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1.
本试验从卵母细胞卵丘多少和初次卵裂时间早晚2个方面进行研究,旨在改善小型猪克隆方案的体外培养环节,提高克隆效率。将卵母细胞按卵丘多少分成3组,比较卵母细胞的成熟效果,取成熟效果好的卵母细胞进行后续试验;PA和SCNT试验均按初次卵裂时间早晚分成3组,在体外培养条件下,比较胚胎的卵裂率和囊胚率。结果表明,卵丘多的卵母细胞体外成熟39～40 h和卵丘少的卵母细胞体外成熟41～42 h,比不区分卵丘多少的卵母细胞体外成熟39～42 h的成熟效果要好;初次卵裂时间发生在26 h以前的胚胎比26 h之后的胚胎在数量和质量上都明显优越,前者胚胎的卵裂率和囊胚率显著高于后者,此结果在孤雌激活(parthenogenetic,PA)胚胎和体细胞核移植(somatic cell nucleartransfer,SCNT)胚胎的体外试验中均得到验证。本研究完善了小型猪克隆方案的体外培养环节,为器官异种移植提供相关技术参考。  相似文献   

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3.
Incomplete cytoplasmic maturation of in vitro matured (IVM) oocytes has been known to cause microtubule and microfilament alterations, which may result in abnormal pronuclear formation and failed embryonic development. We examined the influences of maturation conditions on meiotic spindle morphology at metaphase of meiosis II (MII) in porcine oocytes. Porcine oocytes were matured under various conditions, i.e., in vitro or in vivo, with different amounts of cumulus cells, with or without hormonal supplements, and with various exposure durations to the hormones, to examine the effects on spindle morphology in MII oocytes by immunofluorescence under confocal laser microscopy. Interpolar spindle length (microm) and spindle area (microm2) were compared among these maturation conditions. The spindle length was significantly shorter in IVM oocytes compared to those matured in vivo. Oocytes collected from cumulus oocyte complexes (COCs), which were poor in cumulus cells, showed smaller spindle areas than those from cumulus-rich COCs. The spindle length and area were both significantly reduced in oocytes grown without hormonal supplements. When oocytes were grown with hormonal supplements for either 6 or 22 hours for the first half of culture, there was no difference in the spindle morphology between these oocytes. These results suggested that maturation conditions significantly influence morphogenesis of MII spindles in porcine oocytes. Oocytes matured in poor conditions were more likely to have a shorter spindle length (long axis) and smaller spindle areas.  相似文献   

4.
Two studies were conducted with the overarching goal of determining the extent to which lipolytic changes relate to germinal vesicle breakdown (GVBD) in bovine oocytes matured under thermoneutral or hyperthermic conditions. To this end, cumulus-oocyte complexes underwent in vitro maturation for 0, 2, 4, 6 or 24 h at 38.5 (first study) or 38.5 and 41.0 C (second study; heat stress applied up through first 12 h only, then shifted to 38.5 C). Independent of maturation temperature, triglyceride and phospholipid content decreased markedly by 2 h of in vitro maturation (hIVM; P < 0.0005). Content was lowest at 24 hIVM with no detectable impact of heat stress when exposure occurred during first 12 hIVM. Germinal vesicle breakdown occurred earlier in oocytes experiencing heat stress with effects observed as soon as 4 hIVM (P < 0.0001). Germinal vesicle breakdown was associated with lipolytic changes (R2 = 0.2123 and P = 0.0030 for triglyceride content; R2 = 0.2243 and P = 0.0026 for phospholipid content). ATP content at 24 hIVM was higher in oocytes experiencing heat stress (P = 0.0082). In summary, GVBD occurs sooner in heat-stressed oocytes. Although marked decreases in triglyceride and phospholipid content were noted as early as 2 hIVM and preceded GVBD, lipolytic changes such as these are not likely serving as an initial driver of GVBD in heat-stressed oocytes because changes occurred similarly in oocytes matured at thermoneutral conditions.  相似文献   

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6.
The acquisition of equine oocyte developmental capacity is ensured by the follicular environment, such as granulosa cells, which could reflect the meiotic development potential of immature oocytes. This study evaluated the relationship between DNA fragmentation of granulosa cells, using the chromatin dispersion test, and equine oocyte meiotic development after in vitro maturation. Granulosa cells and cumulus–oocytes complexes (n = 50) were recovered from slaughterhouse‐derived ovaries. Oocytes were in vitro matured, stained and evaluated under fluorescence microscopy. Maturation rates were classified into outstanding, medium and poor levels of maturation using 25th and 75th percentiles as thresholds. For DNA assessment, each sample was processed with the Ovoselect® kit (Halotech DNA). High, low and total DNA fragmentation percentages were compared among levels of maturation rates by ANOVA, followed by Duncan test. Results were expressed as mean ± SE. Total and high DNA fragmentation rates of granulosa cells were significantly higher (p < 0.05) in follicles whose oocytes had reached outstanding maturation level than those originating from follicles whose oocytes had reached poor maturation level. In conclusion, the DNA fragmentation analysis of equine granulosa cells can be a valuable test to identify equine oocytes showing the best meiotic competence after in vitro maturation.  相似文献   

