Chronological changes of bovine follicular oocyte maturation in vitro

Chronological changes of bovine follicular cumulus-oocyte-complexesi were studied after in vitro maturation over a period of 48 h. According to their thickness and compactness of cumulus investments they were classified into 4 groups and cultured in enriched Ham’s F-10 medium with or without human chorionic gonadotrophin (hCG) and estradiolbenzoate (EB) for 0, 6, 12, 18, 21, 24, 27, 30 and 48 h. Representative samples were taken at each time interval for evaluation of nuclear maturation stages, ooplasm quality and size of the peri vitelline space (PVS). The results showed that oocyte nuclear breakdown (ONBD) required 6 to 12 h culture, and the peak of the first polar abstriction occurred at 24 h. The culture period required for ONBD and abstraction of the first polar body were related to the thickness and compactness of cumulus investments with and approximately 6 h delay in heavily compacted complexes. Ooplasm quality evaluation failed to show a clear trend, but the PVS increased in size from 0 h to 30 h and then, retracted again from 30 to 48 h. The overall maturation rate in the presence of hCG and EB was 79.1 %, and a substantial proportion (68.8 %) of nude or partially covered oocytes reached metaphase II stage. In the presence of hCG and EB no block at either metaphase I or at anaphase-telophase I was observed. In the absence of hCG and EB the percentage of oocytes reaching metaphase II was much lower (48.6%) in comparison with oocytes matured in the presence of these hormones (79.1 %). It was concluded a very high proportion of slaughterhouse oocytes could be matured in vitro and that the cumulus investments and addition of certain hormones affected the maturation rate.     

超低温冷冻对牛卵母细胞组蛋白乙酰化和膜蛋白CD9表达的影响

本试验旨在探讨玻璃化冷冻对牛体外成熟卵母细胞(MⅡ期)组蛋白乙酰化和膜蛋白CD9表达的影响。牛MⅡ期卵母细胞采用OPS法冷冻,即卵母细胞于10%EG+10%DMSO溶液中预处理30s,然后再移入玻璃化溶液EDFSF30中处理25s,以OPS为承载器投入液氮中。毒性组卵母细胞未投入液氮,其他过程与冷冻组相同,新鲜牛MⅡ期卵母细胞为对照组。卵母细胞解冻后,利用免疫荧光染色法检测存活细胞DNA组蛋白乙酰化;qRT-PCR和Western blot检测CD9mRNA蛋白的表达。结果表明:冷冻组卵母细胞形态正常率(93.8%)和存活率(92.7%)显著低于毒性组(100.0%,97.2%)和对照组(100.0%,98.5%)(P<0.05),而毒性组与对照组之间无显著差异。超低温冷冻后,卵母细胞DNA组蛋白乙酰化水平显著上升(P<0.05),CD9mRNA与蛋白表达显著降低(P<0.05)。以上显示,玻璃化冷冻不但降低了牛体外成熟卵母细胞的存活率,而且改变了DNA组蛋白乙酰化和膜蛋白CD9表达水平。     

Effects of melatonin on maturation,histone acetylation,autophagy of porcine oocytes and subsequent embryonic development

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1 nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1 nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle‐derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.     

Role of the hyaluronan receptor CD44 during porcine oocyte maturation

Previous our studies have shown that CD44, the principal receptor for hyaluronan, is present on cumulus cells during oocyte maturation. Although hyaluronan-CD44 interaction has been implicated in cumulus expansion and/or oocyte maturation, the full significance of CD44 remains unknown. The objective of the present study was to further investigate the role of CD44 in cumulus expansion and oocyte maturation in pigs. We demonstrate here in that CD44 has a key role in oocyte maturation but not in cumulus expansion. Previous studies have reported the physiological significance of cumulus expansion in oocyte maturation. However, our results suggest that cumulus expansion is a necessary condition for oocyte maturation, but that it is not sufficient on its own. Furthermore, western blot analysis demonstrated that the CD44 of the in vitro-matured cumulus-oocyte complexes (COCs) had a larger molecular weight and more terminal sialic acid, which has been proven to inhibit the hyaluronan-binding ability of the receptor, than the CD44 of the in vivo-matured COCs, indicating that the hyaluronan-CD44 interactions during in vitro maturation might be insufficient compared with those in vivo. The insufficient interactions of hyaluronan-CD44 during in vitro maturation may cause the inferior capacity of fertilization and development of oocytes matured in vitro.     

