Effects of ghrelin on in vitro development of porcine in vitro fertilized and parthenogenetic embryos

The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remarkably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blastocyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.     

NGF和BDNF对猪孤雌激活胚胎体外发育的影响

在NCSU-23改良培养液中分别添加不同质量浓度的脑源性神经生长因子(不rain-derived neurotrophic factor,BDNF)和神经生长因子(nerve growth factor,NGF),观察其对猪孤雌激活胚胎体外生长的影响。结果显示:在培养液中添加40μg/L的BDNF,可促进猪孤雌激活胚胎的囊胚形成;在猪PA胚胎培养液中添加不同质量浓度的NGF,对猪孤雌激活胚胎的在卵裂率、囊胚率和囊胚孵化率影响不明显。结果表明,在NCSU-23改良培养液中添加一定质量浓度的BDNF可促进猪孤雌激活胚胎体外培养过程中囊胚的形成。     

Effects of activation methods on DNA synthesis and development of parthenogenetic porcine embryos

This study investigated the timing of DNA synthesis and patterns of pronuclear (PN) formation during the first cell cycle, and its influence on developmental competence, velocity and proliferation index of porcine parthenote blastocysts produced by different activation treatments. Oocytes were activated as follows: electrical stimulation (EST), EST combined with 7.5 μg/ml cytochalasin B (EST + CCB), 10 μg/ml cycloheximide (EST + CHX) and 1.9 mm 6-dimethylaminopurine (EST + 6-DMAP) for 3 h. DNA synthesis and PN formation were evaluated using 1 mm 5'bromo-2'deoxy-uridne (BrdU) at 2 h intervals from 1 to 13 h or 5 to 13 h of post-activation (hpa), respectively. In EST, DNA synthesis started at 3 hpa, reached the peak at 11 hpa and decreased at 13 hpa. Treatment with 6-DMAP resulted in an early increase of DNA synthesis at 3 hpa, whereas CCB delayed DNA synthesis for 2 h. In EST and EST + 6-DMAP, most of the eggs showed 1PN, whereas, incidence of 2PN in EST + CCB was higher than 1PN. EST + CHX was observed with 1PN, 2PN and multiple PN. Blastocyst rate in EST + CCB and EST + 6-DMAP were significantly (p<0.05) higher than EST + CHX. But, the developmental velocity was not different among groups. Proliferation index of blastocysts, as indicated the number of blastomere at S-phase of the cell cycle was low in all groups. In conclusion, CCB, CHX and 6-DMAP used for producing porcine parthenogenetic embryos induced different onset of DNA synthesis and PN, but they did not affect the subsequent embryo development.     

Ghrelin对水牛体外受精和孤雌激活胚胎体外发育的影响

本研究的目的是探讨Ghrelin对水牛体外受精和孤雌激活胚胎体外发育的影响.体外成熟的水牛卵母细胞经体外受精或离子霉素孤雌激活后.分别在舍0,0.5,5,50和500 μg/L Ghrelin的培养液中进行体外培养,观察各组胚胎的卵裂率和囊胚率.结果显示,在培养液中添加不同浓度的Ghrelin对体外受精和孤雌激活胚胎的卵裂率均无显著影响(P>0.05),但添加500 μg/L的Ghrelin显著提高体外受精胚胎的囊胚发育率(33.5% vs 13.7%,P<0.05),50 μg/L或500 μg/L的Ghrelin均显著提高孤雌激活胚胎的囊胚发育率(32.4%和34.6% vs 14.5%,P<0.05).结果表明,培养液中添加Ghrelin对胚胎的早期卵裂没有影响,但可促进水牛体外受精和孤雌激活胚胎囊胚的形成.     

不同血清类型对水牛胚胎体外培养效果的影响

从屠宰场收集水牛卵巢上抽取的卵母细胞,经体外成熟培养后分别进行孤雌激活(PA)和体外受精(IVF),将获得的PA或IVF胚胎分别培养于添加浓度均为5%的牛血清白蛋白(BSA)、胎牛血清(FBS)、炭情水牛血清(OBS)、发情牛血清(OCS)和混合血清(OCS与FBS按1:1混合)的培养液中培养7～9 d,并观察胚胎的体外发育情况.结果,PA胚胎培养在OCS和混合血清组的卵裂率(73.52%,68.07%)和囊胚率(30.59%,27.73%)均显著高于BSA(38.37 %.6.40%)、OBS(44.97%,10.05%)和FBS(52.02%,17.04%)组(P＜0.05);IVF胚胎的卵裂率和囊胚率以OCS组最高(62.50%,22.62%),显著高于BSA(30.50%,6.38%)、OBS(35.48 %,7.10 %)和FBS组(40.11%,11.76%,P＜0.05),但与混合血清组(60.42%,21.35%;P＞0.05)差异不显著.结果表明,在水牛胚胎体外培养体系中添加5%的OCS或混合血清均能有效地促进水牛胚胎的体外发育.     

