提高牛胚胎体外生产效率的研究进展

作者从采卵率、卵母细胞成熟率、受精率、胚胎发育率、胚胎冷冻解冻后生存率等方面综述了提高牛胚胎体外生产效率的研究进展。     

牛胚胎体外生产技术与前景展望

近年来，现代生物技术突飞猛进的发展给全球畜牧业生产提供了良好的发展机遇，动物胚胎生产技术早已休体内生产阶段发展到休外生产阶段，使得牛胚胎的体外生产这一新兴产业也应运而生了。     

牛胚胎体外生产质量控制

牛胚胎体外生产质量控制就是在常规实验的基础上 ,避免额外因素对胚胎体外生产造成影响。本文从实验记录、卵母细胞、精液、培养条件、胚胎质量等方面分析讨论了牛胚胎体外生产中的质量控制     

牛胚胎的体外生产技术

应用体外受精技术，可以有效地利用淘汰的高产奶牛和屠宰的青年母牛卵巢中的未成熟卵，大量，廉价地实验室生产胚胎，这对加速我国高产奶牛群和良种肉牛群的繁殖，是一项行之有效的胚胎工程技术，借鉴国外实验室生产牛胚胎的经验和根据我们实验室的实践，本文比较详细地叙述了体外受精牛胚胎（试管牛）的生产过程，包括卵母细胞的获得，培养，精子获能，体外受精以及受精后的胚胎培养，超低温冷冻保存与解冻后的胚胎移植等。     

牛胚胎体外生产与早期胚胎发育的探讨

试验对牛胚胎体外生产技术中的体外受精液、精卵共育时间、血清和培乔液等影响早期胚胎体外发育的因素进行了研究。以BO（Brackett ＆ Oliphant）和BM（BO：成熟培养液=3：2）作为受精液，受精卵的囊胚发育率分别为26．0％和15．0％；精卵共育时间以9-12h为宜；受精卵在含血清FBS或OCS培养液中的专胚发育率分别为26．4％或29．9％，明显高于在无血清培养基中的囊胚发育率（10．3％）；TCMl99、mBECMaa、mSOFaa3种胚胎培养液均能支持受精卵的体外发育，在其中培养受精卵囊胚发育率分别为18．O％、30．7％和29．2％。试验结果表明：精卵于BM受精液中共育9～12h后，将假定受精卵置于添加5％OCS的mBECMaa或mSOFaa培养液中培养，能显著提高体外生产胚胎的囊胚发育率。     

牛胚胎体外生产技术的简化研究

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     

哺乳动物体外胚胎染色体分析的现状

所谓动物体外胚胎是指动物的卵母细胞在体外条件下进行发育、成熟、受精 (或核移植 )和培养所产生的胚胎。该过程主要是依靠体外受精、胞质内单精子注射及核移植等技术来实现的。从第一例体外受精 (IVF)犊牛的出生 ,到体细胞克隆羊“多莉”的诞生 ,动物体外胚胎技术自建立以来 ,在医学和农业上己经取得了许多惊人的成就 ,成为了家畜改良的重要技术。但由于该技术还不十分完善 ,存在一些缺陷 ,因而妨碍了它在实际中的应用。因为从目前越来越多的研究发现 ,体外生产的胚胎发育成功率低 ,流产率高。另外 ,人们在研究中还发现 ,通过核移植 (NT…     

不同体外培养条件对家兔早期囊胚贴壁行为的影响

采集家兔自然交配后96h的早期囊胚，以低糖DMEM＋150mL／L胎牛血清＋0．1mmol／L非必需氨基酸＋100IU／mL青霉素＋100IU／mL链霉素为基础培养基，比较了不同胚胎处理方法、不同饲养层以及培养液中添加不同成分对兔早期囊胚贴壁和增殖的影响，以完善兔胚胎干细胞的建系方法。结果表明，以胚胎分割法和链霉蛋白酶-E（proteinaseE）处理掉黏蛋白及部分透明带的胚胎容易贴壁和增殖；在小鼠成纤维细胞饲养层和兔胎儿成纤维细胞饲养层上，胚胎的脱带率差别不大，但在小鼠成纤维细胞饲养层上贴壁率明显提高，且贴壁后内细胞团增殖较快；添加胰岛素、白血病抑制因子和伊巯基乙醇均利于抑制ES的分化和促进内细胞团的增殖。     

体外生产的牛胚胎超微结构的研究

利用两种常规体外培养系统研究了体外培养及不同培养条件对牛体外受精胚胎早期发育各时期超微结构的影响。结果显示：体外发育的牛胚胎在发育的各个时期（从原核期到囊胚）胞质中均含有丰富的脂滴，胚胎中特别是在桑椹胚及囊胚阶段可见到一些异常细胞。两系统下发育的胚胎中脂滴形态不同，从桑椹胚阶段开始，胚胎线粒体的发育在M199-BOEC-FCS培养系统中明显滞后于其在SOFaa-BSA培养系统中的对照。     