兔卵母细胞成熟过程中组蛋白H3Ser10磷酸化表达分析

为研究卵母细胞成熟过程中组蛋白H3第10位丝氨酸(H3Ser10)磷酸化变化规律及其表达水平,本研究以体外成熟免卵母细胞为材料,采用免疫荧光标记方法检测兔卵母细胞成熟各时期组蛋白H3Ser10磷酸化的动态分布,同时探讨不同浓度组蛋白磷酸化激酶抑制剂(ZM447439)处理卵母细胞对组蛋白H3Ser10磷酸化表达的影响.结果显示:(1)兔卵母细胞组蛋白H3Ser10磷酸化始于生发泡破裂期(GVBD),且表达水平最高;第1次减数分裂中期(MⅠ期)磷酸化水平降低,第1次减数分裂后期(AⅠ期)及第2次减数分裂中期(MⅡ期)磷酸化水平呈上升趋势,但均比GVBD期低.(2)低浓度ZM447439对兔卵母细胞核成熟进程影响不大,当其浓度增加到30 μmol/L时,93.5％的卵母细胞停留在GVBD期.(3)ZM447439浓度为5μmol/L时,20.7％卵母细胞H3Ser10去磷酸化,其他卵母细胞H3Ser10磷酸化信号与未处理组相似;当ZM447439浓度增加到30μmol/L时,大多数卵母细胞中的H3Ser10磷酸化消失.以上结果表明:兔卵母细胞组蛋白H3Ser10磷酸化始于GVBD期,且一直持续到MⅡ期,GVBD期磷酸化水平最高.ZM447439可有效抑制兔卵母细胞减数分裂的恢复,且随其浓度的增加,组蛋白H3Ser10磷酸化水平降低;当浓度达30μmol/L时,卵母细胞组蛋白H3Ser10终止磷酸化.     

水牛卵母细胞在不同培养液中成熟与凋亡的比较研究

为了解卵母细胞体外成熟与凋亡过程,进而提高卵母细胞体外成熟率,本试验研究了在培养液中添加不同浓度的表皮生长因子(EGF)、胰岛素样生长因子-1(IGF-1)对水牛卵母细胞体外成熟和凋亡的影响。结果表明:(1)添加各种浓度的EGF(10,20,30,50,100 ng/mL)均可以提高水牛卵母细胞的成熟率,降低卵母细胞的凋亡率,其中50 ng/mL EGF有显著影响(P<0.05);(2)添加各种浓度的IGF-1(10,30,50,100 ng/mL)均能提高水牛卵母细胞体外成熟率,降低卵母细胞的凋亡率,以30 ng/mL效果明显(P<0.05);(3)添加20 ng/mL EGF+30ng/mL IGF-1组卵母细胞体外成熟率和凋亡率分别高于和低于单独添加IGF-1和EGF组。     

Crocin supplementation during oocyte maturation enhances antioxidant defence and subsequent cleavage rate

The purpose of the present study was to assess the effect of crocin supplementation during oocyte maturation on the antioxidant defence and anti‐apoptotic ability and subsequent developmental competence of porcine oocytes. Oocytes were cultured in media containing 0, 300, 400 or 500 µg/ml of crocin. Upon maturation, the maturation rates, reactive oxygen species (ROS) and glutathione (GSH) levels, mRNA expression of genes (SOD, CAT, GPx, Bcl‐2, BAX and Caspase3), expression of cleaved caspase3 and subsequent embryo cleavage rates were measured. Results indicated that the maturation rate of the 400 µg/ml group was 86.80% (p < 0.01). The ROS concentration of the 500 µg/ml group was the lowest (p < 0.01). The GSH concentration of the 400 µg/ml group was the highest (p < 0.01). The SOD, CAT and GPx mRNA expression levels were the highest in the 300, 400 and 500 µg/ml groups, respectively, with the expression levels of all genes being significantly higher than that of the control group (p < 0.01). The Bcl‐2/BAX mRNA expression ratio in 400 and 500 µg/ml groups significantly higher than other groups and significantly decreased caspase3 expression level (p < 0.01). The expression level of cleaved caspase3 in the 500 µg/ml treatment group was the lowest, significantly lower than that of the control group (p < 0.01). The cleavage rate of the 400 µg/ml group was 62.50% (p < 0.01). These experimental results show that the supplementation of in vitro culture medium with 400 µg/ml of crocin significantly enhanced the antioxidant defence and anti‐apoptotic ability and subsequent cleavage rate of porcine embryo.     

