不同化学激活方法和电激活参数对猪体外成熟卵母细胞早期孤雌发育的影响

利用屠宰场猪卵巢卵母细胞,在体外成熟培养44～48 h后,对核成熟卵母细胞进行激活.试验1为不同化学激活方法10% 乙醇 10 mg/L 放线菌酮组、2.5 mmol/L 氯化锶 10 mg/L放线菌酮组、5 μmol/L离子霉素 2.5 mmol/L 6-二甲氨基嘌呤(6-DMAP)组、200 μmol/L 硫柳汞 8 mmol/L二硫苏糖醇组和对照组(不用任何激活剂).试验2为用不同电场强度和脉冲时程进行电激活之后放入胚胎培养液中进行体外培养7 d.结果显示,(1)4种不同化学激活方法处理卵子的1原核形成率、2原核形成率和原核形成率都显著比对照组高(P＜0.05),其中离子霉素 6-DMAP组的2原核形成率和总原核形成率最高,分别为(23.1±3.5)%和(65.2±3.5)%,显著高于其他处理组(P＜0.05);(2)4种不同化学激活方法处理组的囊胚率都比对照组高(P＜0.01),其中离子霉素 6-DMAP组的卵裂率及囊胚率最高,分别为(46.6±18.5)% 和(5.6±4.2)%;(3)本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响;(4)电场强度为1.7 kV/cm、脉冲时程为50、70 μs时猪体外成熟卵母细胞激活效果最好,卵裂率、囊胚率、囊胚细胞数分别为(77.4±9.7)%、(12.4±3.7)%、(17.6±5.9)%和(75.1±10.6)%、(12.3±2.6)%、(19.1±8.1).以上结果说明本试验所用的4种化学激活方法均能有效激活猪体外成熟卵母细胞,其中离子霉素 6-DMAP的激活效果最理想;在本实验室条件下,采用1.7 kV/cm的电场强度、50～70 μs的脉冲时程均能有效地激活猪体外成熟卵母细胞;本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响.     

Effects of cycloheximide on parthenogenetic development of pig oocytes activated by ultrasound treatment

The present study was carried out to examine the parthenogenetic development of pig oocytes treated with different concentrations of cycloheximide for different durations following activation by ultrasound stimulation. When oocytes were treated with 10 microg/ml cycloheximide for different durations, the blastocyst formation rate of oocytes treated for 5 h was significantly (P<0.05) higher than those of oocytes treated for 0-2 h. The blastocyst formation rate of oocytes treated with 10 microg/ml cycloheximide for 5 h was significantly (P<0.05) higher than those of oocytes treated with 0-5 or 15-20 microg/ml cycloheximide for the same duration. When oocytes were treated with different concentrations of cycloheximide for 2 h, however, the blastocyst formation rate of oocytes treated with 40 microg/ml cycloheximide was significantly (P<0.05) higher than those of oocytes treated with 0-10 or 50 microg/ml cycloheximide. The blastocyst formation rate of oocytes treated with 10 microg/ml cycloheximide for 5 h was not significantly different from that of oocytes treated with 40 microg/ml cycloheximide for 2 h. These treatments did not affect the activation status of oocytes compared with controls that were not treated with cycloheximide. The results of the present study showed that cycloheximide improves the parthenogenetic development of pig oocytes activated by ultrasound stimulation.     

Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.     

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.     

Effects of polyethylene glycol and a synthetic ice blocker during vitrification of immature porcine oocytes on survival and subsequent embryo development

We evaluated the effects of polyethylene glycol (PEG) and Supercool X‐1000 (SC) as supplements during the vitrification of immature cumulus‐enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG‐, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG‐ groups; however, all values were lower than those in the non‐vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC‐, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC‐ groups but lower than those in the non‐vitrified control. The percentage of cleavage in the SC‐ group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non‐vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.     

Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos

Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes.     

