猪卵母细胞体外成熟体系研究简述

随着胚胎生物技术的日臻完善并逐步应用于家畜的育种和繁殖领域,人们对动物胚胎工程的认识逐渐加深,一系列更深层次的胚胎生物技术越来越受到人们的重视。20世纪80年代以来,人们在家畜卵母细胞体外培养、体外受精、     

猪卵巢卵母细胞的体外成熟及体外受精

从屠宰猪的狼巢卵包中抽取卵母细胞，在ＣＯ２培养箱中成功地进行了卵母细胞体对成熟和体外受精试验。在５％ＣＯ２和９５％空气的湿润条件下把带有卵丘细胞２的卵母细胞在ＴＣＭ－１９９＋ＰＭＳＧ＋ｅｓｔ．ｒａｄｉｏｌ－１７．     

牛卵母细胞体外成熟和体外受精技术

本文综述了十几年来牛的卵母细胞体外受精和体外成熟技术的研究进展，包括精子的体外获能、卵母细胞采集受成熟培养和体外受精、体外受精早期胚胎的培养等环节，并且对影响该技术的王要原因进行了分析，对体外受精在家畜育种中的应用提出了展望。     

猪卵泡卵母细胞的体外成熟与体外受精

本试验对猪卵泡卵母细胞不同体外成熟培养时间、不同精子获能时间、不同精卵共孵育时间对体外受精的影响进行了研究。结果表明,体外成熟培养44 h左右,精子获能时间在1～2 h之间,精卵共孵育时间在6～8 h之间,受精后卵裂率最高。     

猪卵母细胞体外成熟与体外受精的研究

从猪卵巢2～5mm的卵泡中抽取卵母细胞,用含有FCS,BSA-V,PMSG,HCG的TCM-199液和KRB修正液于37-39℃,5%CO_2,5%O_2和90%N_2的湿润环境中成熟培养和体外受精。实验结果表明:培养40小时后,4组卵丘细胞的扩展率分别为82.96%、89.76%、89.54%、85.57%,组间差异不显著(P>0.05)。第一极体放出率分别为16.90%、6.45%、13.64%、18.46%,第2组明显低于其他各组,但不显著(P>0.05)。用体外获能的精子授精,卵裂率分别为25.93%,18.18%,31.03%,27.03%;授精后29、30小时出现2—细胞,44～45小时出现4—细胞,56小时出现8—细胞。     

猪小腔卵泡卵母细胞体外成熟研究

本实验通过与2～6mm猪卵泡卵母细胞相对比，研究了猪2mm以下卵泡卵母细胞的体外成熟。结果如下(1)由2mm以下卵泡获得的小(直径100.29±7.9μm)、中(109.36±3.71μm)和大(118.90±2.46μm)3类卵母细胞培养48h的成熟率分别为0、22.31％和45.45％。(2)2mm以下卵泡卵母细胞的成熟率(52.63％)显著低于2～6mm卵泡卵母细胞的成熟率(80.39％)。(3)以含PFF的培养液培养2mm以下卵泡卵母细胞其成熟率(53.54％)显著高于不含PFF的对照组(22.77％)。(4)在培养的前24h加入PMSG、后24h去除PMSG，其成熟率(41.86％)与含PMSG的培养液连续培养48h的成熟率(50.0％)无显著差异，而显著高于前24h不加PMSG，后24h加入PMSG培养的成熟率(5.41％)；也显著高于以下不含PMSG的培养液连续培养48h的成熟率(3.3％)。     

