卵巢运输温度及卵母细胞促成熟因子对山羊卵母细胞体外成熟和体外受精的影响

探讨山羊卵母细胞体外成熟培养的影响因素,为优化山羊体外受精程序奠定基础。比较从屠宰场采摘的山羊卵巢运输温度和体外培养促成熟因子对卵母细胞体外成熟培养和体外受精效果的影响。结果表明,在运输温度接近室温(25±2)℃时的卵母细胞成熟率显著高于接近体温(36±2)℃时的运输温度(成熟率分别为53.8%,35.0%,P0.01),两种运输温度的卵巢卵母细胞成熟培养后体外受精率无显著差异(分别为37.9%,36.6%,P0.05)。与在体外成熟基础培养液(M1:M199 media+1μg/mL E2+10μg/mL FSH+10μg/mL LH+20%EGS)中进行成熟培养相比,在基础培养液中仅添加20 ng/mL EGF(M2)和另外添加10%GFF(M3)进行体外成熟培养的卵母细胞成熟率均有明显提高,其中成熟率在M1为53.8%,极显著低于M2的69.5%和M3的72.6%,P0.01;M2和M3间差异不显著;卵母细胞成熟培养后体外受精率在三者间无明显差异分别为37.9%,39.5%和40.6%,P0.05。山羊体外受精时从屠宰场采摘的卵巢宜在室温条件下进行运输,在体外成熟培养体系中加入EGF和GFF均可有效促进山羊卵母细胞的体外成熟,但EGF起关键作用。     

体细胞核移植克隆民猪: 培养液对卵母细胞成熟及胚胎发育的影响

体外培养成熟的卵母细胞是进行克隆猪研究所需受体卵母细胞的主要来源, 卵母细胞成熟质量与体细胞核移植胚胎发育能力关系密切. 为提高卵母细胞体外成熟率和成熟质量, 进而提高体细胞核移植猪的成功率, 本实验以改进的TCM199培养液为基础液(T), 分别添加10%的猪卵泡液(T+pFF)和 10%的胎牛血清(T+FBS)后进行卵母细胞成熟培养, 以成熟率和体细胞核移植胚胎发育率等重要指标为标准, 研究了pFF和FBS对卵母细胞成熟及核移植胚胎发育能力的影响. T, T+pFF和T+FBS组在成熟培养后42 h卵母细胞成熟率分别为(53.2±3.8)%, (69.7±3.8)%和(70.2±3.7)%, 添加10%的pFF和FBS显著(P<0.05)提高了卵母细胞成熟率; 3组不同成熟培养液获得的成熟卵母细胞在体细胞核移植后囊胚发育率差异不显著, 但T+pFF组的囊胚细胞数(34.5±2.24)显著(P<0.05)高于T组的囊胚细胞数(26.6±1.25). 来自T+pFF组的体细胞核移植胚胎经手术法移植入发情周期为第0天或第1天的18头受体母猪输卵管, 其中有3头受体母猪妊娠发育到期, 获得克隆民猪14头, 其中有6头健康成活至今. 实验结果表明, 培养液中添加10%pFF可以有效提高卵母细胞成熟比例和成熟质量, 在含有10% pFF培养液中获得的成熟卵母细胞具有支持核移植胚胎全程发育的能力.     

体细胞核移植克隆民猪: 培养液对卵母细胞成熟及胚胎发育的影响

体外培养成熟的卵母细胞是进行克隆猪研究所需受体卵母细胞的主要来源, 卵母细胞成熟质量与体细胞核移植胚胎发育能力关系密切. 为提高卵母细胞体外成熟率和成熟质量, 进而提高体细胞核移植猪的成功率, 本实验以改进的TCM199培养液为基础液(T), 分别添加10%的猪卵泡液(T+pFF)和 10%的胎牛血清(T+FBS)后进行卵母细胞成熟培养, 以成熟率和体细胞核移植胚胎发育率等重要指标为标准, 研究了pFF和FBS对卵母细胞成熟及核移植胚胎发育能力的影响. T, T+pFF和T+FBS组在成熟培养后42 h卵母细胞成熟率分别为(53.2±3.8)%, (69.7±3.8)%和(70.2±3.7)%, 添加10%的pFF和FBS显著(P<0.05)提高了卵母细胞成熟率; 3组不同成熟培养液获得的成熟卵母细胞在体细胞核移植后囊胚发育率差异不显著, 但T+pFF组的囊胚细胞数(34.5±2.24)显著(P<0.05)高于T组的囊胚细胞数(26.6±1.25). 来自T+pFF组的体细胞核移植胚胎经手术法移植入发情周期为第0天或第1天的18头受体母猪输卵管, 其中有3头受体母猪妊娠发育到期, 获得克隆民猪14头, 其中有6头健康成活至今. 实验结果表明, 培养液中添加10%pFF可以有效提高卵母细胞成熟比例和成熟质量, 在含有10% pFF培养液中获得的成熟卵母细胞具有支持核移植胚胎全程发育的能力.     