Exogenous paraoxonase‐1 during oocyte maturation improves bovine embryo development in vitro

Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml^?1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml^?1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.     

影响哺乳动物卵母细胞体外成熟的因素

卵母细胞体外成熟是哺乳动物体外受精技术的关键步骤之一 ,它直接关系到体外受精技术的成功与否。由于影响哺乳动物卵母细胞体外成熟的因素很多 ,使得这一技术在应用时存在一定难度和问题。人们已经通过大量的试验对许多影响因素进行了分析和研究 ,并获得了有效的结果。本文旨在对影响该技术的几项重要因素进行阐述 ,以供同行们在试验中能形成较全面的考虑 ,从而进一步提高该技术的成功率。1　影响卵母细胞减数分裂的化学物质促减数分裂甾醇 (Ｍｅｉｏｓｉｓ ａｃｔｉｖａｔｉｎｇＳｔｅｒｏｌｓ,ＭＡＳ)是近年来新发现的能够促进哺乳动物生…     

猪卵母细胞体外成熟的影响因素

卵母细胞是胚胎工程和发育生物学的重要组成部分,它是体外受精、性别控制、克隆及转基因等技术成功与否的前提和关键。本文初步探讨猪卵母细胞体外成熟的影响因素。     

All‐trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation

All‐trans retinoic acid (t‐RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t‐RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus‐oocyte complexes (COCs) were matured in vitro in the absence or presence of t‐RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real‐time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t‐RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t‐RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t‐RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t‐RA inhibits the apoptosis of cumulus cells (P < 0.01). t‐RA treatment up‐regulated the expression of B‐cell lymphoma 2 (BCL‐2), catalase (CAT) (P < 0.05) and down‐regulated the expression of Caspase‐8 (P < 0.05). In conclusion, t‐RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.     

Vitrification of pig oocytes induces changes in histone H4 acetylation and histone H3 lysine 9 methylation (H3K9)

In the present study, acetylation status of histone H4 and methylation status of the lysine 9 residue of histone H3 (H3K9) were assessed by immunofluorescence in order to determine the effect of vitrification on epigenetic status of pig MII oocytes. Hyperacetylation of H4 and dimethylation of H3K9 were assessed in control oocytes, after cryoprotectant treatment and after vitrification at two time points, immediately after warming and after a post-warming incubation for 2?h. While no changes in the immunopositivity for both the epitopes were recorded after cryoprotectants, the percentage of negative oocytes for dimethyl H3K9 was observed to increase immediately after devitrification. The influence of vitrification was more evident after 2?h post-thaw incubation when acetylation status of H4 significantly increased and a rise in the percentages of both oocytes exhibiting strong positivity and negative oocytes for dimethyl H3K9 was observed. In conclusion, acetylation of H4 and methylation of H3K9 are altered by vitrification procedure that may lead to an aberrant epigenetic presentation of female chromatin to the fertilizing event and may be, at least in part, responsible for the reduction of developmental competence of vitrified pig oocytes.     

Equine oocyte maturation with epidermal growth factor

Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species. This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods. Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15-25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM-199 supplemented with 50 ng/ml EGF) and EMS Group (TCM 199 supplemented with 10% v/v oestrous mare serum). Each group was divided further into 3 treatments with tyrphostin A-47, a specific tyrosine kinase inhibitor, at 0, 10(-4) and 10(-6) mmol/l. Maturation was determined as the percentage of oocytes reaching metaphase II stage at the end of the culture period. Immunohistochemical detection of EGF-receptor (EGFR) was performed using a streptoavidin-biotin method. The recovery rate and oocyte retrieval were 84.6% (recovered oocytes/follicles aspirated) and 6.55 (oocytes/mare), respectively. Treatment with EGF significantly (P<0.05) increased the incidence of metaphase II stage compared with the control group (69.4 vs. 26.9% in controls, respectively). The specific-tyrosine kinase inhibitor A-47 was effective in suppressing EGF-effect on EGF-cultured oocytes; no significant differences were observed in EMS-supplemented oocytes when cultured with A-47. EGF-receptor was localised in follicles, with localisation being more prominent in the cumulus than in mural granulosa cells. This finding, together with the increase of oocyte nuclear maturation rate when using EGF in culture media and the inhibition of maturation by tyrphostin A-47, suggests a physiological role for EGF in the regulation of equine oocyte maturation. The results should help successful development of assisted reproductive technology in the horse.     

