卵泡大小和颗粒细胞对山羊卵泡卵母细胞体外成熟和体外受精的影响

用切割法采集卵泡液,收集卵丘一卵母细胞复合体（Cumulus oocytes comlexs,COCs）和自然裸卵,将部分COCs去除卵丘细胞获得机械裸卵,COCs放入体外成熟培养液中培养为成熟卵母细胞,加入获能的精子液,进行体外受精。结果表明：卵母细胞的体外成熟率和卵裂率与卵泡直径密切相关,大卵泡（80．95％,P〈0．01）和中等卵泡（75．50％,P〈0．05）的卵母细胞成熟率高于小卵泡（50．27％）;犬卯泡（53．53％）和中等卵泡（47．13％）的卵裂率显著高于小卵泡的32．26％（P〈0．05）。COCs、机械裸卵和自然裸卵的体外成熟率分别为75．0％、54．2％和10．5％,差异极显著（P〈0．01）,卵裂率分别为53．8％、10．8％和0％,差异极显著（P〈0．01）。对照组和1×10^5、1×10^6个／mL颗粒细胞组卵母细胞体外成熟率分别为68．6％、69．6％和67．8％,无显著差异（P〉0．05）,但均显著高于1×10^7个／mL（51．5％,P〈0．05）和1×10^10个／mL（35．5％,P〈0．05）颗粒细胞组,但各组间的体外受精率无显著差异（P〉0．05）。结果提示,大卵泡和中卵泡的卵母细胞的体外成熟率和卵裂率显著高于小卵泡,体外成熟培养液中添加高浓度的颗粒细胞能显著抑制卵母细胞的体外成熟。     

猪分级卵母细胞体外成熟时间规律的研究

试验根据猪卵丘－卵母细胞复合体（COCs）的颗粒细胞层数,把COCs分成A、B、C和D级,A、B级用于体外分别培养24、32、38、44、48和54 h,C级用于体外分别培养24、32、38、44、48、54和60 h,观察它们不同时间排出极体数,然后将排出极体的卵母细胞孤雌激活。结果发现,A和B级COCs培养48 h时成熟率最高（83.2%和78.0%）,明显高于同级水平培养24、32和38 h的成熟率;不同级别COCs体外培养相同时间,A与B级成熟率差异不显著（P＞0.05）,但二者与C级的成熟率差异显著（P＜0.05）;体外培养48 h COCs卵裂率最高（81.7%）,与培养38 h前的卵裂率差异显著（P＜0.05）。结果表明,A、B级卵母细胞更适于体外培养,能获得高的成熟率和卵裂率,COCs周围颗粒细胞对卵母细胞的成熟有显著影响;COCs培养38 h之前,部分虽能看到极体,但激活后卵裂率极低,说明卵母细胞并未真正成熟,通常通过排出极体判断卵母细胞成熟是不够准确的,COCs体外培养44～48 h是成熟的最佳时期,能为体细胞核移植提供大量优质的MⅡ期卵母细胞。     

不同繁殖季节山羊卵母细胞体外成熟及受精效果研究

本试验在2012年11月至2013年5月间进行,笔者从荣昌屠宰场采集了重庆本地黑山羊的卵巢,对于非繁殖季节（冬）和繁殖季节（春）期的卵巢卵母细胞的回收情况作了比较,并观察了不同级别卵母细胞的体外成熟和体外受精情况.结果表明：繁殖季节的卵巢平均回收COCs数、可用COCs数和COCs可用率分别为5.43枚、2.65枚、48.57％,均较非繁殖季节高（P＜0.05）;A、B、C、D级卵母细胞体外成熟率的差异极显著（P＜0.01）,但A、B、C级卵母细胞体外成熟（IVM）卵的体外受精率、卵裂率无显著性差异（P＞0.05）.     