牛不同直径卵泡内卵母细胞体外受精后的卵裂率和染色体异常分析

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。     

Transfer of Bovine Blastocysts Derived from Short-term In Vitro Culture of Low Quality Morulae Produced In Vivo

The aim of this study was to evaluate if blastocysts arising from in vitro culture of Grade 3 bovine morulae produced in vivo can promote acceptable pregnancy rates when transferred into recipients. Embryos of different stages and qualities were recovered from superovulated Bos taurus and B. indicus donors. Grade 3 morulae were cultured in either Holding Plus® or TCM‐199 (supplemented with 10% bovine fetal serum) media for 24 h at 38.5°C. After this culture period, the resulting blastocysts were morphologically classified (Grades 1, 2 and 3) and transferred into recipients previously synchronized with the donors. Non‐cultured Grades 1 and 3 morulae were used as control. Pregnancy diagnosis was carried out 60 days after embryo transfer and the data were analysed by logistic regression, considering variables, such as embryo quality (Grade), donor breed, culture medium, donor‐recipient synchrony and seasonality. Embryo quality was the only variable, showing significant effect on the pregnancy rate. Pregnancy rates for non‐cultured Grade 1 and 3 morulae, and blastocysts arising from cultured Grade 3 morulae were 58.1% (n = 31), 17.1% (n = 35) and 51.1% (n = 47), respectively (p < 0.05). There were no statistical differences between non‐cultured Grade 1 morulae and cultured blastocysts. Pregnancy rates for Grades 1 (65.0%) and 2 (60.0%) were higher than Grade 3 (29.4%) cultured blastocysts (p < 0.05). It was concluded that short‐term in vitro culture is a very convenient method of identifying morphologically low quality morulae with higher chances of continuing development after the transfer into recipients.     

Vitrification of Bovine Blastocysts Produced In Vitro Inflicts Selective Damage to the Inner Cell Mass

In contrast to the embryos derived from live animals, the embryos produced in vitro undergo increased damage and reduced survival after cryopreservation, particularly when produced with serum. In medium containing serum, retinoic acid increases cell numbers in the inner cell mass and the trophectoderm without altering their relative proportions in the bovine blastocyst. In this work, in medium without serum, we analyzed the contribution of retinoic acid to the development of blastocyst and survival to vitrification, and found a strong cell reduction in the inner mass when compared to the trophectoderm. Day-6 in vitro -produced morulae were treated for 24 h with retinoic acid (0.7 and 1.4 μ m ) and subsequently cultured without additives for a further 24 h period. Day-8 blastocyst production and cell counts in hatched blastocysts were unaffected by retinoic acid. However, Day-7 expanded, vitrified embryos produced with retinoic acid 1.4 μ m survived at lower rates than controls when cultured after warming. Vitrification greatly reduced cell numbers in the inner mass (p < 0.0001), while cells in the trophectoderm remained unaltered. Differential cell counts analysis in blastocysts should be taken up to replace unspecific determination of total cells to appreciate substantial modifications in their exact terms. The strong reduction we found in the inner cell mass could explain why in vitro survival to cryopreservation is sometimes scarcely informative on the viability of the embryo after transfer to recipients.     

牛体外受精胚的玻璃化保存及细胞遗传学研究

用25％的乙二醇和0.2mol/L蔗糖与25％忆二醇混合的玻璃化溶液对牛体外受精的早期囊胚用1步法冷漠保存，融解后，在D－PBS液（含有0.5mol蔗糖）中15min除去冷冻保护剂，培养48和96h。结果表明：用含有0.2mol的蔗糖和25％的乙二混合的玻璃化溶液保存的早期囊胚的生态力明显高于不含蔗糖的玻璃化溶液（P＜0.001）。染色体分析结果表明：含有蔗糖的玻璃化溶液保存的囊胚的卵裂细胞数明显高于单独使用25％乙醇保存的囊胚（P＜0.05）。染色体异常发生率无明显变化。     

Cell Counts and Survival to Vitrification of Bovine In Vitro Produced Blastocysts Subjected to Sublethal High Hydrostatic Pressure

This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp‐70) were also examined. Day 7 and 8 bovine in vitro‐produced blastocysts were submitted to an HHP treatment (60 MPa, at 32°C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post‐warming) and hatching (48 h post‐warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP‐treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32°C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP‐treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp‐70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post‐warming.     

