抗氧化剂对家畜胚胎体外发育影响的研究进展

现在研究普遍认为活性氧自由基是导致胚胎体外发育阻滞的主要原因。本文主要综述了不同抗氧化剂对牛胚胎体外发育的影响,并分析其作用机制。     

活性氧对哺乳动物早期胚胎体外发育阻滞的研究进展

哺乳动物早期胚胎体外培养过程中常发生体外发育阻滞现象，活性氧是引起发育阻滞的重要原因之一，作者综合国内外研究报道，综述了活性氧对哺乳动物早期胚胎体外发育的影响及克服发育阻滞现象的方法，为哺乳动物胚胎体外培养提供一些参考。     

哺乳动物早期胚胎体外发育阻滞的产生与克服

哺乳动物早期胚胎发育阻滞是体外培养条件下普遍存在的现象。文中就离体情况下由于生长环境的变化使早期胚胎所遭受到外界因素的影响进行了探析。综合国内外研究报道，提出通过改善培养条件和利用体细胞共培养，克服早期胚胎的体外发育阻滞。旨在为哺乳动物胚胎的体外培养提供一些参考。     

活性氧对兔早期胚胎体外发育的影响

研究旨在探讨活性氧-过氧化氢(H2O2)对兔2-细胞期胚胎体外发育的影响。在添加不同浓度过氧化氢的含胎牛血清的TCM-199培养液中,培养兔2-细胞期胚胎的对照组、≤88μmol/L、94μmol/L、103μmol/L≥118μmol/L组的囊胚发育率分别为86%、75%、34%、26%和5%;不同发育时间添加H2O2时对兔2-细胞期胚胎体外发育的影响为第1天处理组(37.14%)、第2天处理组(54.29%)与对照组(91.42%)差异显著。不同发育时间停止H2O2处理对兔2-细胞期胚胎体外发育的影响为第1天不处理组(82.26%)与对照组(97.14%)无显著性差异,但与第2天不处理组(52.50%)和第3天不处理组(30.00%)有差异。可以抑制兔2-细胞期胚胎体外发育的最低过氧化氢浓度为94μmol/L。兔2-细胞胚胎体外发育过程中,16-细胞之前的胚胎对环境中的过氧化氢都敏感,尤其是8-细胞之前的胚胎对环境中的过氧化氢更为敏感。     

昆明小鼠早期胚胎体外发育阻滞原因分析

为了分析小鼠早期胚胎在体外发育阻滞的原因 ,建立早期胚胎体外培养系统 ,应用几种不同的培养系统对昆明小鼠单细胞胚胎在体外培养了 96～ 12 0 h。结果表明 ,小鼠单细胞胚胎在 M1 6 和 BWW培养液中卵裂均受到阻滞 ,且两者之间差异不显著 (P>0 .0 5 ) ;CZB和 HTF培养液能有效地克服小鼠胚胎的 2 -细胞发育阻滞 ,与 M1 6 和 BWW相比 ,2 -细胞卵裂率和囊胚率均存在显著性差异 (P<0 .0 1) ;在 M1 6 培养液中添加牛磺酸对早期胚胎发育有促进作用 (P<0 .0 1) ;小鼠早期胚胎在 CZB和牛输卵管上皮细胞 (COEC)共培养体系中的卵裂率较对照组明显提高 (P<0 .0 5 )     

氨基酸对胚胎体外发育的影响

1 前言 氨基酸是胚胎培养液的重要成分,在培养液中添加氨基酸可促进胚胎发育[1],提高囊胚率、孵化率和胚胎质量,使胚胎的细胞数增加[2].但不同氨基酸对胚胎发育的作用不同,不同添加方式其效果也不一样.Bavister和他的同事对小鼠的研究工作,第一次揭示了氨基酸在体外胚胎培养中的作用.     