马鹿卵母细胞体外培养与体外受精的超微结构

用FSH 对马鹿进行超数排卵,通过抽吸法和切割法采集卵泡卵母细胞,用M199 为基础的培养液在38.5℃、5%CO₂ 和饱和湿度条件下对马鹿卵母细胞进行体外培养与体外受精培养,利用透射电镜观察不同时期马鹿卵母细胞的超微结构,旨在揭示马鹿卵母细胞体外培养前、培养后及受精后超微结构的变化规律。结果表明,培养前卵丘细胞紧紧包围卵母细胞,卵母细胞表面的微绒毛细长,伸入透明带内,皮质区及细胞中心分布大量的细胞器。培养后卵丘细胞与卵母细胞结合松散,卵母细胞表面微绒毛短粗,倒伏于卵表面,第一极体无核,皮质颗粒在皮质区成层排列,细胞质中细胞器分布均匀。受精后卵母细胞表面的微绒毛由倒伏而竖起,第二极体有核,细胞质中细胞器丰富,主要分布于细胞中心。     

马卵母细胞孤雌激活与体细胞核移植技术

在马(Equus caballus)的繁殖和非繁殖季节,本研究探讨马扩展型(Ex)和紧凑型(Cp)卵丘-卵母细胞复合体(COCs)卵母细胞的孤雌激活效率。在繁殖季节,探讨马驹和成年马成纤维细胞核移植(SCNT)的成功率。孤雌激活实验结果显示,在繁殖季节,发育到2-细胞、4-细胞和桑椹胚的比例,扩展型(Ex)卵丘-卵母细胞复合体分别是52.8%(19/36)、38.9%(14/36)和5.6%(2/36),紧凑型(Cp)卵丘-卵母细胞复合体分别是47.9%(23/48)、33.3%(16/48)和6.2%(3/48)。在非繁殖季节,发育到2-细胞、4-细胞的比例,扩展型(Ex)分别是37.2%(16/43)和16.3%(7/43),紧凑型(Cp)的比例分别是35.1%(27/77)和11.7%(9/77),都没有获得桑椹胚。同一季节,扩展型(Ex)与紧凑型(Cp)胚胎发育的比率差异不显著(P 0.05),不同季节,两者差异显著(P 0.05)。体细胞核移植实验结果显示,以马驹成纤维细胞作为核供体细胞,胚胎发育到2-细胞、4～8细胞和桑椹胚的比例分别是41.5%(22/53)、33.9%(18/53)和15.1%(8/53),以成年马成纤维细胞作为核供体细胞,比例分别是38.9%(7/18)、22.2%(4/18),没有获得桑椹胚。综上所述,季节和卵丘-卵母细胞复合体(COCs)类型影响马卵母细胞孤雌激活的效率,不同核供体细胞影响克隆胚胎构建的成功率。     

蛋白质合成抑制剂亚胺环己酮(CHX)对猪卵母细胞体外成熟的影响

研究了蛋白质合成抑制剂亚胺环己酮 (CHX)对猪卵母细胞体外成熟过程中的GVBD、染色质凝集、MⅡ期成熟及卵丘细胞扩展的作用。结果表明 :( 1)培养液中添加CHX ,可抑制卵母细胞GVBD的发生 ,而且此作用是浓度依赖性的 ,但CHX的抑制效果是完全可逆的 ;( 2 )在含 10 μg/mlCHX液中分别培养 0、 6、 12和 2 4h后转入正常培养液再继续培养至 4 8h ,卵母细胞成熟率分别为 84 1%、 77 1%、 4 8 9%和 2 7 8% ;( 3 )正常培养液中培养 0、 6、 12、 2 4、 3 6和 4 8h后 ,再转入浓度为 10 μg/mlCHX液中继续培养至 4 8h ,卵母细胞成熟率分别为 0、 0、 0、 3 1 3 %、 65 4 %和 79 5 % ;( 4 )CHX对卵丘细胞扩展的影响随培养时间延长而增强 ,在CHX中处理时间为 16h或更长 ,完全抑制卵丘细胞的扩展     