Comparative IFN-τ Secretion after Hatching by Bovine Blastocysts Derived Ex Vivo and Completely Produced In Vitro

The interferon-tau (IFN-tau) secretion levels after hatching by bovine blastocysts derived from in vitro maturated oocytes (Group A) and from in vivo (Group B) were investigated considering embryo quality. Only very homogeneous blastocysts of excellent or good quality were considered from day 7 of culture (Group A) and day 7 after artificial insemination with frozen-thawed from the same bull used for in vitro fertilization (Group B). All embryos were individually cultured into a 50 microl droplet of synthetic oviduct fluid medium with 10% fetal calf serum. After 24-h culture both Group A (n=44) and B (n=40) secreted <54 pm IFN-tau. After 48-, 72-, 96- and 120-h culture, Group A daily secreted 143 +/- 24 pm IFN-tau (n=19) vs 85 +/- 12 pm IFN-tau (n=21) for Group B (p < 0.01), 491 +/- 128 pm IFN-tau (n=29) vs 216 +/- 37 pm IFN-tau (n=23) (NS), 499 +/- 135 pm IFN-tau (n=26) vs 353 +/- 93 pm IFN-tau (n=21) (NS), 559 +/- 136 pm IFN-tau (n=22) vs 333 +/- 75 pm IFN-tau (n=20) (NS), respectively. Taken all together during 5 days, Group A produced per embryo 1690 +/- 290 pm IFN-tau (n=22) vs 982 +/- 182 pm IFN-tau (n=20) for Group B (p < 0.05). For all culture time there were sizable percentages of embryos that did not produce concentrations of IFN-tau above a certain cut-off level, and as such were not used to compute the means. In respect of the embryo quality whatever the groups after days 7-12 of culture, IFN-tau secretions were 1815 +/- 453 pm (n=10) for the embryos of excellent quality vs 1356 +/- 200 pm (n=28) for those of good quality (NS) and 360 +/- 188 pm (n=4) (p < 0.05) for embryos of fair quality. A positive relationship between IFN-tau production and in vitro development of quality I embryos was observed, whatever the embryos origins and, the embryos completely produced in vitro secreted more IFN-tau than the embryos produced in vivo.     

牛腔前卵泡的体外培养

通过在α- MEM和 D- MEM/ F1 2 基础液中添加不同比例的 ITS、丙酮酸钠、谷氨酰胺、次黄嘌呤和血清 ,观察了不同基础液对牛腔前卵泡体外生长发育的影响 ,从而筛选出较好的组别。在筛选出的基础液组别中添加 3个水平的FSH、L H、E2 ,观察了其对牛腔前卵泡体外生长发育的影响。结果表明 ,α- MEM和 D- MEM/ F1 2 2种基础液中以α-MEM为好 ,不同的基础培养液组合对牛腔前卵泡的体外培养有一定的影响。添加不同水平的激素对牛腔前卵泡体外培养也有显著影响。试验初步筛选出最佳的培养液成分为α- MEM ITS(I- 5 m g/ L ,T- 5 mg/ L ,S- 5μg/ L ) 丙酮酸钠(0 .2 3mmol/ L) 谷氨酰胺 (1.5 m mol/ L) 次黄嘌呤 (2 m mol/ L) 血清 (7.5 % ) FSH(0 .2 5 mg/ L) L H(5 IU/m L ) E2 (0 .5 mg/ L )。     

Effects of Serum-Free In Vitro Maturation of Bovine Oocytes on Subsequent Embryo Development and Cell Allocation in Two Developmental Stages of Day 7 Blastocysts

Maturation of oocytes and the subsequent outcome of the in vitro production (IVP) are affected by the composition of in vitro maturation (IVM) medium. To determine the use of serum interfering with effects of single molecules, we aimed at developing simplified IVM medium. The experimental IVM media were: (1) M199-medium supplemented with hormones and serum (control), (2) as 1 but serum was substituted with fatty acid-free serum albumin (FAFBSA) and (3) M199-medium without hormonal and serum supplementation (M199). The quality of embryos was assessed on day 7 by morphology and cryotolerance, as well as by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and differential staining. Results showed that the nuclear maturation was suppressed in M199 group alone. Embryo cleavage and development rates, and the proportion of quality 1 blastocysts were lower in the FAFBSA and M199 groups compared to the control. Differences in the cell allocation of fresh embryos were observed at the blastocyst stage, but not at the expanded blastocyst stage. The control group blastocysts had larger number of cells allocated to the inner cell mass (ICM), and the FAFBSA group blastocysts larger apoptotic cell proportion compared to the blastocysts derived from other groups. After cryopreservation, the reduction of ICM proportion and increase of apoptotic cell proportion of embryos were equal between the experimental groups. In conclusion, exclusion of serum from the IVM media reduces embryo development and may cause perturbations in blastocyst development. Differences in the cell allocation of blastocysts between IVM media may appear only when the developmental stages are taken into account.