显著高于对照组(P<0.05)。结果提示,在换液的基础上添加FBS有利于猪IVF和PA胚胎的体外发育;氯化钠调节的高渗透压可以促进猪IVF胚胎的早期发育;添加100 μmol/L VE可以改善猪IVF胚胎的体外发育体系。 相似文献
20.
按照多房棘球绦虫幼虫-泡球蚴培养的培养基（RPMI-1640、M199和MEM）分为3组：Ⅰ组为含10％胎牛血清的RPMI-1640;Ⅱ组为含10％胎牛血清的MEM;HI组为含10％胎牛血清的M199。将泡球蚴在3种细胞培养液中进行培养,观察其存活、生长以及发育情况。结果显示,培养9d的泡球蚴的成活率分别为：Ⅰ组90．10％、Ⅱ组50．25％、Ⅲ组22．03％;成囊率分别为：Ⅰ组57．12％、Ⅱ组63．15％、Ⅲ组48．17％;头节外翻率分别为：Ⅰ组98．28％、Ⅱ组88．65％、Ⅲ组75．50％。可见,大多数虫体在早期向囊发育,一部分虫体头节外翻,并伴有规律的伸缩运动,但随时间的延长虫体运动减缓,又向囊蚴发育。通过对多房棘球绦虫泡球蚴的体外培养,初步表明合有10％小牛血清的细胞培养基RPMI-1640较适合泡球蚴的生长发育,为研究寄生虫发育提供了最基本的数据资料。 相似文献