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8.
为提高体外成熟卵母细胞的发育能力,本实验采用卵泡内存在的减数分裂抑制剂次黄嘌呤(Hypoxanthine,HX)在体外维持小鼠GV期卵母细胞减数分裂阻滞,探讨了次黄嘌呤对卵母细胞减数分裂抑制作用的时效性、可逆性以及对卵丘扩展和解除抑制后的发育能力的影响。结果表明(1)4mmol/LHX维持减数分裂阻滞的作用具有时效性,GV%在18h时显著下降。(2)HX处理时间短于24h时,解除抑制后再成熟时卵母细胞的成熟率不受影响,抑制24h再成熟14h时成熟率仍可达86%。(3)HX处理会抑制卵丘扩展,解除抑制后再成熟时卵丘扩展程度跟抑制时间长短有关。(4)HX处理6h后,卵母细胞的孤雌激活率上升(42%vs20%,P<0.05),但胚胎的发育能力下降。这证明HX维持小鼠卵母细胞减数分裂阻滞的作用具有时效性和可逆性的特点,为建立提高体外成熟卵母细胞的发育能力的培养体系打下了基础。  相似文献   

9.
Growing porcine oocytes from early antral follicles can acquire meiotic and developmental competence under suitable culture conditions, but at lower rates compared to full‐grown oocytes. We postulated that estradiol‐17β (E2) supported the acquisition of meiotic and developmental competence as well as cumulus‐expansion ability during growth culture. Growing oocytes from early antral follicles (1.2 to 1.5 mm in diameter) were grown in vitro for 5 days in a medium containing 0, 10?7, 10?6, 10?5 or 10?4 mol/L E2; after in vitro maturation, 35, 58, 47, 74 and 49% of oocytes matured to metaphase II, 25, 79, 77, 90 and 97% acquired cumulus‐expansion ability, and 23, 54, 63, 89 and 64% were fully surrounded by cumulus cells, respectively. Following maturation, electro‐stimulation was applied to the oocytes grown with 10?5 mol/L E2. After 6 days of culture, in vitro‐grown oocytes developed to the blastocyst stage at a rate similar to that for full‐grown oocytes (31% and 40%, respectively). Therefore, we suggest that the use of E2 during growth culture improves the meiotic and developmental competence of oocytes, cumulus‐expansion ability, and cumulus cell attachment to the oocytes.  相似文献   

10.
The objective was to investigate the RNA synthesis in porcine blastomere nuclei upon transplantation into in vitro matured enucleated oocytes. Nuclei from 2- to 8-cell porcine embryos were introduced into the ooplasm of in vitro matured and enucleated porcine oocytes by electrofusion, and the resultant reconstructed embryos were cultured in vitro. Before fusion or at different intervals after this event embryos were incubated with [3H]-uridine, fixed, and histologically processed for autoradiography in order to detect RNA synthesis. About two thirds of the embryos were considered to depict normal development. All blastomeres displayed pronounced RNA synthesis before fusion, at 3 and 9 h after fusion the synthesis decreased or ceased, and at 24-49 h some embryos resumed synthesis at the 1- to 2-cell stage.  相似文献   

11.
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.  相似文献   

12.
The objectives of the present study were to investigate the relationship between the morphological status of cumulus cells surrounding canine oocytes after maturation culture and the meiotic stage of the oocytes. In addition, the effect of the removal of cumulus cells from canine cumulus-oocyte complexes (COCs) during maturation culture on their meiotic competence was examined. Canine COCs were collected from bitches at the anoestrous and dioestrous stages and only COCs with >110 microm in vitelline diameter were cultured in medium 199 with 10% canine serum for 72 h. In the first experiment, the relation between the morphological status of cumulus cells surrounding oocytes cultured for 72 h and their meiotic stages was examined. At the end of maturation culture, the proportions of intact, partially nude and completely nude oocytes were 65.2%, 22.9% and 11.9%, respectively. The proportion of maturation to metaphase II of completely nude oocytes was highest among the oocytes with different morphological status of cumulus cells. In the second experiment, the cumulus cells were partially or completely removed from COCs at 48 h after the start of maturation culture and the oocytes were cultured for a further 24 h. The proportion of oocytes reaching metaphase II in the completely denuded oocytes was significantly higher than that in the control oocytes without the removal treatment of cumulus cells. The results indicate that morphological status of cumulus cells surrounding oocytes may be related to the nuclear maturation of canine oocytes, and the removal of cumulus cells from COCs during maturation culture can promote the completion of oocyte meiotic maturation.  相似文献   