影响猪卵母细胞体外受精效率因素的研究

通过比较参与受精的卵母细胞颗粒细胞存在与否、精子上浮时间、精卵共孵育时间、不同受精液等4个方面的因素,研究这些因素对猪卵母细胞体外受精后胚胎发育能力的影响,以求找到最佳的猪卵母细胞体外受精体系。将选择带有不同颗粒细胞的卵丘卵母细胞复合体分为3组：含全部颗粒细胞、2~3层颗粒细胞和裸卵;调整精子在受精液里的上浮时间为0、30、60、120min研究其受精能力;比较3、6、20h精卵共孵育时间对体外受精的影响;结果表明：在本试验体系下,在mTBM受精液中,将精子上浮处理60min,与含2~3层颗粒细胞的卵丘-卵母细胞复合体共孵育6h的IVF体系最为有效,其卵裂率为（77.6±2.3）%,囊胚率为（25.7±2.6）%。     

影响猪卵母细胞体外成熟的因素

猪卵母细胞体外成熟培养为胚胎体外生产、体细胞克隆等研究领域提供了大量廉价实验材料。本文就影响猪卵母细胞体外成熟的因素及其研究进展作简要综述。     

牛卵母细胞体外成熟和体外受精技术

本文综述了十几年来牛的卵母细胞体外受精和体外成熟技术的研究进展,包括精子的体外获能、卵母细胞采集及成熟培养和体外受精、体外受精早期胚胎的培养等环节,并且对影响该技术的主要原因进行了分析,对体外受精在家畜育种中的应用提出了展望。     

Effect of Lycopene on Cytoplasmic Maturation of Porcine Oocytes In Vitro

The aim of the present study was to improve cytoplasmic maturation of porcine oocytes by the addition of lycopene into in vitro maturation (IVM) media. We designed six experimental groups; IVM medium was supplemented with 10 IU/ml FSH, FSH and 10 IU/ml human chorionic gonadotrophin (hCG), or FSH and 7 μm lycopene in the first half of the IVM culture (0–22 h) followed by further culture (22–44 h) with or without hCG. The addition of lycopene into IVM media delayed the interruption of communication between an oocyte and the cumulus cells. Although meiotic competence was similar among the six groups, the glutathione level of matured oocytes was significantly higher in the lycopene‐supplemented group (9.89 pmol per oocyte) than that in other groups (7.25 and 7.81 pmol per oocyte). Fertilization rate was significantly improved in lycopene‐supplemented groups (58.3%) more than that in the group supplemented with FSH only (43.1%), whereas there were no differences in developmental competence among the groups (blastocyst rate: 20.1–29.5%). These results indicate that insufficient cytoplasmic maturation during conventional IVM resulted by disconnection of the gap junction between an oocyte and the cumulus cells in the early phase during IVM culture. We concluded that lycopene induced a prolonged sustainment of gap junctional communication between an oocyte and the cumulus cells during porcine IVM culture, which was an effective cytoplasmic maturation of porcine IVM oocytes.     

Effect of Sericin Supplementation During In Vitro Maturation on the Maturation,Fertilization and Development of Porcine Oocytes

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus‐oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA‐fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.     

Effect of Porcine and Ovine FSH on Nuclear Maturation of Pig Oocytes In Vitro

The effect of porcine or ovine FSH on the maturation rate of porcine oocytes and on the time course of meiotic progression was studied. Groups of 20 grade‐A cumulus oocyte complexes, aspirated from slaughterhouse cycling‐gilt ovaries, were cultured in vitro in 400 μl of Modified Parker's Medium supplemented with oestrous cow serum and porcine FSH (Folltropin®‐V, 0.50 mg/ml) or ovine FSH (Ovagen^TM, 0.44 iu/ml), in four‐well dishes under mineral oil, at 38.5°C, 5% CO₂ in humidified air. At the end of each 3‐h interval, from 3 to 42 h of culture, the nuclear status of oocytes was assessed microscopically (1000×), after fixation (methanol/acetic acid: 3/1) and orcein (2%) staining. Oocytes were classified as (i) immature (IMM), i.e. oocytes at germinal vesicle stage, germinal vesicle break down and prophase I, (ii) metaphase I (MI) and (iii) metaphase II (MII), i.e. oocytes at anaphase I, telophase I and metaphase II. Data were analysed using regression analysis, chi‐square and t‐test. Nuclear status was assessed in 1610 oocytes (porcine FSH: 787, ovine FSH: 823). Most of the oocytes were at MI from 24 to 33 h (porcine FSH 60.27%, ovine FSH 42.80%, p < 0.001) and at MII from 36 to 42 h (porcine FSH 80.38%, ovine FSH 67.45%, p < 0.01) of culture. Significantly higher maturation rate was observed in porcine FSH than in ovine FSH treated oocytes (86.69 ± 12.97%, 71.34 ± 9.86%, mean ± SD, p < 0.05), after 42 h of culture. In conclusion, under the specific culture conditions, porcine FSH seems to support pig oocyte maturation better than ovine FSH.     

