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1.
为了通过比较培养筛选出能改进和提高山羊卵母细胞体外成熟效率的培养体系 ,培养的山羊卵母细胞以 TCM-199为基础培养液 ,添加 :(1) 10 %血清 (胎牛血清 (FBS)或发情山羊血清 (EGS) ) 2 0 m g/ L促黄体素 (L H) 10 mg/ L促卵泡素 (FSH) 1m g/ L 雌二醇 (E2 ) ;(2 ) 10 % EGS 促性腺素 (L H∶ FSH=5 mg/ L∶ 0 .5 m g/ L 或 2 0 mg/ L∶ 10mg/ L )或者 0 .0 75 IU / m L人绝经期促性腺素 (HMG) 1mg/ L estradiol 17β;(3) 10 % EGS 0 .0 75 mg/ L HMG 10～ 2 0 μg/ L EGF。此外 ,以 M199 10 % EGS 0 .0 75 mg/ L HMG 10～ 2 0 μg/ L EGF为培养基 ,溶解于自制超纯水或储存的商品化超纯水来培养卵母细胞。培养条件为 38℃ ,5 % CO2 。培养 2 4 h后 ,在体式显微镜下统计处于 M 期的卵母细胞比例。结果显示 :在卵母细胞成熟液中添加 10 % EGS比 10 % FBS的成熟培养效果好 ;添加 HMG能够促进卵母细胞的成熟 ,其效果比添加不同比例的 L H/ FSH好 ;成熟液中添加 10～ 2 0μg/ L EGF能促进卵母细胞的成熟 ,但成熟率没有明显提高 ;新鲜的超纯水对于卵母细胞的培养是必要的。结论 :新鲜超纯水配制的 M199 10 % EGS 0 .0 75 IU / m L HMG 10～ 2 0μg/ L EGF培养液可以获得最佳卵母细胞培养效果  相似文献   

2.
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3.
Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.  相似文献   

4.
In general, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in the regulation of cumulus cell expansion and oocyte maturation. We investigated the effects of supplementation of FSH or LH in in vitro maturation (IVM) medium on the incidence of cumulus cell expansion and nuclear maturation in canine oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM-199 supplemented with 10% foetal bovine serum (FBS), 1 mg/ml cysteine, 0.2 mm pyruvic acid and different concentrations of FSH or LH (control, 0.5, 5 or 50 microg/ml) at 38.5 degrees C, 5% CO(2) in air for 72 h. The cumulus cell expansion was measured by microscopic visualization, and nuclear maturation of denuded oocytes was determined by staining with 10 microg/ml Hoechst33342 for 30 min. The cumulus cell expansion in the 5 microg/ml FSH group (397.2 +/- 64.3 microm) was significantly higher than those in the control, 0.5, and 50 microg/ml FSH groups (168.3 +/- 19.1, 286.0 +/- 69.7 and 300.0 +/- 84.3 microm, respectively; p < 0.05). However, there was no difference in cumulus cell expansion among the control, 0.5, 5 and 50 microg/ml LH groups (165.6 +/- 20.2, 160 +/- 26.5, 172 +/- 20.5 and 168 +/- 23.1 microm, respectively; p > 0.05). After 72 h of IVM, the proportion of nuclear development to the MI-MII stage in the 0.5 microg/ml FSH group (15.1%) was higher than those in the control, 0.5 and 50 microg/ml FSH groups (0.9%, 6.5% and 8.0%, respectively; p < 0.05). However, there was no significant difference in nuclear maturation to the MI-MII stage among control, 0.5, 5 and 50 microg/ml LH groups (4.6%, 2.3%, 5.4% and 8.6%, respectively; p > 0.05). This study indicated that a FSH supplement in IVM medium can increase cumulus cell expansion and nuclear maturation, while the nuclear maturation rate remained low. Further studies are required to improve the nuclear development to the MI-MII stages in canine oocytes.  相似文献   

5.
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.  相似文献   

6.
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7.
Cysteine supplementation to in vitro maturation (IVM) media of bovine oocytes increases cellular glutathione production. Beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mm cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/ml); EGF (10 ng/ml); and IGF-I + EGF (100 + 10 ng/ml). Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the maturation media (12 h C IGF-I + EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I + EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in embryos was assessed. No treatment effect was observed on the expression of these genes. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.  相似文献   