13.
The aim of this study was to induce the cold‐inducible RNA‐binding protein (CIRBP) expression on cumulus–oocyte complexes (COCs) through exposure to a sub‐lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold‐inducible RNA‐binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5°C) and cold‐stressed oocytes (33.5°C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold‐stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold‐stressed groups, cold‐stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.  相似文献   

14.
Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non‐breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes because of their important role in oocyte growth and maturation. We also compared the effect of supplementing the maturation medium with bovine serum albumin (BSA) or oestrus mare serum (EMS). A total of 645 oocytes isolated from 158 and 154 ovaries collected during the breeding and the non‐breeding season, respectively, were used in this study. Oocytes were matured for 30 h in TCM 199 supplemented either with 10% EMS or with 4 mg/ml BSA. The presence of permeable GJs between cumulus cells and oocytes was investigated with the injection of a 3% solution of the fluorescent dye Lucifer yellow into the ooplasm. No differences in efficiency of oocyte retrieval or oocyte meiotic competence were detected between oocytes collected during the breeding and non‐breeding season. The vast majority (90%) of the oocytes collected during the breeding season had fully functional communications with their surrounding cumulus cells but such communications were completely interrupted in 55.3% of the oocytes collected during the non‐breeding season. During the non‐breeding season, the proportion of oocytes whose communications with cumulus cells were classified as closed or intermediate at the end of maturation was lower in the group matured with BSA than with EMS (71.4 vs 97.7, p < 0.05). The same trend, although not statistically significant, was observed during the breeding season also. The presence of BSA caused an incomplete cumulus expansion during both seasons. Our data indicate that oocytes collected during the non‐breeding season do not show any meiotic deficiency but lack active communication with the surrounding cumulus cells at the time of their isolation from the ovary. No data are available at present for determining the consequences on the developmental competence even if data from other species suggest that this is likely.  相似文献   

15.
Recent studies have shown that factors from adipose tissue influence and regulate the reproductive system. Hormones such as leptin and resistin are now known to regulate several reproductive processes. Adiponectin is the most abundant protein secreted by adipose tissue, and its circulating concentration is inversely related to adiposity and body mass index. Little is known about the involvement of adiponectin in reproduction. In the present study, the effect of recombinant adiponectin on the meiotic maturation and early embryo development in vitro was investigated, using porcine oocytes. Adiponectin receptors, AdipoR1 and AdipoR2, were found to be expressed in porcine oocytes and cumulus cells of both small and large follicles. Both AdipoR1 and AdipoR2 were immunolocalized to cumulus-oocyte complexes (COCs), oocytes, and early developing embryos. When included in oocyte maturation medium for 46 h, adiponectin significantly decreased the frequency of meiotic immature oocytes derived from large follicles (3-6 mm) but not from small follicles (<3mm). From studies of oocytes matured in the presence of adiponectin and mitogen-activated protein kinase (MAPK) pathway inhibitors MEK1 (PD98059), MEK1/2 (U0126), and p38MAPK (SB203580) it was concluded that adiponectin enhances oocyte maturation thought the p38MAPK pathway. Finally, a superior rate of embryo development to the blastocyst stage was achieved by embryos cultured in the presence of adiponectin. These results indicate that adiponectin has a positive effect on the meiotic maturation and in vitro embryo development of porcine oocytes and suggests a physiological role for this adipokine in early development in mammals.  相似文献   

16.
This study was conducted to determine whether meiotic maturation could be induced in ovarian oocytes from the American brown bear (Ursus arctos), a model for gamete "rescue" techniques for endangered ursids. The bears were euthanized, and their ovaries were transported to the laboratory within 4 h. The mean ovarian size was 2.4 x 1.8 cm (range: 2.0-3.3 x 1.5-2.2 cm). The ovaries obtained from the 2 brown bears yielded 97 oocytes (48.5/female), and 88 (90.7%) of them were morphologically classified as normal quality. Oocytes were in vitro matured at 38.5 C in 5% CO2 for 24 or 48 h in TCM-199 supplemented with 10% FBS, 1 microg/ml estradiol-17beta, and 10 microg/ml FSH. In Exp. 1, morphologic evaluation of matured oocytes was conducted by measuring the diameters of oocytes with a zona pellucida (ZP) or cytoplasm without a ZP. In Exp. 2, activation was induced by applying two 20 microsec DC pulses of 2.0 kV/cm delivered by an Electro Cell Fusion Generator. The activated oocytes were cultured in TCM-199 containing 2 mM of 6-dimethylaminopurine for 4 h, in Charles Rosenkrans (CR) 1 for 3 days and the in CR2 for another 4 days. The diameters of the matured bear oocytes with a ZP and with cytoplasm without a ZP (161.8 +/- 6.0 and 135.3 +/- 7.5 microm, respectively) were significantly (P<0.05) larger than those of bovine oocytes (150.7 +/- 4.9 and 118.7 +/- 7.5 microm). The maturation rates of the bear oocytes were 17.6 and 59.4% at 24 and 48 h of in vitro maturation, the percentage of activated oocytes that developed to the 2 or 4-cell stage was 31.6%; however, no blastocysts were observed. These results indicate that bear oocytes can develop to metaphase II in an in vitro culture system and that activated oocytes can develop to the 2 or 4-cell stages.  相似文献   