影响犊牛早期胚胎体外发育的因素

犊牛超数排卵不但可为科研和生产提供大量可用卵母细胞,而且对于加快育种进程、缩短育种间隔、基因优良个体扩繁等存在巨大的潜力和意义。但是,多数研究表明犊牛卵母细胞在体外发育方面还不及成年牛卵母细胞,文章就诱导犊牛卵泡发育取得的主要研究成果和犊牛卵母细胞及其胚胎在发育等方面存在的问题及其影响因素进行了综述。     

Iloprost对猪早期胚胎体外发育的影响

本研究旨在探讨PGI2类似物iloprost对猪胚胎体外发育的影响。试验以IVF胚胎为研究对象,分别在不同时期(0、24、48、72h)将不同浓度(0、0.2、0.5、1.0、2.0、5.0、10.0μmol.L-1)iloprost加入到猪胚胎培养液中,于156h时记录囊胚发育率和囊胚细胞数,筛选获得最佳添加方案,检测胚胎脂肪代谢速度和代谢相关基因(cox2、creb、pparδ、pdk、cpt2)的表达水平,分析iloprost影响胚胎发育的机制。结果显示,最佳的添加方案为,在受精后48h加入2.0μmol.L-1 iloprost,胚胎囊胚发育率(28%)和囊胚细胞数(49.42)显著高于(P<0.05)对照组的囊胚发育率(16%)和囊胚细胞数(28.22);添加iloprost后,胚胎脂肪酸降解速度也显著加快(P<0.05),脂肪酸代谢相关基因cox2、creb、pparδ、cpt2的表达量上升,糖代谢相关基因pdk表达量无显著变化。结果表明,PGI2类似物iloprost可以促进胚胎降解脂肪酸,为胚胎发育提供能量,提高了胚胎体外发育能力。     

胚胎干细胞的体外诱导分化

胚胎干细胞是从早期囊胚内细胞团或桑葚胚中分离的一种多潜能细胞，具有体外无限增殖和自我更新能力，而且还能被定向诱导分化形成机体几乎所有的细胞类型，如肝细胞、造血细胞、成骨细胞、神经细胞、神经胶质细胞、肌肉细胞、胰岛细胞、内皮细胞、黑色素细胞、淋巴细胞、脂肪细胞、成纤维细胞等。     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

活性氧对哺乳动物早期胚胎发育的影响

活性氧(ROS)是自然界普遍存在的含氧化合物的总称,它对哺乳动物早期胚胎发育具有不良影响。本文综述了活性氧对哺乳动物早期胚胎发育的影响以及它与胚胎体外发育阻滞的内在联系,对深入探讨哺乳动物早期胚胎体外发育阻滞的机理具有一定的意义。     

Relationship between Oxygen Consumption and Sex of Bovine In Vitro Fertilized Embryos

The present study was conducted to examine the relationship between the oxygen consumption rate and sex ratio of bovine in vitro fertilized embryos on each day of blastocyst formation. The quality of blastocysts collected on day 7, 8, and 9 after in vitro fertilization (IVF) were categorized as ranks A and B (excellent and good, respectively) based on microscopic observation of the morphology. The oxygen consumption rate and sex of individual blastocysts were evaluated using two novel techniques: scanning electrochemical microscopy (SECM) and loop-mediated isothermal amplification (LAMP), respectively. The oxygen consumption rates of embryos of rank A were significantly higher (p < 0.05) than those of rank B, irrespective of the day of blastocyst appearance after IVF. Neither did the proportion of male embryos of ranks A and B differ significantly from each other at any of the days examined, nor from the average proportion (53%). The oxygen consumption rate of embryos of rank B collected on day 8 was significantly higher (p < 0.05) in female embryos than in male embryos collected on the same day. However, there were no apparent differences of oxygen consumption rates at each day of blastocyst appearance between male and female embryos of rank A. These results indicate that the oxygen consumption rate of individual embryos reflects their quality but does not correlate with the sex ratio of embryos of excellent quality.     

Bovine Immunodeficiency Virus in Relation to Embryos Fertilized In Vitro

The association of bovine immunodeficiency virus (BIV) with embryos derived by in vitro fertilization from oocytes of experimentally infected heifers or oocytes/embryos exposed to the virus in vitro was investigated. Using a nested-PCR assay, proviral DNA of BIV was not detected in follicular fluid or in embryos derived from BIV-infected donors. In vitro exposure of oocytes to BIV during maturation or insemination with BIV-infected semen resulted in zona pellucida-intact embryos testing negative for BIV provirus. However, exposure of zona pellucida-free day-7 embryos to the virus resulted in a positive BIV assay for 28% of the batches of embryos, suggesting that the zona pellucida has a role in protecting against BIV infection. The presence of BIV in the IVF system had no apparent effect on the development of bovine embryos to the blastocyst stage.     