冷冻保存能降低绵羊卵母细胞受精能力

本实验研究了体外成熟绵羊卵母细胞在舍抗冻保护荆DMS()和EG的冷冻液中暴露和OPS法玻璃化冷冻保存后对体外受精卵裂率、精子入卵率、皮质颗粒分布、酶溶解透明带时间及雌原核形成的影响.将体外成熟24 h的绵羊卵母细胞分为3组:(1)对照组,卵母细胞不进行处理;(2)毒性组,卵母细胞在冷冻液中进行暴露但不投入液氮中冷冻;(3)冷冻组,卵母细胞利用OPS法进行玻璃化冷冻.处理组卵母细胞在浓度递减的蔗糖溶液中脱除抗冻保护剂.结果,卵母细胞体外受精的卵裂率和单精子入卵率,毒性组(62.3%和29.3%)和冷冻组(67.6%和28.2%)显著低于对照组(78.4%和45.0%)(P＜0.05),毒性组和冷冻组间无显著差异(P＞0.05).为了研究冷冻保存导致卵母细胞受精能力降低的机制,处理组及对照组一部份卵母细胞分别于处理后0 h(IVM24 h)、2 h(IVM26 h)和体外受精后12 h(IVFl2 h)测定皮质颗粒分布和用0.1%链霉蛋白酶溶解透明带时间,另一部份卵母细胞则在不含有精子的受精液中孵育12 h后测定雌原核形成率.结果,在IVM24 h和IVM26 h,皮质颗粒呈完全释放的比例,毒性组(41.2%和40.8%)和冷冻组(41.7%和51.8%)显著性高于对照组(7.1%和18.4%)(P＜0.05);IVM26 h酶溶解透明带时间,毒性组(435.6±16.6)s和冷冻组(422.3±14.6)s显著长于对照组(381.6±15.3) s(P＜0.05);雌原核形成率,毒性组(58.7%)和冷冻组(63.9%)组显著高于对照组(8.2%)(P＜0.05).上述结果表明,舍DMS()和EG的冷冻液对体外成熟绵羊卵母细胞具有孤雌激活作用,引起皮质颗粒的提前释放,导致透明带变硬,降低卵母细胞的受精能力.     

Treatment of porcine oocytes with MEM vitamins during in vitro maturation improves subsequent blastocyst development following nuclear transfer

This study was carried out to investigate the effects of minimum essential medium (MEM) vitamins during in vitro maturation (IVM)/in vitro culture (IVC) of porcine nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. Porcine cumulus-oocyte complexes (COCs) were divided into five groups, matured for 44 h in maturation medium with various concentrations of MEM vitamins (0, 0.05, 0.1, 0.2 and 0.4%), and observed for maturation rate. Also, COCs were matured in NUSU-23 media without MEM vitamins for 44 h and cultured in PZM-3 media with various concentrations of MEM vitamins (0, 0.05, 0.4 and 1.0%) for 6 days following nuclear transfer. Factorial (IVM/IVC) experiments were also performed in NCSU-23 medium with or without 0.05% MEM vitamins and PZM-3 medium with or without 0.4% MEM vitamins. They were then tested by examining in vitro development of the porcine reconstructed embryos. The maturation rates of the COCs treated with the MEM vitamins did not differ significantly among the MEM vitamin-treated groups. Addition of vitamins to culture medium did not affect development of porcine reconstructed embryos in vitro. However, addition of low concentrations of MEM vitamins only to maturation medium increased (P<0.05) the proportion of NT embryos developing into blastocysts compared with the control group. Addition of MEM vitamins to IVC medium did not enhance the developmental rate compared with the control group. Thus, addition of MEM vitamins to IVM medium could improve subsequent blastocyst development of porcine NT embryos.     

不同激活条件及半胱氨酸处理对猪ICSI胚胎发育影响研究

本试验探讨了不同辅助激活方法(Calciumionophore A23187激活、Calciumionophore A23187+6-DMAP联合激活和电激活)、不同精子预处理方法(液氮冻融处理和0.1%Triton X-100处理)和在添加半胱氨酸的胚胎培养液中培养不同时间(0 h、4 h、12 h和168 h)对猪卵母细胞内单精子注射(ICSI)胚胎体外发育的影响。结果显示:与无辅助激活相比,A23187+6-DMAP联合激活和电激活均能显著提高ICSI卵母细胞的激活率、卵裂率和囊胚率(P0.05),A23187+6-DMAP联合激活能显著提高ICSI卵母细胞的受精率(P0.05)。液氮冻融精子组ICSI卵母细胞的雄原核形成率显著高于活精子组(P0.05)。在添加半胱氨酸的胚胎培养液中培养4 h的ICSI卵母细胞受精率、雄原核形成率和囊胚率显著高于0 h组(P0.05)。以上结果表明,猪卵母细胞在ICSI后需要辅助激活来启动胚胎顺利发育,A23187+6-DMAP激活效果较好。液氮冻融精子可以促进ICSI后雄原核的形成。半胱氨酸处理4 h对猪ICSI卵母细胞受精和发育均有促进作用。     

Effects of resveratrol on in vitro maturation of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer

As a natural plant‐derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 μM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti‐apoptotic B‐cell lymphoma 2 (BCL‐2) gene and significant downregulation of the pro‐apoptotic BCL2‐associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 μM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 μM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.     