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。     

In vitro development of porcine embryos in coculture with endometrial cell monolayers or culture supernatants

The objective of this study was to determine if porcine endometrial cell contact would enhance the in vitro development of porcine embryos relative to endometrial cell culture supernatants or ovarian fibroblast contact. One hundred forty-nine embryos of various developmental stages were collected from 10 excised gilt reproductive tracts. Embryos were washed three times and placed randomly into one of four treatments: Minimal Essential Medium + 5% heat-treated fetal calf serum (complete MEM), porcine endometrial cell coculture (pEM), porcine endometrial cell supernatant (pEMS) and porcine ovarian fibroblast coculture (pOF). All cultures were conducted in 96 well microtiter plates, one embryo per well, at 37 C in a 5% CO2 in humidified air atmosphere. Observations for stage of development were made every 12 h by phase contrast microscopy (100X). Embryos in the pEM and pOF treatments had significantly greater (P less than .05) mean embryonic development scores (1.59 +/- .22 and 1.44 +/- .18, respectively) than embryos cultured in complete MEM only or pEMS (.63 +/- .13 and .48 +/- .15, respectively). The results of this study indicate that cell to embryo contact enhances in vitro development of porcine embryos.     

Effects of cycloheximide treatment on in-vitro development of porcine parthenotes and somatic cell nuclear transfer embryos

This study aimed to verify the beneficial effect of cycloheximide (CHX) treatment on the development of porcine somatic cell nuclear transfer (NT) embryos, which were constructed with enucleated oocytes and cumulus cells by using a single direct current (DC) pulse. In the first experiment, a single DC pulse applied to the induction of fusion and activation of NT embryos gave a high fusion rate. However, cleavage and subsequent development of fused couplets (NT embryos) to the blastocyst stage were poor. Experiment 2 was conducted to determine whether CHX treatment could enhance metaphase II (M II) oocyte activation and improve the subsequent parthenogenetic development. After giving the DC pulse and incubation with or without CHX, M II oocytes incubated with CHX showed higher cleavage and development to blastocysts compared with those incubated without CHX (P < 0. 05). Experiment 3 was carried out to verify the beneficial effect of CHX on the development of NT embryos. The NT embryos treated with the DC pulse and CHX treatment showed higher cleavage and subsequent development compared with those treated with the DC pulse alone (P < 0.05) . The present study demonstrates that CHX treatment enhances the electrical stimulus-induced activation of oocytes and NT embryos, and improves the subsequent development of parthenotes and NT embryos. The results indicate that protein synthesis inhibition treatment required for the induction of oocyte activation promotes the development of NT embryos.     

Effects of bovine serum albumin and estrous cow serum on development and ultrastructure of in vitro-produced porcine embryos

This study evaluated the effects of bovine serum albumin (BSA; 4 mg/ml) and estrous cow serum (ECS; 10%) in North Carolina State University (NCSU) 23 medium on the development of in vitro-matured and in vitro-fertilized porcine oocytes. Early cleavage rate was significantly (P < 0.05) higher in NCSU/ECS (71.3 +/- 14.7%) vs. NCSU/BSA (60.6 +/- 4.7%). Cleavage beyond the four-cell stage was not different between the two culture media (43.5 +/- 9.5% and 41.4 +/- 17.7%, respectively). The proportion of development to blastocysts was--with borderline significance (P = 0.05)--higher in NCSU/BSA (28.0 +/- 4.4%) than in NCSU/ECS (20.4 +/- 7.3%). Blastocysts produced in NCSU/BSA had significantly (P < 0.001) higher cell numbers than those cultured in NCSU/ECS (29.5 +/- 20.1 vs. 16.9 +/- 10.8). The ultrastructure of in vitro-produced blastocysts from both culture systems was compared vs. in vivo-derived blastocysts. The latter showed a clear differentiation between trophectoderm (TE) and inner cell mass (ICM) cells. The TE cells were anchored to other TE cells or ICM cells by long, well-developed junctional complexes. The apical membrane of trophoblast cells was covered with numerous microvilli. Mitochondria were abundant, round to elongated in shape, and showed clear transverse cristae. The ultrastructure of blastocysts cultured in NCSU/BSA mimicked that of in vivo-derived embryos closely. In contrast, blastocysts from the NCSU/ECS culture system displayed an irregular ultrastructure with reduced numbers of organelles and numerous cytoplasmic inclusions, such as lipid-yolk-vacuoles and vacuoles with lipid content. In some sections of these embryos, cellular debris was detected in cytoplasm. The shape of mitochondria was more ovoid and cristae were not visible. In summary, our results demonstrate a beneficial influence of ECS in the culture medium on initial cleavage of in vitro-produced porcine embryos. Clearly negative effects of ECS in the subsequent culture period are associated with marked ultrastructural changes of embryonic cells.     