促卵泡素与促黄体素对猪卵母细胞体外成熟的影响

本试验探讨了促性腺激素（FSH、LH）对猪卵母细胞体外成熟的影响,最终找到了其合理的使用剂量。在成熟液中添加LH浓度分别为1、5、10IU／mL时,与没有添加FSH与LH的对照组（15．9％）相比,成熟率有显著提高,成熟率分别为49．1％、30．0％、36．3％。LH浓度为1IU,mL的实验组成熟率最高,显著高于对照组（P〈0．05）;然后以成熟液中只添加1IU/mLLH为对照组,试验组分别添加1、5、10IU/mLPSH。试验结果表明：添加FSH浓度为5和10IU/ml时其成熟率分别为40．1％、27．6％,低于对照组（49．0％）。添加FSH浓度为1IU／mL时其成熟率（49．1％）高于对照组（49．0％）,但差异不显著（P〉0．05）。两组试验同时得出,随着LH与FSH添加浓度的增加,成熟率呈下降趋势。通过本试验结果得出,在体外成熟培养猪卵母细胞时,各添加1IU／mL LH与FSH时,得到最佳的成熟效果。     

促卵泡素与促黄体素对猪卵母细胞体外成熟的影响

本试验探讨了促性腺激素(FSH、LH)对猪卵母细胞体外成熟的影响,最终找到了其合理的使用剂量。在成熟液中添加LH浓度分别为1、5、10 IU/mL时,与没有添加FSH与LH的对照组(15.9%)相比,成熟率有显著提高,成熟率分别为49.1%、30.0%、36.3%。LH浓度为1 IU/mL的实验组成熟率最高,显著高于对照组(p<0.05);然后以成熟液中只添加1 IU/mLLH为对照组,试验组分别添加1、5、10 IU/mLFSH。试验结果表明:添加FSH浓度为5和10 IU/ml时其成熟率分别为40.1%、27.6%,低于对照组(49.0%)。添加FSH浓度为1 IU/mL时其成熟率(49.1%)高于对照组(49.0%),但差异不显著(p>0.05)。两组试验同时得出,随着LH与FSH添加浓度的增加,成熟率呈下降趋势。通过本试验结果得出,在体外成熟培养猪卵母细胞时,各添加1 IU/mL LH与FSH时,得到最佳的成熟效果。     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

山羊卵母细胞体外成熟的研究

在38℃和39℃不同温度条件下培养山羊卵母细胞,结果表明:在38℃和39℃下成熟率分别为63.6%和57.3%,二者之间差异不显著,说明只要培养温度在山羊正常体温的变动范围内,对其卵母细胞的成熟率没有影响;在38℃温度条件下用10?S的M199培养液成熟率为62.5%,而10 mg/ml BSA的M199培养液成熟率为58.7%,二者之间差异不显著,说明无血清培养可以代替血清培养.     

FSH 、LH对猪卵母细胞核成熟及体外成熟时间对去核效率的影响

克隆动物(核移植)的原理就是将卵裂球或体细胞核移人去核的卵母细胞或受精卵中,采用各种激活方法使之激活,发生卵裂,移植受体后产生个体。猪的体细胞克隆效率与其他动物(牛、羊)相比较低,需