Effect of recombinant human follicle-stimulating hormone and luteinizing hormone on in vitro maturation of porcine oocytes evaluated by the subsequent in vitro development of embryos obtained by in vitro fertilization, intracytoplasmic sperm injection, or parthenogenetic activation

The aim of this work was to study the effect of recombinant human (rh) FSH and LH on in vitro maturation of pig oocytes compared with a conventional hormonal supplement based on equine (PMSG) and human chorionic gonadotropins (hCG), as evaluated by the developmental ability of 3 types of pig embryos obtained by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or artificial activation (ATA). In Exp. 1, one cumulus-oocyte complex group (A group) was supplemented with rh-FSH and rh-LH (0.1 IU/mL each), and the other group (B group) was supplemented with PMSG and hCG (10 IU/mL each). No differences in nuclear maturation between the A and B groups were observed (68.5 vs. 71.4%, respectively). No differences were detected between hormonal treatments in the rates of cleavage or blastocyst formation of ATA, IVF, and ICSI embryos. Total cell number of the embryos was not significantly different in any experimental group (A: 31.1, 28.5, and 19.8 vs. B: 25.2, 25.5, and 20.6 for ATA, IVF, and ICSI embryos, respectively). In Exp. 2, the effects of different concentrations of rh-FSH and rh-LH (0.5, 0.1, or 0.05 IU/mL) in maturation medium on nuclear maturation and in vitro development of embryos obtained by IVF were studied. No effect of different hormonal concentrations on blastocyst formation rates was observed (8.5, 13.0, and 5.7%, respectively). Blastocyst cell number was not different in any experimental group. In conclusion, the results obtained here permit us to substitute PMSG and hCG with rh-FSH and rh-LH and to produce pig embryos obtained by IVF, ICSI, or ATA.     

Effects of maturational age of porcine oocytes on the induction of activation and development in vitro following somatic cell nuclear transfer

To determine the effect of maturational age of porcine oocytes on the induction of activation and development following nuclear transfer, we investigated maturation rate and efficacy of oocyte activation treatment with an electrical stimulus (ES) and cycloheximide (CHX) at various timing of maturation culture. Most oocytes developed to the metaphase II (MII) stage after 32 hr of maturation culture. Both in newly matured (32 and 36 hr of age) and MII-arrested (42 and 48 hr of age) oocytes, ES followed by exposure to CHX for 6 hr caused higher rates of pronuclear formation than ES alone. Effect of maturational age of oocytes on the development of parthenotes activated with ES and CHX was then examined. The highest percentage of parthenotes developed to blastocysts was obtained when ES was added at 42 hr of culture. Finally, newly matured (33 hr of age) and MII-arrested oocytes (44 hr of age) were enucleated, fused with serum-starved pig fetal fibroblasts and activated with ES and CHX around suitable timing (39 to 40 hr of age) or later (50 to 51 hr of age), respectively. The frequencies of cleavage (58.8%) and blastocyst formation (13.6%) of newly matured oocytes were significantly (P<0.05) higher than those of MII-arrested oocytes (38.9 and 1.8%, respectively). These results demonstrated that the development of porcine nuclear transfer embryos can be improved by using complete matured oocytes as cytoplasts and activation treatment with ES followed by CHX treatment.     

Effect of exposure duration of ovaries and oocytes at ambient temperature on parthenogenetic development of porcine follicular oocytes

This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on in vitro maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.     

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 µg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 µg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 µg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 µg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.     

罗同丘细胞对小鼠卵母细胞体外成熟及其后续发育的影响

观察了卵丘细胞共培养对小鼠生发泡期部分裸露（PNO）和裸卵（NO）成熟和发育能力的影响；分别用小鼠、大鼠、猪的卵丘细胞与小鼠PNO和NO进行了共培养。检测了小鼠PNO和NO的减数分裂能力、生发泡构型、受精和胚胎发育能力。结果，PNO、NO的减数分裂能力显著（P〈0．05）低于卵丘完整复合体（COCs）的减数分裂能力。COCs中SN型卵母细胞比率显著高于PNO和NO中SN型卵母细胞比率（P〈0．05），大部分PNO和NO的卵母细胞核型为NSN型。与对照组相比，与小鼠或大鼠卵丘细胞共培养的小鼠PNO和NO的减数分裂能力没有显著提高，但是与猪卵丘细胞共培养却可以提高小鼠PNO和NO的减数分裂能力。结果表明，猪卵丘细胞能促进小鼠卵母细胞的体外成熟，但并不影响小鼠卵母细胞的受精和胚胎发育。     

Stage‐specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment

Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium‐3 (mPZM‐3) with increased NaCl to 138 mmol/L (mPZM‐3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM‐3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM‐3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM‐3 for 7 days. The cleavage and blastocyst formation rates of SCNT embryos cultured in mPZM‐3+NaCl for the first 2 days and then cultured in mPZM‐3 for 5 days were also significantly (P < 0.05) higher than those of embryos cultured in mPZM‐3 for 7 days. These results showed that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound.     