不同屠宰方式山羊卵巢卵母细胞的体外成熟比较

从屠宰场采集的卵巢567枚，用抽吸法获COCs2851枚，其中适宜体外成熟培养的A、B级COCs1129枚，获可用COCs2．0枚／卵巢；繁殖季节的卵巢平均回收COCs数、COCs可用率和平均可用卵数分别为5．8枚、49．2％、2．9枚，均较非繁殖季节高（分别为4．7、34．5％、1．6），山羊发情较为集中的秋季卵巢卵母细胞的回收数和可用数均较其他季节高。对2种来源的屠宰场卵母细胞进行体外成熟培养，结果显示来源于“烫毛＋烤皮”屠宰方式的卵母细胞体外成熟率为84．5％；来源于“屠宰＋烫毛”，不经过烤皮处理的屠宰场卵母细胞体外成熟率为90．4％，二者无显著性差异（P〉0．05），说明屠宰时经高温处理羊只的卵巢卵母细胞也能在体外培养中继续发育成熟；试验采用的成熟液和培养体系较适合山羊卵母细胞的体外成熟。     

猪卵丘包裹层数与猪卵母细胞质量的相关性研究

为探讨如何从大量的未成熟卵母细胞中挑选出合适的卵母细胞用于体外成熟培养,本研究根据猪卵丘-卵母细胞复合体(COCs)的颗粒细胞层数和胞质情况,把挑选的COCs分成A、B、C3个等级;体外成熟培养42 ～44 h后,所获得的成熟卵母细胞做孤雌激活,比较孤雌胚的发育能力.结果表明:A、B、C3级卵母细胞的成熟率之间没有差异(P＞0.05),但是A级卵母细胞与B级卵母细胞的孤雌胚胎的囊胚率显著高于C级卵母细胞(P＜0.05).说明卵丘细胞包裹层数不会影响卵母细胞的成熟率,但会影响其潜在的发育能力.     

猪卵巢卵母细胞采集方法的比较研究

本试验利用抽吸法和剖切法两种采集方法采集猪卵巢卵母细胞,对获得可用卵丘卵母细胞数和卵丘完整程度的影响进行比较研究,并分析在卵巢收集时不同的保存温度、运输时间对卵母细胞体外成熟的影响。结果表明：剖切法采集细胞数目显著高于抽吸法（P〈0．01）;A、B级卵母细胞的比例在剖切法中显著高于抽吸法（P〈0．05）;抽吸法所采集的c级卵母细胞显著高于剖切法（P〈0．05）;剖切法中卵母细胞卵丘完整率高于抽吸法（P〈O．05）;离体卵巢保存的最佳温度为36℃-38℃;其保存时间为1～1．5h时成熟率57．6％与2—2．5h时成熟率52．8％相比无显著差异（P〉O．05）;保存时间超过6h时卵母细胞的成熟率显著下降。     

牦牛卵泡发育及卵母细胞成熟过程中CYP19A1表达差异分析

旨在探索CYP19A1在牦牛卵泡发育过程及不同成熟阶段卵母细胞中的表达水平。采用实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)检测CYP19A1在牦牛不同级别卵泡(≤3 mm、3～5 mm、5～8 mm及≥8 mm)和卵丘—卵母细胞复合体(Cumulus-oocyte complex,COCs)体外成熟的不同阶段(成熟0 h、12 h、18 h及24 h)中的表达水平,免疫荧光技术(Immunofluorescence,IF)检测CYP19A1蛋白在COCs中的表达定位。结果显示:CYP19A1基因在不同级别的卵泡中均有表达,在卵泡发育早期表达量最高,随卵泡发育呈低水平表达;CYP19A1基因在COCs中的相对表达量显示,未成熟(0 h)COCs的表达量最低,成熟18 h的COCs表达水平达到最高值,之后呈下降趋势。通过免疫荧光技术发现COCs中CYP19A1蛋白的表达主要集中在卵丘细胞上。推测CYP19A1在卵巢颗粒细胞中的表达有助于卵泡发育和卵母细胞成熟雌二醇(Estradiol,E_2)的分泌,对卵泡和卵母细胞早期发育有积极的调控作用,为进一步探索CYP19A1在牦牛雌性生殖中的作用机制提供了理论支撑。     