成熟培养液中胎牛血清添加量对山羊卵母细胞体外培养成熟的影响

本研究以屠宰奶山羊的新鲜卵巢为材料,回收可培养卵丘细胞-卵母细胞复合体(COC)。将可培养COC置于M199+FBS+100 IU/mL FSH+100 IU/mL LH培养微滴中,在5%CO2、95%空气、饱和湿度的CO2培养箱中39℃下培养。进行二因子三水平设计,比较基础培养液中添加FBS体积分数为0%、10%和15%时,及培养时间16 h、24 h、26 h的成熟率。结果表明:不添加FBS组的成熟率极显著(P0.01)低于其他两组,而10%FBS组与15%FBS组差异不显著(P0.05);体外培养16 h与培养24 h、26 h的卵母细胞成熟率分别为67.6%与81.7%、81.7%,差异极显著(P0.01),而后二者间未表现差异。     

山羊体外受精的研究

通过在山羊卵母细胞体外成熟培养液中添加不同的血清和不同浓度的卵泡液 ,在体外成熟培养液中培养不同的时间 ,以及采用不同的精子获能方法来摸索效率较高的体外受精方法体系。在山羊卵母细胞体外成熟培养液中添加FCS、EGS及不同浓度的卵泡液 ,成熟培养时间分别为 16、2 0、2 4和 2 7h ,山羊新鲜精液用钙离子载体法和肝素法进行获能处理后用于体外受精 ,比较其体外成熟率和体外受精率。添加FCS、EGS和 2 0 %卵泡液组的成熟率无显著差异 ,显著高于添加 10 %和 3 0 %卵泡液组的成熟率 ;但添加FCS和EGS组的受精率显著高于添加10 %、2 0 %、3 0 %卵泡液组的受精率。培养 2 4h组和 2 7h组的成熟率显著高于另外两组 ,而培养 2 7h组的受精率显著高于其余各组。用钙离子载体法处理的山羊精子的顶体反应率和体外受精率显著高于肝素法。EGS可以代替FCS添加于成熟培养液中 ,对COCs的成熟率和受精率没有明显的影响 ,但卵泡液不能完全代替FCS的作用。培养时间为 2 7h ,精子用钙离子载体法处理获能后能得到较高的体外受精率     

处理方式不同的颗粒细胞和卵泡液对牛卵母细胞体外受精后卵裂率和囊胚率的影响

将牛的卵母细胞置于添加有不同预处理颗粒细胞及含有卵泡液的培养液或不舍有卵泡液的培养液中进行体外成熟、受精及胚胎发育培养,研究了颗粒细胞、卵泡液及颗粒细胞与卵泡液交互作用对牛卵母细胞成熟、受精后卵裂率、囊胚率的影响。2178枚卵母细胞体外成熟受精后胚胎发育的对比观察结果表明：颗粒细胞、卵泡液及颗粒细胞与卵泡液交互作用对卵母细胞成熟受精后胚胎的卵裂具有显著影响（P〈0．05）;颗粒细胞对囊胚的发育有显著影响（P〈0．05）;卵泡液及颗粒细胞与卵泡液交互作用对囊胚的发育无显著影响（P〉0．05）。不同因素对卵裂率、囊胚率的影响表现为：颗粒细胞因素〉培养液因素〉培养液&#215;颗粒细胞交互作用。结论：TCM199培养液中添加卵泡液和单层颗粒细胞组成的培养系统用于牛卵母细胞体外成熟及胚胎发育的效果较好。共培养体系中的单层颗粒细胞用经酶消化分散处理后在培养箱中孵育10min的颗粒细胞替代时,胚胎的发育效果并不受影响。     