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation

Background

Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation.

Methods

Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution.

Results

The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct.

Conclusions

Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.     

DRP1 deficiency induces mitochondrial dysfunction and oxidative stress-mediated apoptosis during porcine oocyte maturation

Background: Environmental pollution induces oxidative stress and apoptosis in mammalian oocytes, which can cause defects in reproduction;however, the molecular regulation of oxidative stress in oocytes is still largely unknown. In the present study, we identified that dynamin-related protein 1(DRP1) is an important molecule regulating oocyte mitochondrial function and preventing oxidative stress/apoptosis. DRP1 is a member of the dynamin GTPase superfamily localized at the mitochondrial-endoplasmic reticulum interaction site, where it regulates the fission of mitochondria and other related cellular processes.Results: Our results show that DRP1 was stably expressed during different stages of porcine oocyte meiosis, and might have a potential relationship with mitochondria as it exhibited similar localization. Loss of DRP1 activity caused failed porcine oocyte maturation and cumulus cell expansion, as well as defects in polar body extrusion.Further analysis indicated that a DRP1 deficiency caused mitochondrial dysfunction and induced oxidative stress,which was confirmed by increased reactive oxygen species levels. Moreover, the incidence of early apoptosis increased as detected by positive Annexin-V signaling.Conclusions: Taken together, our results indicate that DRP1 is essential for porcine oocyte maturation and that a DRP1 deficiency could induce mitochondrial dysfunction, oxidative stress, and apoptosis.     

Experimental studies of the function of cumulus cells during extracorporeal oocyte maturation in the pig]

In order to study in more detail the functional significance of the granulosa cell reaction for the in vitro oocyte maturation in the pig, 412 oocytes taken from Graafian follicles and 510 taken from tertiary follicles were grown on different culture media. 147 oocytes, which during in vitro maturation showed a granulosa cell reaction, were transplanted and were utilized to study their embryonic developmental potentialities. It was possible to induce the granulosa cell reaction in about two-thirds of oocytes by the addition of the fluid taken from Graafian follicles. Denudation of oocytes led to a severe inhibition of maturation. Only one-third of the transplanted oocytes showed normal embryonic developmental potentialities. The findings suggest that maturation of the oocyte nucleus is related directly to the granulosa cell reaction, while the maturation of the cytoplasm is independent of it.     

Steroidogenesis and the influence of MAPK activity during in vitro maturation of porcine cumulus oocyte complexes

The aim of this study was to investigate steroidogenesis within porcine cumulus oocyte complexes during in vitro maturation and to examine the possible influence of the mitogen-activated protein kinase (MAPK). Porcine cumulus oocyte complexes were matured in vitro with and without the MAPK kinase inhibitor U0126 for 0, 5, 26 and 46 h. The 17β-estradiol and progesterone concentration in the culture medium were then determined. In addition, the mRNA levels of StAR, Cyp11A1, 3β-HSD and Cyp19A1 in cumulus cells were analysed by RT-PCR. Using an immunoblot, the MAPK phosphorylation in cumulus cells and oocytes was examined. During the first 26 h of in vitro maturation, 17β-estradiol secretion was predominant, whereas, after a culture period of 46 h, the progesterone secretion decreased conspicuously. Under the influence of U0126, the secretion of 17β-estradiol increased progressively during the complete maturation period, while progesterone secretion was completely inhibited. The mRNA levels of StAR and Cyp11A1 were not altered by U0126; however, corresponding to the hormone secretion, the gene expression of Cyp19A1 was up-regulated and the expression of 3β-HSD down-regulated. The results suggested an influence of the MAPK on steroidogenesis in cumulus cells comparable to a luteinization factor. Hormone synthesis in cumulus cells during oocyte maturation seems to be regulated by altering expression of Cyp19A1 and 3β-HSD.     