不同级别绵羊卵母细胞在春季体外培养条件下的成熟率比较

为了对在春季屠宰场所采绵羊卵母细胞的分布及不同分布的成熟率有明确的认识．2003年3月至4月共进行15次试验，收集卵母细胞1485枚。按卵母细胞外层所含卵丘细胞及完整程度分戍A、B、C、D、E5个级别。A级：卵丘细胞层完整且紧密，一般为4层以上；B级：卵丘细胞层完整，一般为1～3层；C级：有卵丘细胞包被，但不完全；D级：裸卵，但细胞质均匀，细胞形态完好；E级：退化裸卵，胞质不均匀，细胞形态消失。选择A，B，C，D级卵母细胞单独进行体外成熟培养，培养液为TCM-199 5％FCS 5μg/mL LH 5μg／mL FSH 1μg／mL β-estradiol 2％penstrep，培养奈件为5％CO2,38．5℃,饱和湿度。结果显示：A，B，C,D级的分布分别为19．8％、28．1％、19．6％和32．4％．成熟率分别为81．3％、65．3％、61．5％和17．0％。总体成熟率为52．1％,卵丘一卵母细胞复合体(COCs)的成熟率为68．9％。     

卵丘细胞对辽宁绒山羊卵母细胞体外成熟与孤雌发育的影响

为了进一步探索辽宁绒山羊卵母细胞体外成熟的方法,本试验以屠宰场绒山羊卵巢为材料,采用抽吸法收集直径大于2 mm卵泡的卵母细胞,研究卵丘细胞对卵母细胞体外成熟和孤雌发育的影响。试验1,将一部分COCs经机械吹打脱除卵丘细胞成为机械裸卵(DOs),然后以4种方式培养,即COCs单独培养、DOs与COCs共培养(DOs(COCs))、DOs与卵丘颗粒细胞共培养(DOs(CCs)),以及DOs单独培养。试验2,根据包裹卵母细胞的卵丘细胞的完整性分为3组,有3层卵丘细胞紧密包围的卵母细胞复合体COCs3,有1-3层卵丘细胞包围的卵母细胞COCs1-3,无卵丘细胞包围的裸露卵母细胞NOs,3组各自单独培养。结果表明:COCs组的成熟率、孤雌卵裂率和囊胚率最高,分别为83.25%、41.75%和29.25%,卵丘细胞的存在有利于卵母细胞体外成熟和随后的发育,COCs3组成熟率、孤雌卵裂率和囊胚率分别为83.25%、42.50%和29.75%,显著高于COCs1-3和NOs组,卵丘细胞的完整性也影响着卵母细胞的体外发育,自然裸卵已失去体外发育能力。     

昆明小鼠卵母细胞体外成熟培养条件优化

对不同的培养液（M199、mDPBS、TYH）和培养液添加物对小鼠C级卵母细胞体外发育和成熟的影响进行了研究。结果表明,mDPBS培养液的培养效果（GVBD：19．8％,PBI：12．3％）好于（P〈0．05）M199培养液（GVBD：13．3％,PBI：8．9％）和TYH培养液（GVBD：12．3％,PBI：7．4％）;mDPBS中添加丙酮酸钠和FCS（GVBD：100％,PBI：86．3％）明显优于（P〈0.01）单纯添加丙酮酸钠（GVBD：42．9％,PBI：23．8％）,也明显优于（P〈0．01）添加丙酮酸钠＋BSA（GVBD：63．2％,PBI：53．8％）;就A、B和C级3种卵母细胞相比,C级卵母细胞的成熟发育最佳,GVBD的发生率为100．0％,PBI的排出率为86．2％。     

Optimization of recovery and maturation methods for cumulus-oocyte complexes in jennies