活性氧对仔猪睾丸支持细胞微丝的影响

为阐明活性氧(ROS)对睾丸支持细胞(SC)的影响,并深入探讨精子发生调节机制,通过体外培养的仔猪睾丸支持细胞研究ROS对SC中微丝结构的影响.利用H2O2模拟细胞内的ROS环境,使用细胞松弛素B(CytB)破坏细胞中的微丝并利用维生素E(VE)抑制细胞内的ROS,通过细胞活性检测、免疫荧光、酶活检测、免疫印迹等方法检测ROS对睾丸支持细胞微丝的影响.结果表明,H2O2和细胞松弛素B均能提高SC内ROS的水平和细胞外信号调节激酶(ERK)的活性,降低抗氧化酶的活性,破坏细胞内微丝的结构和分布,二者具有协同作用,VE能降低ROS对SC的作用.上述证明,H2O2通过不依赖于微丝途径和降低细胞内抗氧化能力使细胞内ROS处于较高水平,并通过ERK级联,使细胞中微丝发生多聚化,破坏微丝的结构.     

Culture of Bovine Embryos in Polyester Mesh Sections: The Effect of Pore Size and Oxygen Tension on In Vitro Development

The purpose of this study was to assess the feasibility of polyester mesh culture for the in vitro production of bovine embryos, as polyester mesh is an alternative way for tracking individual embryos throughout culture using time‐lapse cinematography (TLC). Bovine embryos were isolated during in vitro culture using sections of three different polyethylene terephthalate (PET) mesh products. In vitro matured and fertilized bovine oocytes were cultured in the 217 × 217, 230 × 230 or 238 × 238‐μm openings of PET mesh sections or in simple micro‐drops (control) for 7 days under either 20% or 5% O₂ tensions. No difference in embryo developmental rates was found between the culture groups in terms of cleavage, blastocyst formation and blastocyst expansion irrespective of O₂ tension. In contrast, under 20% O₂ tension, blastocysts that developed in PET mesh with 217 × 217‐μm opening had significantly higher numbers of total and trophectoderm (TE) cells than control embryos; however, the numbers and proportions of inner cell mass (ICM) cells did not differ. Under 5% O₂ tension, no difference was found among the culture groups in the numbers of total, ICM and TE cells in embryos. All three PET mesh products investigated in this study were proven to be effective to prevent embryo movement. The results demonstrate that bovine embryos can be cultured in PET mesh sections without negative side‐effects and suggest that embryo distance determined by the mesh affects embryo quality at atmospheric oxygen tension. Polyethylene terephthalate mesh with 217 × 217‐μm openings was found to be the most suitable for further application in TLC.     

几种冷冻条件对牛体外受精卵发育率的影响

试验比较了乙二醇 (EG)、丙二醇 (PG)、二甲基亚砜 (DMSO)、甘油 (G)和不同的投液氮温度 (- 33℃或 - 4 0℃ )对牛体外受精卵冷冻后发育率的影响。结果 :投液氮温度无论是 - 33℃或 - 4 0℃ ,均以 1.5 mol/ L EG的冷冻效果为最好 ,与 1.5 mol/ L PG相比 ,受精卵的发育率差异显著 (39.7% vs19.2 % ;4 7.8% vs2 4 .7% ,P <0 .0 5 ) ;同 1.5mol/ L DMSO和 1.5 mol/ L G相比 ,受精卵的发育率差异极显著 (39.7% vs 16 .1% or 13.3% ;4 7.8% vs 19.2 % or17.3% ,P <0 .0 1)。 - 4 0℃投液氮 ,受精卵冷冻后的发育率略高于 - 33℃ ,但差异不显著 (P >0 .0 5 )。以 2 5 % EG 2 5 % PG作为细胞外玻璃化溶液对牛体外受精卵进行冷冻 ,冷冻后受精卵的发育率达 5 8.9% ,高于用 1.5 mol/ L EG冷冻在 - 4 0℃投液氮这一处理 (47.8% ) ,但无统计学上差异 (P >0 .0 5 )。然而 ,试验组冷冻后受精卵的发育率均极显著低于对照组 (80 .6 8% ,P <0 .0 1)。结果表明 ,投液氮温度以 - 4 0℃为较好 ,防冻剂以 EG的冷冻效果为最佳。玻璃化冷冻法完全可以应用于冷冻牛体外受精卵     