亚牛磺酸对猪胚胎体外培养效果的影响

为了探讨亚牛磺酸的浓度对猪胚胎体外培养效果的影响,试验以孤雌激活胚胎为研究对象,将NCSU-23中的亚牛磺酸浓度从原来的5 mmol/L降低到2.5,1.0,0.5 mmol/L,检测猪卵母细胞激活后的分裂率和囊胚发育率.结果表明:各种浓度亚牛磺酸处理后的分裂率和囊胚发育率(30.7%、27.6%、22.8%、26.5%)差异不显著(P>0.05);但不使用亚牛磺酸则导致囊胚发育率极显著下降(30.7%vs1.4%,P<0.01).结果说明亚牛磺酸是猪胚胎培养液必不可少的重要成分,但其浓度可下调至0.5mmol/L.     

Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

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。...     

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.     

Effects of meiotic inhibitors and gonadotrophins on porcine oocytes in vitro maturation,fertilization and development

This study evaluated the effect of three reversible meiotic inhibitors (MINs) and their interaction with gonadotrophins (Gns) on the meiotic maturation and developmental competence of porcine oocytes. In experiment 1, the oocytes were matured for 22 hr in the presence or absence of dbcAMP (1 mM), cycloheximide (7 μM) or cilostamide (20 μM) with or without Gns, and for an additional 22 hr in the absence of MINs and Gns. At 22 hr of maturation, regardless of the presence of Gns, a higher proportion (p < .001) of oocytes cultured in the presence of MINs were effectively arrested at the germinal vesicle stage compared with the oocytes cultured without MINs. At 44 hr of maturation, the proportion of oocytes that reached MII was higher (p < .05) in groups with Gns compared with groups without Gns. In experiment 2, oocytes that were matured as in experiment 1 were inseminated and cultured for 7 days to evaluate fertilization parameters and blastocyst formation. Only oocytes from the dbcAMP + Gns group had higher (p < .05) efficiency of fertilization compared with the other treatment groups. The presence of dbcAMP during maturation also increased (p < .05) blastocyst formation and efficiency of blastocyst formation in both the presence and absence of Gns. These results indicate that the interaction of Gns with the tested MINs improved meiotic progression. In addition, regardless of supplementation with Gns, the presence of dbcAMP during the first maturation period increased and even doubled the capacity of oocytes to develop to the blastocyst stage.     

猪体细胞核移植胚体外发育初步研究

采用胞质内注射法进行猪体细胞核移植，对去核、激活和培养等关键技术过程进行研究。结果表明：（1）点压法、挤压法对卵母细胞的去核率明显高于盲吸法（三者分别为62．5％、64．6％、50．7％，P〈0．05）。对早成熟的卵母细胞（36-44h）进行去核可明显提高去核效率（P〈0．05），在36-38h、39-41h、42-44h去核率分别为60．9％、67．8％、64．3％，而45-48h为48．4％。（2）体细胞预激活有助于提高核移胚卵裂率（28．0％、20．1％，P〈0．05）。钙离子载体A23187单独或与6-二甲基氨基嘌呤（6-DMAP）联合作用能使猪体细胞核移胚激活继续发育。（3）核移胚以胚胎培养液NCSU23及卵丘单层共培养体系进行分别培养，核移胚卵裂率无明显差异（30．06％、31．5％，P〉0．05）。但NCSU23培养4细胞后发育能力更高（13．5％、3．9％，P〈0.05）。