聚乙烯醇和卵泡液添加方式对猪卵母细胞体外成熟及孤雌胚胎发育的影响

本试验研究目的在于探讨在猪卵母细胞体外成熟(IVM)过程中,聚乙烯醇(polyvinyl alcohol,PVA)和猪卵泡液(porcine follicular fluid,PFF)添加方式对猪卵母细胞体外成熟及孤雌激活早期胚胎发育的影响。卵母细胞、卵丘细胞复合体(COCs)在含有HCG和PMSG的改良TCM-199+10%FBS+10%猪卵泡液(PMH-PFF)或改良TCM-199+10%FBS+0.1%PVA(PMH-PVA)成熟液中培养22~23 h;再移至无HCG和PMSG的PM-PFF或PM-PVA的成熟液中成熟培养至44 h。试验:(1)处理组1:使用PMH-PFF培养22~23 h后,更换PM-PFF继续培养至44 h;(2)处理组2:使用PMH-PFF培养22~23 h后,更换PM-PVA继续培养至44 h;(3)处理组3:使用PMH-PVA培养22~23 h后,更换PM-PVA继续培养至44 h;(4)处理组4:使用PMH-PVA培养22~23 h后,更换PM-PFF继续培养至44 h。将在不同成熟体系中IVM 44 h后的卵母细胞进行固定染色,鉴定卵母细胞核和细胞质成熟情况;对在不同处理组的成熟液中成熟培养44 h的卵母细胞进行孤雌激活后放入PZM-3中培养,分别于第2天、第7天统计分裂率和囊胚发育率。结果表明:经44 h成熟培养后,处理组1卵母细胞核成熟率(42.00%)显著低于处理组2(64.67%)、处理组3(69.00%)、处理组4(64.33%)核成熟率(p0.05)。处理1、处理2、处理4的卵母细胞皮质颗粒分布类型Ⅲ比例(62.00%/54.67%/46.67%)均高于处理组3(28.67%),且处理组1、处理组2卵母细胞皮质颗粒分布类型Ⅲ比例显著高于处理组3卵母细胞皮质颗粒分布类型Ⅲ比例(p0.05)。处理组1、处理组2、处理组4孤雌分裂率(81.92%/76.00%/73.33%)均高于处理组3(69.00%)。处理组3孤雌激活囊胚率(9.67%)显著低于处理组1(23.33%)、处理组2(25.00%)、处理组4(23.00%)(p0.05)。结果表明,在本研究条件下,处理组2的各项指标均优于其它组,IVM液中添加猪卵泡液培养22~23 h后更换添加有PVA的IVM液继续培养至44 h,更有利于促进IVM猪卵母细胞核成熟、胞质成熟以及早期孤雌胚胎发育。     

Effect of ovary storage and oocyte transport method on maturation rate of horse oocytes

Two experiments were conducted to determine the effects of storage on equine ovaries or isolated oocytes. Ovaries were collected at an abattoir and were maintained at room temperature during collection and transport (3-9h total). After arrival at the laboratory, ovaries were divided into three groups: immediate oocyte collection (control), storage at room temperature overnight (15-18 h) before oocyte collection, or storage at 4 degrees C overnight before oocyte collection. Collected oocytes were cultured in maturation medium for 24h. There was a significant increase in the proportion of oocytes classified as having compact cumuli in the two storage groups when compared with the controls. For oocytes originally having expanded cumuli, the rate of maturation to MII was significantly higher in the control group (72%) than in either storage group, and the maturation rate for oocytes from ovaries stored at room temperature (27%) was significantly higher than that for ovaries stored at 4 degrees C (10%). A similar trend was seen for oocytes originally having compact cumuli (24, 11, and 3% in MI-II for control, room temperature, and cold groups, respectively). In Experiment 2, we evaluated the effect of different packaging systems on the maturation of horse oocytes within a portable incubator. Use of 1 ml of equilibrated maturation medium in a 1 ml glass vial was associated with maturation equivalent to that for standard incubation.     

Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles 相似文献   

Methods for collecting follicular oocytes from mares

A series of experiments was conducted to develop a procedure for consistent, repeatable collection of oocytes from the preovulatory follicle of the mare. In one experiment, in situ follicular aspiration with a needle and syringe was performed on 19 mares. From 37 aspirations, four oocytes were recovered (10% recovery rate). In a second experiment, ovaries were visualized via standing flank laparotomy during which two different aspiration techniques were used. Use of a needle and syringe as in the first experiment resulted in successful oocyte recovery in one of seven (14%) attempts. Aspiration via a continuous irrigation vacuum system (CIV), developed for use during laparotomy, resulted in collection of oocytes from six of 10 (60%) attempts. In the third experiment, oocytes were recovered from seven of 18 (38%) attempts at in situ follicular aspiration using a double-lumen needle attached to the CIV. In each experiment, some mares were subjected to stimulation of follicular maturation by exogenous hormones. Oocyte recovery was significantly increased in treated mares as compared with nontreated mares. Results indicate that collection of equine follicular oocytes by in situ aspiration is possible with moderate success. Oocytes apparently are not physically damaged by the procedure, as most retained either the corona radiata or the entire cumulus cell mass.     

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.     

Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes

There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes were fixed immediately after collection to assess whether the collection method influenced the initial chromatin configuration of oocytes. In Experiment 2, in vitro maturation rates of oocytes recovered by aspiration or scraping were compared. In Experiment 3, oocytes were matured in vitro and submitted to intracytoplasmic sperm injection (ICSI). Initial chromatin configuration differed according to collection method in that there was a significantly higher prevalence of diffuse chromatin within the germinal vesicle in oocytes recovered by scraping than in oocytes recovered by aspiration (29/87, 33% and 28/166, 17%, respectively; P < 0.01). Maturation of oocytes to metaphase II did not significantly differ between scraped and aspirated oocytes (56/101, 55.4 % vs. 65/106, 61.4%, respectively). The overall pronucleus formation rate after ICSI of oocytes recovered by scraping was not significantly different than that of oocytes recovered by aspiration (50/99, 52.6% vs. 50/85, 68.5 %, respectively); however, the rate of abnormal fertilization was significantly higher for oocytes collected by aspiration (14/73, 19% vs. 6/94, 6%, respectively; P <0.05). These results demonstrate that the collection method affects the population of recovered oocytes and may contribute to differences in results observed among laboratories working with horse oocytes.     

In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture

Viability of equine embryos produced by oocyte maturation, intracytoplasmic sperm injection and embryo culture to the blastocyst stage in vitro was evaluated after transfer of embryos to recipient mares. No pregnancies were produced after transfer of five blastocysts that had been cultured in G media. Transfer of 10 blastocysts cultured in modified DMEM/F-12 medium produced five pregnancies and three live foals; the two lost pregnancies developed only trophoblast (based on transrectal ultrasonography). To evaluate the status of the inner cell mass, equine blastocysts produced in vivo and in vitro were assessed after differential staining. A discrete inner cell mass could not be appreciated in blastocysts of either source after staining; this was attributed to the presence of a network of cells within the trophoblastic vesicle. Because increased medium calcium concentrations have been reported to decrease the incidence of trophoblast-only pregnancy after transfer of equine nuclear transfer embryos, we investigated the effect of increased calcium concentrations during oocyte maturation or during embryo culture. Increasing calcium concentration of culture medium from 2 to 5.6mM during in vitro oocyte maturation did not affect maturation rate (75 and 68%, respectively) or blastocyst development after fertilization (23 and 27%). However, increasing calcium concentration (from 1.3 to 4.9 mM) of medium used for embryo culture significantly decreased blastocyst development (27% versus 13%, respectively) and adversely affected embryo morphology. More work is needed to optimize culture systems for in vitro production of equine embryos.     

Methods for collecting and maturing equine oocytes in vitro

The objective of our study was to develop an effective method for collecting and maturing equine oocytes. In Experiments 1 and 2, oocytes were collected from excised ovaries obtained via colpotomy. In Experiment 3, oocytes were collected from ovaries obtained after slaughter. Follicles were aspirated and flushed with various treatments to recover the oocytes, which were then cultured and stained to observe the stage of meiosis. In Experiment 1, the aspiration treatments consisted of 0.5 ml of modified Dulbecco's PBS with 0, 100 or 500 lU/ml hyaluronidase. There was no increase (P>0.05) in oocyte recovery with the addition of hyaluronidase. The oocytes were cultured in either TCM-199 or Ham's F-10 medium containing 0.5 ug/ml FSH, 1 ug/ml LH, 1 ug/ml estradiol 17β, 250 uM Na-pyruvate and 10% estrual mare serum for 0, 24, 36 or 48 h. Maturation rates were higher (P<0.05) at 36 h for oocytes cultured in TCM-199 (79%) than for those in Ham's F-10 (21%). There was no difference (P>0.05) in the percentage of maturation of oocytes between the 2 media at 48 h of culture. In Experiment 2, a single aspiration was performed with no flushing medium (dry aspiration) in 0.5 ml of PBS or in PBS with 1000 IU/ml hyaluronidase. The oocytes were then cultured in TCM-199 for 24, 30 or 36 h. There was an increase (P<0.05) in oocyte recovery when follicles were flushed with PBS, with or without hyaluronidase. There was also a difference (P<0.05) in the percentage of maturation of oocytes between 30 and 24 h (86 vs 48%), but no further increase was seen by 36 h (84%). In Experiment 3, follicles were aspirated with PBS 5 to 6, 6 to 7 or 7 to 8 h after slaughter. The oocytes were cultured for 30 h in TCM-199 either with or without 100 IU/ml eCG. There was no effect of eCG or time from slaughter on oocyte maturation or cumulus expansion (P>0.05).     

Equine oocyte in vitro maturation: influences of sera, time, and hormones.

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 micrograms/ml), equine luteinizing hormone (eLH; 100 micrograms/ml), bovine follicle-stimulating hormone (FSH; 5 micrograms/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P less than 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P less than 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.     

Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2–5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs, 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.