牛卵泡液对牛卵母细胞体外成熟及受精胚发育力的影响

研究了牛卵母细胞体外成熟液和胚胎培养液中添加不同浓度的牛卵泡液对其体外成熟率和受精胚发育力的影响。结果表明:添加10%牛卵泡液的实验组,卵母细胞的成熟率与血清对照组没有显著差异(P>0.05);添加10%卵泡液的实验组与血清对照组相比,卵裂率和囊胚率没有显著差异,却显著高于添加5%和20%牛卵泡液的实验组(P<0.01),且各实验组囊胚内细胞数差异不大(P>0.05)。因此,用10%的牛卵泡液可以取代成熟液和胚胎培养液中的血清,并可降低实验成本。     

牛卵母细胞的体外成熟

在卵母细胞体外成熟液（含FSH）中分别添加10、30、50μg/L表皮生长因子（EGF）,24 h检查成熟率。结果表明,添加10、30μg/L EGF组牛卵母细胞成熟率为79.8%、71.5%与对照组（未添加EGF）成熟率（70.4%）无明显差异（P〉0.05）,但EGF添加至50μg/L时牛卵母细胞第一极体（PB1）排出率显著提高,达85.4%（P〈0.01）;在此基础上,比较了成熟液中添加尿促性素（HMG）对牛卵母细胞体外成熟的影响,结果发现在含FSH和EGF的成熟液中添加HMG未能显著提高牛卵母细胞体外成熟效果（P〉0.05）;最后,比较了HMG对牛卵母细胞体外成熟的效果,结果发现单独添加HMG成熟效果显著高于FSH（P〈0.05）,表明成熟液中如不含其他生长因子,则单独添加HMG较FSH对牛卵母细胞体外成熟的效果好。     

激素和血清对牛卵母细胞体外成熟的影响

卵母细胞的体外成熟直接关系到体外受精胚胎的数量和质量,从而影响到胚胎移植的成功率。本文就促性腺激素、类固醇激素和血清对牛卵母细胞体外成熟的影响,进行了较为详尽的阐述。     

Pregnancies from bovine oocytes matured and fertilized in vitro

Production of transgenic animals and embryo cloning are only a few examples of new biotechnological methods applied to animal embryos. All these techniques require large amounts of oocytes and early embryos. In many laboratory animals, embryos matured and fertilized in vivo are easily obtained, but with larger domestic species it requires laborious surgical procedures and the number of embryos obtained remains relatively small (Bracken et al. 1982). The in vitro maturation of follicular oocytes derived from slaughterhouse ovaries and their in vitro fertilization provides large numbers of oocytes and embryos with considerably less effort. The final proof of the success in the in vitro maturation and fertilization procedure is the birth of healthy progeny. Also the normal preimplantation development of the embryos gives useful information about the efficiency of the method employed.     

牛卵母细胞体外成熟

卵母细胞体外成熟培养研究主要集中在提高体外成熟卵母细胞支持胚胎发育的能力上。本文主要从成熟机理、卵母细胞的来源、卵母细胞的成熟培养环境及培养时间等方面进行了综述,介绍了目前卵母细胞体外成熟培养的进展情况。     

Establishment of an advanced chemically defined medium for early embryos derived from in vitro matured and fertilized bovine oocytes

A chemically defined medium would be useful for analyzing promoters or inhibitors in in vitro culture (IVC) of bovine embryos. However, an IVC system for bovine embryos in a chemically defined medium has not been fully established. The present study was carried out to establish an advanced chemically defined medium for bovine embryos that supports a high rate of embryo development to the blastocyst stage. In the first experiment, we examined the effects of addition of Medium RD (RPMI1640 and Dulbecco's MEM, 1:1 v/v) to mKSOM/aa on developmental competence. The addition of 10% RD to mKSOM/aa with BSA improved the rate of development to the blastocyst stage; however, 10% RD-mKSOM/aa with PVP, which is a chemically defined medium, caused a reduction in the percentage of hatching blastocysts. In the second experiment, embryos were cultured in the chemically defined medium of 10% RD-mKSOM/aa containing 11.7, 23.4, 46.8, 70.2 or 96.8 μM inositol. Inositol at the concentration of 70.2 μM improved the rate of development to the hatching blastocyst stage. In the third experiment, the optimal RD concentration in the IVC medium was evaluated. Embryos were cultured in the chemically defined medium supplemented with 10, 20 or 30% (v/v) RD. The rate of development to the blastocyst stage was highest with 20% RD. In the fourth experiment, the effects of N-acetylglucosamine (GlcNAc) as an IVC medium supplement on developmental competence were examined. The rate of development to the blastocyst stage with 1.0 mM GlcNAc was significantly higher than that without GlcNAc, but the rate of development with 1.2 mM GlcNAc was not different from that without GlcNAc. We also evaluated the ability of blastocysts produced in RD-mKSOM/aa to develop to normal calves after being transferred into recipients. Ten of the 16 recipients became pregnant, with 9 delivering normal calves. These results indicate that 20% RD-mKSOM/aa containing 70.2 μM myo-inositol and 1 mM GlcNAc is useful as a chemically defined medium for IVC of bovine embryos.     

Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

牛卵泡卵母细胞的体外成熟和冷冻保存

在简化牛卵母细胞体外成熟的基础上,观察了保护剂、平衡温度、预平衡方法、解冻方法以及冷冻方法对牛卵泡卵母细胞冷冻的影响。结果表明:在体外成熟培养液中添加26.2 mmol/L的NaHCO3和对屠宰场卵巢进行选择更能促进牛卵母细胞的体外成熟;无论是程序冷冻还是玻璃化冷冻,乙二醇(EG)与甘油(GLY)相比更能促进牛卵泡卵母细胞的冷冻效果;在程序冷冻中,冷冻液中添加0.1 mol/L蔗糖比添加0.3mol/L的蔗糖更能促进牛卵泡卵母细胞的冷冻;在玻璃化冷冻中,37℃和25℃的平衡温度比4℃更适合牛卵泡卵母细胞的冷冻;在10%、5%、1%的预平衡浓度之间,5%的预平衡浓度即可达到预平衡的效果;在解冻时,多步脱除保护剂更能保护冷冻的卵母细胞;比较了几种最小样本量(minimum size sample,MSS)玻璃化冷冻方法,解冻成熟培养后的成熟率,拉细毛细玻璃管冷冻法为(41.67±3.19)%,极显著高于未拉细毛细玻璃管冷冻法(glassmicropipette,GMP)的(30.19±1.93)%和固体表面冷冻法(solid surface vitrification,SSV)的(28.33±2.89)%(P<0.01)。     

Co-culture of in vitro fertilized bovine embryos with different cell monolayers.

The effects of different cell monolayers on in vitro development of early bovine embryos derived from in vitro maturation and fertilization were examined in this study. Early embryos (four to eight cells) were randomly allocated to bovine granulosa cell (GC), oviductal cell (OC), or uterine cell (UC) monolayers in Exp. 1 and to GC, skin cell (SC; from 10-d-old chicken embryos), testicular cell (TC; from 10-d-old mouse), and liver cell (LC; from 10-d-old chicken embryos) monolayers in Exp. 2, and cultured for 6 d at 38.6 degrees C in a humidified atmosphere of 5% CO2 in air. The culture medium was 12.5 mM HEPES TCM 199 supplemented with 1% calf serum and 1 mM sodium pyruvate. In Exp. 1, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, OC, and UC monolayers was 26.9 (28/104), 37.5 (39/104), and 39.2 (40/102), respectively. In Exp. 2, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, SC, TC, and LC monolayers was 53.3 (40/75), 42.9 (33/77), 49.3 (37/75), and 44.3 (35/79), respectively. There were no significant differences in development among groups in either experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-肉碱对水牛卵母细胞体外成熟的影响

以水牛为模型,通过探讨L-肉碱(L-carnitine)对水牛卵母细胞体外成熟的影响,同时深入研究添加L-肉碱后卵母细胞抗氧化能力的变化情况,以进一步阐明L-肉碱影响卵母细胞成熟的作用机制。水牛卵丘-卵母细胞复合体(COCs)在添加有不同质量浓度的L-肉碱(0,1,10,100 mg/L即对照组、Ⅰ组、Ⅱ组和Ⅲ组)的成熟液中体外成熟培养24h后,统计卵母细胞第一极体排出率。结果显示:Ⅰ组、Ⅱ组和Ⅲ组卵母细胞第一极体排出率分别为(64.44±1.93)%、(66.17±2.76)%和(63.27±1.19)%,与对照组(56.60±1.56)%相比差异显著(P<0.05)。免疫荧光染色观察显示:添加L-肉碱的各处理组的卵母细胞中活性氧的含量显著低于对照组(P<0.05)。同时,利用酶联免疫法测定卵母细胞中脂质过氧化物丙二醛的含量,结果显示Ⅰ组、Ⅱ组和Ⅲ组的卵母细胞中脂质过氧化物丙二醛的含量分别为0.125 797,0.125 217,0.124 155μmol/L,极显著低于对照组(0.142 609μmol/L)(P<0.01)。此外,采用实时荧光定量PCR方法检测卵母细胞周围的卵丘细胞中卵丘扩展相关基因ptx3、has2以及卵母细胞抗氧化相关基因gpx4的表达情况,分析结果显示:Ⅰ组、Ⅱ组和Ⅲ组卵丘细胞中has2的表达量均显著高于对照组的表达量(P<0.05),其中以Ⅱ组的最高;而Ⅱ组和Ⅲ组的卵丘细胞中ptx3基因和卵母细胞中gpx4基因的表达量显著高于对照组(P<0.05)。综上可知,水牛卵母细胞体外成熟液中添加适合质量浓度的L-肉碱能显著提高卵母细胞体外成熟率,推测L-肉碱可以通过提高卵母细胞抗氧化能力来促进卵母细胞的体外成熟。     