Embryo production in donkeys is inefficient compared with that in other livestock. Obtaining a sufficient number of MII oocytes is the first step to solving this problem. In this study, the number, morphology and maturation rates of cumulus-oocyte complexes (COCs) obtained from abattoir-derived ovaries or live jennies were compared. The diameter of follicles from abattoir-derived ovaries was measured and divided into group 1 (2–6 mm), group 2 (6–10 mm), group 3 (10–20 mm), group 4 (20–28 mm) and group 5 (>28 mm). The results showed that the number of follicles per ovary in group 2 (3.6 ± 0.28) and 3 (4.2 ± 0.90) was higher than that in the other groups (p < .05). The recovery rate in group 3 was higher than group 1 (48.8% vs. 26.8%, p = .00), but lower than group 5 (48.8% vs. 76.5%, p = .025). The percentage of grade A COCs in group 3 was higher than group 2 (59.3% vs. 39.5%, p = .00) and group 1 (59.3% vs. 26.7%, p = .00). Moreover, the percentage of grade A COCs in group 4 (55.0%, p = .710) and group 5 (46.2%, p = .351) was reduced compared with that in group 3. From the above results, the developing follicles (group ovum pick-up [OPU], 10–20 mm) and preovulation follicles (group OPU-Preov, >35 mm) were aspirated from live jennies using OPU. Although there was no difference in the recovery rates of COCs between group 3 and OPU (48.8% vs. 43.0%, p = .184), the percentage of grades A COCs in group OPU was higher than group 3 (72.5% vs. 59.3%, p = .036). There was no difference in the maturation rate between group 3 and OPU (60.3% vs. 69.3%, p = .171) after the COCs matured in vitro. The rates of recovery (72.2%) and maturation (92.3%) in group OPU-Preov were higher than those in other groups (p < .05). Moreover, the effects of maturation time and serum type on maturation rates were evaluated in groups B44 (44 h, FBS), B36 (36 h, FBS) and D44 (44 h, foetal donkey serum, FDS). These results indicated that the maturation rate in group B36 was lower than group B44 (13.1% vs. 47.0%, p = .00) and group D44 (13.1% vs. 53.3%, p = .00). In conclusion, the quality of donkey COCs from OPU was higher than that from abattoir-derived ovaries, the suitable time of donkey in vitro maturation (IVM) was 44 h, and FBS could be replaced with FDS in donkey IVM medium.     

培养介质、卵丘细胞和卵泡直径对猪卵母细胞体外成熟的影响

A类卵母细胞在mTCM 199、NCSU2 3和NCSU37体系中培养 4 4～ 5 2小时后 ,成熟率分别为 76 .1%、78.1%和 6 5 .2 %。前两者差异不显著 (P >0 .0 5 ) ,但显著高于后者 (P <0 .0 5 )。卵母细胞在添加eCG和hCG的NCSU2 3体系中的成熟率 (75 .6 % )明显高于添加FSH的LH和成熟率 (6 5 .2 % ) (P <0 .0 5 )。A、B、C三类卵母细胞在NCSU2 3的成熟率分别为 73.3%、6 0 .4 %和 11.0 % ,三者间差异显著 (P <0 .0 5 )。大 (ф >6mm)、中 (ф =3～ 6mm)和小 (ф <3mm)三种卵泡中的卵母细胞在NCSU2 3中培养后 ,成熟率分别为 5 6 .2 % ,78.1%和 5 1.9% ,中等卵泡中卵母胞的体外成熟率显著高于其他两组 (P <0 .0 5 )。     

牦牛卵巢卵母细胞体外培养成熟条件的建立

研究了不同采集方法和不同培养液对牦牛卵母细胞体外成熟的培养效果。结果:在牦牛乏情期,每个卵巢平均回收卵数为(9.33±4.30),可用卵数为(5.63±4.19)。将牦牛卵丘卵母细胞复合体分别置于5种成熟培养液中培养,成熟率分别为75.56%、71.11%、81.33%、77.33%和77.78%,卵裂率分别为42.22%、33.33%、49.33%、46.67%和42.22%,其中C的成熟液效果最好。来自卵巢表面卵泡的COCs的成熟率(81.33(vs69.33(,P>0.05)高于来自卵巢内卵泡的COCs,卵裂率(49.33%vs34.67%,P<0.05)显著高于来自卵巢内卵泡的COCs。来自明亮卵泡的COCs的成熟率(81.33%vs33.33%,P<0.01)和卵裂率(49.33%vs3.33%,P<0.01)极显著高于来自混浊卵泡的COCs。     

Quality and Developmental Ability of Dromedary (Camelus dromedarius) Embryos Obtained by IVM/IVF, In Vivo Matured/IVF or In Vivo Matured/Fertilized Oocytes