Effects of ovary storage time and temperature on DNA fragmentation and development of porcine oocytes

The present study was conducted to investigate the effects of storage time and temperature of porcine ovaries on the quality and nuclear maturation in vitro of oocytes obtained from stored ovaries and their subsequent development after in vitro fertilization. The ovaries were stored in physiological saline for 0, 3, 6, 9 and 12 h at various temperatures (4, 15, 25 and 35 C). The pH of follicular fluid obtained from the ovaries, DNA fragmentation of the oocyte nucleus and meiotic competence of oocytes were examined. Some oocytes from ovaries stored at 15, 25 and 35 C for 6 h were fertilized in vitro, and then cultured for 7 days to examine the ability of embryos to develop to the blastocyst stage. When the ovaries were stored at 35 C, the pH of follicular fluid decreased and the proportions of oocytes with DNA fragmented nuclei increased as the storage time was prolonged, and the storage of ovaries for 6, 9 and 12 h resulted in lower maturation rates of oocytes. When the ovaries were stored at 4, 15, 25 and 35 C for 6 h, the storage at higher temperatures (> or =15 C) decreased the pH of follicular fluid and induced nucleic DNA fragmentation in higher proportions of oocytes. None of the oocytes from ovaries stored at 4 C reached metaphase II. The storage of ovaries at 15 C reduced the rates of in vitro fertilized oocytes and subsequent embryo development, but there were no significant differences in the rates of fertilization and blastocyst formation between oocytes from ovaries stored at 25 C and 35 C. Our findings indicate that the storage of ovaries at 25-35 C for 6 h is effective for maintaining the developmental competence of porcine oocytes even though the development rates were lower than those of ovaries stored at 35 C for 3 h.     

Embryo development of porcine oocytes after injection with miniature pig sperm and their extracts

This study examined embryo development of porcine oocytes after microinjection of sperm extracts (SE) in porcine intracytoplasmic sperm injection (ICSI). SE was prepared from miniature pig sperm by a nonionic surfactant, and various concentrations (0.02, 0.04 and 0.08 mg/mL) of SE were injected into the matured oocytes with a first polar body. In the pronuclear stage, the rate of oocytes with two pronuclei and a second polar body (21.4%) in the sperm and SE (0.04 mg/mL) injection group was significantly higher (P < 0.05) compared to other groups. The rate of 2–4‐cell stage in sperm and SE (0.04 mg/mL) injection group was 38.1%, and it was significantly higher than that in the sperm injection group (22.9%). The rate of blastocyst stage in sperm and SE (0.04 mg/mL) injection group was 21.4%, the value was significantly higher than those in SE (0.08 mg/mL) injection group (0%), sperm injection group (5.7%), and sperm and SE (0.08 mg/mL) injection group (2.6%). These results suggest that SE induces activation of porcine oocytes and their further embryonic development, and that SE is effective for porcine ICSI.     

Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 μs each) followed by 6‐dimethylaminopurine (6‐DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6‐DMAP + CHX (12.07%) activation was higher than that of ION + 6‐DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6‐DMAP + CHX and DC + 6‐DMAP + CHX groups. The blastocyst rate of ION + 6‐DMAP + CHX‐activated oocytes in the basic rabbit culture medium (M‐199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M‐199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3–5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6–9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.     

高浓度葡萄糖对猪卵母细胞体外成熟和早期胚胎发育的影响

试验旨在探究高浓度葡萄糖对猪卵母细胞体外成熟及早期胚胎发育能力的影响。取体外分离处于生发泡期的猪卵丘卵母细胞复合体(COCs),分为3个处理组。分别用含葡萄糖浓度为5.6 mmol/L(C组)、10 mmol/L(G-1组)、15 mmol/L(G-2组)的培养液,进行体外成熟(IVM)处理,42 h后观察,并统计卵丘细胞扩散情况和第一极体排出率;对体外成熟42 h后的卵母细胞孤雌激活,统计2-细胞、4-细胞和第7天囊胚发育。结果发现,G-1组和G-2组卵丘细胞扩散度显著低于C组(P<0.05);G-1组和G-2组的MII期卵母细胞死亡率和存活率与C组相比无显著差异(P>0.05),但G-1组极体率显著降低(P<0.05),G-2组极体率极显著低于C组(P<0.01)。孤雌激活后,与C组相比,G-1组和G-2组的2-细胞分裂率显著降低(P<0.05),4-细胞分裂率以及囊胚发育率均极显著降低(P<0.01),但G-1、G-2组囊胚细胞数量与C组相比无显著性差异(P>0.05)。进一步线粒体染色发现,G-1组和G-2组的线粒体与C组相比分布不均。...