Improvement in In Vitro Fertilization Rate,Decrease in Reactive Oxygen Species and Spermatozoa Death Incidence in Rams by Dietary Fish Oil

Our aim was to evaluate the effects of fish oil feeding on sperm classical parameters, level of reactive oxygen spices (ROS), spermatozoa death incidence and in vitro fertilization (IVF) rate in rams. We randomly assigned nine rams, into two experimental groups (isoenergetic and isonitrogenous rations with constant level of vitamin E supplement): control (CTR; n = 5) and fish oil (FO; n = 4, 35 g/day/ram). Diets were fed for 70 days during the physiological breeding season. After a 21‐day dietary adaptation period, semen was collected weekly from each ram by an artificial vagina. Sperm classical parameters were determined by the computer‐assisted sperm analyzer system (CASA), and it was prepared for IVF process by swim‐up technique. These evaluations were performed during the first and last weeks of sampling. Intracellular ROS level and spermatozoa death incidence were detected by flow cytometry on a weekly basis after adaptation. Data were analysed with SPSS 15. The volume, concentration (3.6 and 2.7 × 10⁹/ml) and sperm progressive motility (60 and 48%) were significantly improved in the FO group compared with the CTR (p < 0.05). A comparison of two‐cell stage embryos following IVF in the two groups showed a significantly higher fertilization rate in the FO group (56%) compared with the CTR (49%). Superoxide anion (O₂^?) rate was significantly lower (p < 0.05) at the third week of sampling in the FO. Although the H₂O₂ rate was numerically lower in the FO group compared with the CTR, this difference was not significant. In addition, apoptosis showed a significant difference in the third week of sampling (15 and 30% for FO and CTR, respectively; p < 0.05). Overall, adding fish oil to the ram diet not only improved sperm quality and IVF results, it also could reduce oxygen‐free radicals and the incidence of spermatozoa death.     

Long-Time Exposure of Mouse Embryos to the Sperm Produces High Levels of Reactive Oxygen Species in Culture Medium and Relates to Poor Embryo Development

Small amounts of reactive oxygen species (ROS), metabolites of oxygen, are necessary for sperm-fertilizing capability. However, in excessive levels, their role in infertility has been extensively studied. The conventional in vitro fertilization (IVF) method employs a prolonged co-incubation of gametes for 16–18 h to reach fertilization. However, it has been shown that this long period might create high levels of ROS. We aimed at finding out whether ROS increases in vitro during prolonged incubation with fertilized oocytes and whether high level of ROS relates to poor embryo development. To confirm if levels of ROS relate to length of time, we measured the ROS levels in fertilization medium (FM), which contained mouse embryos exposed to spermatozoa. To evaluate the contribution of sperm in production of ROS, we measured the ROS in the medium with only sperm. The measurements were performed by chemiluminescence assay using luminol as a probe after 4 and 18 h of incubation separately. The ROS levels were significantly increased after 18 h as compared with 4 h (p < 0.0001). Moreover, ROS in the medium with only sperm was also increased after 18 h (p < 0.0001), demonstrating that they were generated either by spermatozoa or as a result of possible reaction of sperm with medium during prolonged incubation. In addition, we compared embryo development after 2, 4, 6, 8, 10, 12 and 18 h of incubation. The number of degenerated embryos exposed to sperm for 12 and 18 h was significantly higher than those exposed for 4 or 6 h (p < 0.01). These results demonstrate that ROS concentrations appear to be related to the length of incubation time, and their excessive levels have a negative effect on embryo development. We suggest reducing incubation time to at least 4 h.     

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.