The effect of source of cumulus-oocytes-complexes (COCs), maturation and fertilization conditions on developmental competence of dromedary embryos was examined. Thirty-six adult females were superovulated with equine Chorionic Gonadotropin (eCG) injection (3500 IU, IM) and divided in three groups of 12 females each. Group 1 provided 138 COC's collected from follicles >or= 5 mm 10 days after stimulation prior hCG treatment and matured in vitro for 30 h. Group 2 provided 120 in vivo matured oocytes which were aspirated from their follicles 20 h after hCG (3000 IU, IV) given on day 10 follow eCG injection. Group 3 provided 65 in vivo matured/fertilized oocytes. Females in Group 3 received hCG on day 10 following eCG treatment and then were mated 24 h later. Fertilized oocytes were collected from the oviducts of females 48-h post-mating. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of COCs. All cultures were performed in three replicates (n = 3) at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. Nuclear maturation rate for Groups 1 and 2 was determined by epifluorescence microscopy in a sample of COCs (n = 30) denuded, fixed and stained with Hoechst 33342. To study the viability of obtained embryos, hatched blastocysts from each group were transferred to recipients followed by pregnancy diagnosis using ultrasonography at 15, 60 and 90 days. The percentage of COCs reaching metaphase II (MII) after 30 h of maturation was slightly but not significantly higher for in vivo matured oocytes (28/30; 93%) than those in vitro matured (25/30; 84%). The total rate of cleavage (2 cells to blastocyst stage) was not different for the three groups. However, significantly (p < 0.05) more blastocyst and hatched blastocysts were obtained from in vivo matured and in vivo fertilized oocytes (Group 3; 52% and 73%) than from in vitro fertilized oocytes whether they were matured in vitro (Group 1; 35% and 32%) or in vivo (Group 2; 32% and 45%). Pregnancy rates were not significantly different amongst all groups for the three first months following embryo transfer. All pregnancies were lost after day 90 follow transfer except for in vivo matured and in vivo matured/fertilized groups. Only in vivo matured/in vitro fertilized and in vivo matured/fertilized produced embryos continued normal development until term and resulted in the birth of normal and healthy live calves. Six claves (29%; 6/21) were born from Group 3 and one (8%; 1/13) calf was born from Group 2. This study shows that the IVC system used is able to support camel embryo development. However, developmental competence and viability of dromedary embryos may be directly related to the intrinsic quality (cytoplasmic maturation) of oocytes.     

Effect of eCG Superstimulation and Buserelin on Cumulus–Oocyte Complexes Recovery and Maturation in Llamas (Lama glama)

The aim of the present study was two fold. Experiment I: evaluate the effect of buserelin on llama's oocyte maturation after exogenous follicular activity suppression, followed by ovarian superstimulation with different doses of equine chorionic gonadotropin (eCG). Experiment II: compare the number of follicles aspirated and the number of cumulus–oocyte complexes (COCs) recovered according to different doses of eCG followed by buserelin. Experiment I consisted in a control group (without buserelin) and a treatment group (with buserelin), both subdivided according to eCG dose administered: A: 500 IU; B: 1000 IU; C: 1500 IU. The treatment group received a single i.v. dose of 8 μg of buserelin when two or more dominant follicles were found at ultrasound evaluation and 20 h later were subjected to surgery. In group A, 83% of the llamas did not respond to superstimulation. In groups B and C differences were observed between the control and the treatment groups for the degree of COCs maturation (p < 0.05). In experiment II animals were divided into two groups according to the eCG dose administered: 1000 and 1500 IU. Twenty hours before surgery females received a single i.v. dose of 8 μg of buserelin. Average number of follicles aspirated and COCs recovered was higher (p < 0.05) with the administration of 1500 IU of eCG. A larger number of expanded COCs were obtained from follicles ≥7 mm in diameter. We conclude that buserelin aids the recovery of a larger number of expanded COCs. Administration of 1500 IU of eCG produces a higher number of follicles for aspiration and number of COCs recovered.     

Predictive value of the area of expanded cumulus mass on development of porcine oocytes matured and fertilized in vitro

The objective of the present study was to investigate the correlation between the degree of cumulus expansion and in vitro development of porcine cumulus-oocytes complexes (COCs) matured and fertilized in vitro. The COCs were matured in the maturation medium (IVMM) supplemented with 15% or 5% of porcine follicular fluid (PFF) from small, medium and large follicles (<2 mm, 2-5 mm and >5 mm, respectively). COCs cultured in IVMM with PFF for 48 h displayed less expansion than those cultured in IVMM alone (P<0.05), irrespective of follicle size. After culture for 24 h in IVMM with PFF and for another 24 h in IVMM alone, the degree of cumulus expansion was more prominent than culture in the presence of PFF for the entire 48 h period (P<0.05), but the percentages of oocytes with PB I showed no significant difference between the control and experimental groups (P>0.05). After in vitro fertilization, the oocytes failed to develop to the morula/blastocyst stages except for those matured in IVMM supplemented with 15% or 5% PFF obtained from >5 mm follicles for the first 24 h and followed by in IVMM alone for the second 24 h (12.5% and 11.1% of the embryos developed to morulae and blastocysts, respectively). The expanded cumulus areas of COCs were significantly positively correlated with their in vitro development (p=0.0058, 0.0001 and 0.0348 for the percentages of embryos developed to 2-4 cell, beyond 4 cell and morula and blastocyst stages, respectively). In conclusion, PFF had an inhibiting effect on cumulus expansion, and the inhibitory effect decreased progressively with the increase in size of follicles from which PFF was obtained, and the action of PFF on cumulus expansion was affected by the PFF culture time. The areas of the expanded cumulus mass may be used as a parameter to predict development of porcine oocytes matured and fertilized in vitro.     