牛有腔卵泡卵母细胞体外成熟的研究

试验从屠宰场收集了 4 8头青年黄牛、34头青年水牛的卵巢共 16 4个 ,共回收可用卵母细胞 86 4枚。水牛平均每个卵巢可回收 3.2 2枚可用卵母细胞 ,相当于黄牛 (6 .72枚 )的一半。试验结果表明 :水牛卵巢生长卵泡少 ,卵母细胞产量少、质量差 ;3种采集黄牛卵泡卵母细胞的方法 ,用抽吸加切割法平均从每个卵巢回收的可用卵数 (7.71枚 )都极显著高于抽吸法 (6 .19枚 )和切割法 (6 .4 4枚 ) (P <0 .0 1) ,但是该法手续繁杂 ,适于一次且只能收集少量卵巢时使用 ;在黄牛和水牛卵母细胞成熟培养液中用 0 .3%PVP代替 10 %FBS ,牛卵母细胞的体外成熟率无显著差异 (P >0 .0 5 )。     

Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification and in-straw dilution

In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.     

牛体外受精胚的发育速度和染色体研究

利用不同个体的牛冷冻精液对屠宰场废弃牛卵巢内卵母细胞进行体外受精,对体外受精胚胎的发育速度和染色体的异常发生进行了研究。结果显示,冷冻精液A组体外受精48h后的卵裂率显著高于冷冻精液B组(P<0.05),2组之间的囊胚形成率差异显著(P<0.05);冷冻精液A体外受精后的胚胎发育速度显著快于冷冻精液B组(P<0.05);染色体分析结果表明,2组胚胎染色体异常发生率无显著差异,异常胚胎均表现为多倍体的发生率较高。     

pH值对猪卵母细胞体外成熟培养的影响

通过用不同pH值的培养观察分析对猪卵母细胞体外成熟培养的影响。结果表明：猪卵母细胞在培养液pH值低于6．5时几乎全部死亡，而pH值达到8，9时死亡率仅为31.7％，经分析，猪卵母细胞对碱性条件的耐受能力比对酸性条件的耐受能力要强。     

The influence of follicular factors on the in vitro maturation of bovine oocytes]

Immature oocytes from antral follicles of cattle were tested for the effect of follicular factors on maturation. In vitro maturation was accomplished by use of follicular fluid from small (2--5 mm) and large (above 15 mm) follicles and by addition to the medium of a granulose factor (GF) which had been isolated from the surface of granulosa cells. The parent material, with 84% (72/86) of oocytes at the germinal vesicle stage (GV-S) at the beginning of culturing, could be rated immature. 46% of all oocytes (41/89) had reached telophase I or metaphase II (full maturation) after 24 hours of maturation in hormone-free control medium (TCM 199 + 10% of foetal calf serum). 36% of oocytes (53/84), on the other hand, stayed between GV breakdown (GVBD) and anaphase I (incipient maturation). Full maturation was reached by as little as 14%. GF and follicular fluid from small antral follicles were found to inhibit GVBD in the oocytes. 59% (36/61) or 48% (61/127) of oocytes were blocked at GV stage. Positive determination of maturation inhibiting action of the above follicular components may provide a chance for their target-oriented use in control of the maturation process. The pool of immature oocytes of the ovaries, under such circumstances, might be more systematically utilised for in vitro manipulations.     

密闭气袋在牛卵母细胞体外成熟培养中的应用

通过简易避光密闭气袋在人体呼出的气相环境下（CO2浓度约5%,O2浓度约15%）,利用生化培养箱进行牛卵母细胞的体外成熟培养。结果显示,牛卵母细胞在密闭气袋中培养与对照组（正常培养）相比成熟率（74.79%比71.69%,P〉0.05）、孤雌激活后卵裂率（83.07%比81.39%,P〉0.05）和囊胚发育率上（26.75%比22.87%,P〉0.05）均无显著差异,但是密闭气袋培养各项数据均略高于对照组。本试验验证了该技术的方便性和有效性,为此技术在生产实践中的推广应用提供依据。