17.
Freezing technologies are very important to preserve gametes and embryos of animals with a good pedigree or those having high genetic value. The aim of this work was to compare immature and in vitro matured porcine oocytes regarding their morphology and ability to be fertilised after vitrification by the open pulled straw (OPS) method. In four experiments 830 oocytes were examined. To investigate the effect of cumulus cells on oocyte survival after OPS vitrification, both denuded and cumulus-enclosed oocytes were vitrified at the germinal vesicle (GV) stage, then after vitrification they were matured in vitro. Besides, in vitro matured oocytes surrounded with a cumulus and those without a cumulus were also vitrified. The survival of oocytes was evaluated by their morphology. After in vitro fertilisation the rates of oocytes penetrated by spermatozoa were compared. Our results suggest that the vitrification/warming procedure is the most effective in cumulus-enclosed oocytes (22.35 +/- 1.75%). There was no difference between the order of maturation and vitrification in cumulus-enclosed oocytes, which suggests the importance of cumulus cells in protecting the viability of oocytes during cryopreservation.  相似文献   

18.
主要探讨无卵丘水牛卵母细胞体外成熟的可行性,以便为研究卵母细胞成熟机理提供模型。无卵丘的水牛卵母细胞随机分为5组,然后分别进行直接成熟培养（M1）,与卵丘细胞单层共培养（M2）,用未扩展的卵丘细胞块包围培养（M3）,与扩展的卵丘细胞团共培养（M4）和用卵巢组织包围培养（M5）。无卵丘的水牛卵母细胞体外成熟培养24 h后检查第一极体（PB1）排出率,随后对这些卵母细胞进行孤雌激活,评定其成熟质量。结果发现,M4组的第一极体排出率明显高于M1组和M5组,其它各组间没有显著差异（P〉0.05）;M5组的孤雌激活卵裂率显著低于M1组和M4组（P〈0.05）,而与M2组和M3组没有显著差异（P〉0.05）,但M3和M4两组的囊胚发育率显著高于M1组和M5组（P〈0.05）。这些研究结果表明：（1）未扩展卵丘细胞包围法和扩展卵丘细胞团支撑法可促进无卵丘水牛卵母细胞的体外成熟,但与卵丘细胞单层共培养没有作用;（2）卵巢组织包围培养不利于水牛卵母细胞的体外成熟。  相似文献   

19.
胰岛素对犬卵母细胞体外成熟的影响   总被引：2，自引：2，他引：0  
研究不同浓度胰岛素及不同培养时间对犬卵母细胞体外成熟率的影响,为改善犬卵母细胞体外培养体系提供参考,采用切割法收集卵巢表面卵丘—卵母细胞复合体(cumulus oocyte complexes, COCs),在含有0.6%葡萄糖的TCM199中添加不同浓度的胰岛素（0、3、6、9 IU/mL）,38.5 ℃、5% CO2培养箱内成熟培养,观察卵丘扩散程度,剥离卵丘细胞获得裸卵后,室温下固定15 min;Hoechst 33342染色,压片,荧光显微镜下观察核形态,用SPSS 14.0软件统计试验数据。不同浓度胰岛素培养48 h后,各组卵丘细胞扩散效果都不明显;卵母细胞核成熟期没有达到减数分裂中期（MⅡ）,但是6 IU/mL胰岛素组生发泡破裂期（GVBD）比率（35.88%±14.63%）显著高于对照组（11.25%±9.75%）;6 IU/mL胰岛素组延长培养时间至72和96 h后,MⅠ-MⅡ期卵母细胞成熟率分别为13.33%±1.5%、20.8%±1.9%。以上结果表明,犬体外成熟培养基中添加胰岛素既没有提高犬卵母细胞核成熟到MⅡ期,也没有改善犬卵母细胞卵丘扩散效果。但是,在6 IU/mL浓度下延长培养时间,相对增加了成熟率。  相似文献   

20.
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.  相似文献   

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