猪卵巢卵母细胞体外成熟体系研究进展

猪卵母细胞体外成熟培养中存在的问题是如何进一步提高卵母细胞的采集效率、急待解决的问题是成熟率不高和成熟质量差的问题。综合论述了猪体外受精技术所包含的猪卵母细胞体外成熟(IVM)培养和受精卵的体外培养两项密切相关的技术与方法，以及完善现有的成熟体系及成熟过程。     

Molecular mechanisms underlying pig oocyte maturation and fertilization

Since the pig is not only an important farm animal, but also a model animal for biomedical applications, the development of reproductive technologies in this species has been very important. In vitro oocyte maturation and fertilization (IVM-IVF) are basic techniques for a number of oocyte- or embryo-related technologies. The practical aspects for pig oocyte IVM-IVF have been reviewed, while the molecular mechanisms underlying oocyte meiotic maturation and fertilization have not been well summarized, although accumulating data have been obtained in recent one decade. This review will focus on what is known about the molecular mechanisms of porcine oocyte maturation and fertilization such as first meiosis resumption, meiotic spindle assembly, second meiosis metaphase (MII) arrest during oocyte maturation, sperm-egg recognition and fusion, sperm acrosome reaction, second meiosis resumption, sperm chromatin decondensation, and pronucleus formation during fertilization, as well as the establishment of polyspermy block.     

Effects of cumulus cells on rabbit oocyte in vitro maturation

Cumulus cells (CCs) are of great importance in oocyte development and maturation in many species, but detailed influence of CCs has not been extensively examined, especially on rabbit. The present study was designed to investigate the effects of CCs and the elongation of in vitro maturation (IVM) time on rabbit oocyte nuclear and ooplasmic maturation and survival. Cumulus oocyte complexes (COCs) and naked oocytes (NOs) were recovered directly from rabbits super-ovulated with eCG. Corona-enclosed oocytes (COs) and denuded oocytes (DOs) were obtained from COCs after removing a part or whole of CCs. The oocytes were cultured in the following seven groups. (i) Cumulus cell enclosed oocytes (CEOs) were cultured alone (CEOs); (ii) COs were cultured alone (COs); (iii) DOs were cultured alone (DOs); (iv) NOs were cultured alone; (v) DOs were co-cultured with COCs [DOs(COCs)]; (vi) DOs were co-cultured with CCs [DOs(CCs)]; (vii) NOs were co-cultured with CCs [NOs(CCs)]. After the oocytes were cultured for 24 and 30 h, the nuclear maturation was evaluated by first polar body (PB1) extrusion while the ooplasmic maturation was evaluated by the cleavage rate after parthenogenetic activation. The results showed that the nuclear maturation rate of CEOs, COs, DOs(COCs) and DOs(CCs) after 24 h incubation were significantly different from each other (p < or = 0.05), the rate of DOs(CCs) was similar to that of DOs (p > or = 0.05). The cleavage rates in the first two groups were significantly higher than those of the others (p < 0.05). For oocytes cultured for 30 h, the nuclear maturation rates were significantly different for each culture model (p < 0.05). The cleavage rates in first two groups were significantly higher than those of others (p < 0.05). Both the nuclear and cleavage rates significantly increased when the culture time of DOs(COCs) was prolonged from 24 to 30 h. DOs(CCs) nuclear maturation was significantly improved when the culture time was prolonged from 24 to 30 h, but the ooplasmic maturation was not. Few NOs incubated with or without CCs accomplished nuclear maturation (approximately 2% both), even when the culture time was prolonged from 24 to 30 h. The oocyte degeneration rates were significantly different for each culture model after both 24 and 30 h incubation (p < or = 0.05). There was no significant difference in oocyte degeneration in the same groups between 24 and 30 h incubation (p > 0.05). The results suggest that rabbit CCs affect oocyte nuclear and ooplasmic maturation, and their survival. The prolongation of the culture time of rabbit oocyte from 24 to 30 h improves the nuclear and ooplasmic maturation differently in the present system. Rabbit oocytes free of CCs, especially NOs, show weak meiotic resumption potential and compromised viability, which cannot be improved by co-culture with dispersed CCs. The degeneration mostly happens at early time of IVM.