输卵管和颗粒细胞单层对牛体外受精胚胎发育的影响

以屠宰场牛卵巢为试验材料,研究输卵管细胞单层(OCM)和颗粒细胞单层(GCM)对牛卵母细胞体外成熟(IVM)、体外受精(IVF)和体外培养(IVC)后胚胎发育能力的影响。(1)从卵泡抽取卵丘卵母细胞复合体(COCs),并根据卵母细胞外面卵丘细胞的层数将其分为3类:1级(≥4层);2级(2～3层);3级(0～1层)。作分别在IVM和IVC培养液中添加GCM(1×106个/mL)与不添加的对比试验。结果显示:添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无明显影响(P>0.05);但添加GCM的2级、3级卵母细胞,受精后的卵裂率、6～8细胞发育率和囊胚率分别高于未添加组(P<0.05)。(2)所有卵母细胞(包括COCs和裸卵)被随机分为3个组,在其IVM和IVC培养液中分别添加OCM、GCM或不添加体细胞(对照组)。结果显示:OCM和GCM组的卵裂率、6～8细胞发育率和囊胚率均高于对照组(P<0.05),而两试验组之间差异不显著。     

Collection and in Vitro Maturation of Equine Oocytes from Estrus,Diestrus and Pregnant Mares

Two experiments were conducted to evaluate oocyte collection rates and in vitro nuclear maturation rates of equine oocytes obtained during diestrus and pregnancy, and to compare these rates with maturation rates in oocytes derived from preovulatory follicles. In Experiment I, transvaginal ultrasound-guided aspiration of follicle was performed during estrus and diestrus in 14 mares over four consecutive cycles. Follicular aspirations during estrus were performed 24 to 27 hours after injection of 2500 IU of hCG given when the largest follicle reached 35 mm in diameter. Oocyte recovery rate from preovulatory follicles was 51% (33/65) in 49 aspiration sessions. Cumulus-oocyte complexes from preovulatory follicles were cultured for 12-15 hours in TCM199 + 10% (NCS) at 38.5°C in 5% CO₂ in air, and 22/33 (67%) were in metaphase II. During diestrus, mares were treated (Group I) or not treated (Group II) daily with equine pituitary extract (EPE) during alternate cycles from days 1 to 14 after the preovulatory aspiration. Diestrous follicles were aspirated when four or more follicles greater than 12 mm in diameter were present. EPE had no effect on the number of follicles that developed during estrus or diestrus (p>0.05). Oocytes were recovered from 119 of 383 diestrous follicles (31 %)in 75 sessions. There was no difference in recovery rates between Groups I and II (p>0.05). Maturation rates for oocytes collected during diestrus, after 42 hours of culture in TCM 199 + 1 μg/ml of Estradiol and lnl/ml of EPE, were not significantly different (p>0.05) between Groups I and II (49% vs. 53%). In Experiment II, mares between 50 and 85 days of pregnancy were used as oocyte donors. The oocyte recovery rate was 53% (66/125). After in vitro maturation for 40 hours (compact COC) or 15 hours (expanded COC), 22% (7/32) and 22% (7/32), respectively, of the oocytes were in metaphase II. It was concluded that: 1) Preovulatory follicles yield a higher percentage of oocytes with a higher rate of maturation to metaphase II than follicles of diestrus and pregnant mares. 2) Diestrous follicles yielded fewer oocytes than follicles of pregnant mares but with a higher percentage of oocyte maturation. Further studies are necessary to determine if oocytes recovered from diestrous follicles and matured in vitro can be fertilized successfully.