8.
In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid.  相似文献   

9.
The objective of this study was to determine the effect of the presence of recombinant ovine growth hormone either alone or together with follicle stimulating hormone (FSH) during ovine oocyte in vitro maturation (IVM) on nuclear maturation and subsequent embryo development. Moreover, the effect of growth hormine (GH) on embryo development whether influenced by the presence of foetal bovine serum (FBS) was assessed. The abattoir‐derived oocytes were randomly divided into four treatment groups and cultured in maturation medium supplemented with: (i) 0.05 IU/ml FSH; (ii) 300 ng/ml roGH; (iii) FSH + roGH; and (iv) no FSH and GH (control). The percentages of germinal vesicle‐stage oocytes in GH‐treated group after 8 h of culture was significantly higher than the FSH and FSH + GH groups and lower than control (22.4%, 8.7%, 9.1%, and 32% respectively). The percentage of MII‐stage oocytes was significantly increased in the presence of GH after 16 and 24 h of culture compared to the control (44.7% and 83.1% vs 32.6% and 73.6% respectively). There was no significant synergism between GH and FSH in terms of nuclear maturation. The blastocyst rates in serum‐supplemented groups were enhanced by the presence of FSH and GH compared to the control (35.4% and 31.3 vs 11.4% respectively). Compared with either GH or FSH alone, the subsequent embryo development (blastocyst rate), however, was negatively influenced by co‐presence of both hormones (22.8%). In contrast, the corresponding values were not affected in the absence of serum. In conclusion, GH had positive effect on nuclear maturation of sheep oocytes. Moreover, the pattern of the effect of GH on embryo development was influenced by the presence of FBS during IVM.  相似文献   

10.
Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.  相似文献   

11.
表皮生长因子对水牛卵母细胞体外培养核质成熟的影响   总被引：1，自引：0，他引：1  
为了探讨表皮生长因子（EGF）对水牛卵泡卵母细胞体外培养核质成熟的影响，在以TCM199为基础的成熟液中加入不同浓度的EGF（0、10、25、50、100 ng/ml），体外成熟培养24~26 h，观察第一极体（PB1）的排放；随后进行孤雌激活检测其分裂率、囊胚发育率、囊胚孵化率，并用Hoechst33342染色后计算囊胚的细胞数。结果发现添加EGF各组的卵母细胞第一极体排放率显著提高（P<0.05）；成熟液中添加25 ng/ml EGF时，卵裂率及囊胚发育率（分别为80.0%、44.8%）明显高于对照组（分别为69.3%、31.9%，P<0.05），但对囊胚的细胞数影响不大。EGF不仅促进水牛卵母细胞体外培养的核成熟，而且有利于卵母细胞的胞质成熟，其中EGF的最佳浓度为25 ng/ml。  相似文献   

12.
The effects of adding cysteamine, EGF, and glucose as an energy substrate under low oxygen tension during in vitro maturation (IVM) were examined to find ways of improving the individual in vitro production (IVP) system in individually cultured bovine oocytes. The basic medium was mSOFaa containing 1 mg/ml polyvinyl alcohol. Immature oocytes were individually cultured in an IVM medium with 10 ng/ml EGF, 100 microM cysteamine, or EGF plus cysteamine under 20% or 5% O(2). Cleavage and blastocyst rates were significantly higher (P<0.05) in IVM culture was under 20% O(2) than in culture under 5% O(2). Under 5% O(2), neither EGF nor cysteamine improved embryonic development. The proportion of matured oocytes was significantly higher (P<0.05) in the presence of 1.5 mM glucose under 20% O(2) (68.6%), and 5.5 mM (66.7%) and 10 mM (65.5%) glucose under 5% O(2). The presence of 5.5 mM glucose significantly (P<0.05) increased the maturation rate compared with the absence of glucose, irrespective of addition of EGF and cysteamine. The addition of cysteamine alone in the maturation medium significantly (P<0.05) increased the intracellular GSH concentration in the oocytes. Also, under 5% O(2) cysteamine and/or EGF significantly (P<0.05) improved the proportions of penetrated oocytes, cleavage and blastocyst formation, which were similar levels to those of oocytes matured under 20% O(2). After vitrification, the re-expanding and hatching rates of blastocysts derived from the individual IVP system containing cysteamine under 5% O(2) were significantly (P<0.05) higher than those of blastocysts derived from the individual IVP system without cysteamine under 5% O(2) and the group IVP system under 20% O(2). The present study showed that a high glucose level (5.5 or 10 mM) was optimal in IVM culture under low (5%) oxygen tension. The addition of EGF and/or cysteamine to the maturation medium had no positive effect on nuclear maturation, but improved fertilizability, developmental competence and cryoresistance following vitrification, probably due to increased GSH synthesis during the IVM process.  相似文献   

13.
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   

14.
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.  相似文献   

15.
Manganese (Mn) is a trace element present in forages and cereals, and its concentration depends on soil status. Manganese deficiency in cattle, goats and ewes not only impairs oestrous cycle but reduces calf birth weight. The achievement of the first oestrus is delayed, and more attempts are necessary to obtain a successful conception. This study was conducted to investigate the effect of the availability of supplemental Mn during IVM on DNA damage of cumulus cells and total glutathione (GSH) content in oocytes and cumulus cells. The effect of supplementary Mn during IVM on subsequent embryo development was also studied. The results reported here indicate (i) DNA damage in cumulus cells decreased with 0, 2, 5 and 6 ng/ml Mn supplementation during IVM (p < 0.05). (ii) Intracellular GSH‐GSSG content increased (p < 0.01) with different Mn concentrations in oocytes and cumulus cells. Also, cumulus cell number per cumulus oocyte‐complexes (COC) did not differ either before or after IVM. (iii) Addition of Mn to maturation medium resulted in similar cleavage rates (p > 0.05) at 0, 2, 5 and 6 ng/ml Mn. However, subsequent embryo development to blastocyst stage was significantly higher (p < 0.01) in oocytes matured with 5 and 6 ng/ml Mn. (iv) There was also an increase (p < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 5 and 6 ng/ml respect to zero Mn (IVM alone) and 2 ng/ml Mn. This study provides evidence that optimal embryo development to the blastocyst stage was partially dependent on the presence of Mn during IVM. Moreover, the availability of Mn during oocyte maturation ensures ‘normal’ intracellular GSH content in COCs and protects DNA integrity of cumulus cells.  相似文献   

16.
With the goal of establishing experimental protocols for cloning sika deer, various conditions for in vitro maturation (IVM) and artificial activation of sika deer oocytes were examined. In vitro maturation was evaluated in seven different culture media. The highest rate of oocyte maturation was 75.4% in 10 μg/ml follicle‐stimulating hormone (FSH), 1 μg/ml LH, 0.2 mm cysteamine and 50 ng/ml epidermal growth factor (EGF) after 24 h of IVM. The efficiency after 24 h of IVM did not differ significantly (p > 0.05) from that observed after 20 h. Cysteamine (0.2 mm ) significantly increased the maturation rates after 20 h (from 59.1% to 67.2%, p < 0.05) and after 24 h (from 63.2% to 71.6%, p < 0.05) of IVM. The IVM rates of oocytes collected during the oestrous season (75.4%) and the anoestrous season (23.3%) were significantly different at 24 h. The 20 μg/ml FSH, 2 μg/ml LH, 0.4 mm cysteamine and 100 ng/ml EGF significantly increased the maturation rates (from 23.3% to 54.2%, p < 0.01) at 24 h during the anoestrous season. For the activation experiments, the most effective method was chemical activation [ionomycin + 6‐dimethylaminopurine (6‐DMAP)], which promoted the development of sika deer oocytes to the blastocyst stage (32.4%). Our results indicate that in vitro matured sika deer oocytes are good candidates for parthenogenetic activation and that chemical treatment is needed for relatively efficient activation of the oocytes. These optimized conditions for IVM and parthenogenetic activation may be useful for efforts to restore populations of the endangered sika deer using the somatic cell nuclear transfer technique.  相似文献   

17.
Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.  相似文献   

18.
Current in vitro embryo production protocols in the Iberian red deer (Cervus elaphus hispanicus) need to be optimized; oocyte harvesting in situ followed by overnight holding could reduce the human effort and shipping costs. In our work, post‐mortem ovaries were retrieved, and the oocytes were harvested and allocated to G1 group (good quality) or G2 + G3 group (low quality). The oocytes were separately subjected to immediate in vitro maturation (IVM) or held overnight in a holding medium composed of 40% of TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts and 20% fetal bovine serum (FBS), at room temperature (16 hr). In vitro maturation was carried out in a basal medium supplemented or not with 50 ng/ml of epidermal growth factor (EGF). Our data showed that addition of EGF to the maturation medium increases the percentage of G1 oocytes reaching metaphase II (3.9% vs. 50%, basal vs. EGF; p < .001) and decreased their degeneration rate (69.9% vs. 22.2%, basal vs. EGF; p < .01) when oocytes were immediately matured. Overnight holding increased the meiotic competence of G1 oocytes (37.5% matured in basal medium) and EGF increased prophase arrest in G2 + G3 oocytes (16.1% vs. 38.8% in germinal vesicle [GV] stage in basal medium vs. EGF added medium; p < .05). Our data demonstrate that oocyte holding can be used in Iberian red deer oocytes. Interestingly, EGF addition increases the oocytes' meiotic competence in immediately matured oocytes but not after oocyte holding depending upon initial oocyte quality.  相似文献   

19.
The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro‐matured Caprine oocytes. A total of 470 in vitro‐matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48–72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro‐matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm‐injected in vitro‐matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non‐activated oocytes.  相似文献   

20.
BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.  相似文献   

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