Mouse embryo co‐culture with autologous cumulus cells and fetal development post‐embryo transfer

This study was conducted to examine the potential for implantation and sustainable fetal development of mouse embryos cultured from the pronuclear to blastocyst stage. Pronuclear embryos from ICR mice (Harlan Sprague‐Dawley) were cultured in Sydney IVF sequential media (Cook) to the blastocyst stage in medium only or co‐cultured with autologous cumulus cells. We also experimented with co‐culture in 100 µL drops. Drop co‐culture produced blastocyst formation rates with a mean of 47.0%, which was significantly higher (P < 0.05) compared to embryos cultured in identical culture conditions except without cumulus cells at 27.3%. Blastocysts obtained in vitro in Cook medium only and co‐cultured in Cook medium with cumulus cells were transferred to pseudopregnant females of ICR strain. The day of blastocyst transfer into surrogate females was designated as post‐transfer of blastocyst day 1 (PT 1). The implantation and fetal development was compared to embryo transfer of in vivo derived blastocysts, which served as controls. There were no statistical differences for implantation and fetal development rates for blastocysts cultured in vitro in either Cook medium only or co‐culture in Cook medium with cumulus cells compared to in vivo‐derived blastocysts. The advantage of the co‐culture system is in generating more blastocysts available for transfer.     

Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo

The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.     

Effects of cumulus cells on in vitro maturation of oocytes and development of cloned embryos in the pig

The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.     

牛体外受精胚胎培养技术研究

在COCs体外培养过程中添加共培养物质和采用不同体积的液滴进行培养,以探讨牛卵母细胞的体外培养效果。结果表明,卵丘细胞、卵丘细胞+bFF共培养系统对胚胎发育均有促进作用,其中以卵丘细胞+bFF共培养系统对桑椹胚和囊胚的发育最好,其囊胚率为10.63%,显著高于对照组的7.23%(P<0.05),高于卵丘细胞组的8.50%(P>0.05)。用培养皿微滴培养(50μL)和用4孔板大体积培养(500μL)的试验表明,大体积培养比微滴培养有优势,桑椹胚和囊胚率均较高,其中囊胚率为10.23%,显著高于培养皿微滴培养的9.07%(P<0.01)。     

Effect of phosphonoformic acid in the development of bovine embryos in vitro

This research evaluated the ability of phosphonoformic acid to inhibit bovine herpesvirus 1 (BHV-1) in cumulus cells commonly used in co-culture with bovine in vitro-produced embryos. At 200 and 400 microg/ml, phosphonoformic acid inhibited 4 logs of BHV-1. Subsequently, phosphonoformic acid (200 and 400 microg/ml) added to both in vitro fertilization and culture medium resulted in a decrease in the proportion of developed blastocysts, and the number of cells per blastocyst was lower in the treated embryos. Therefore, while phosphonoformic acid can effectively inhibit replication of BHV-1 in co-culture cells, it also inhibits development of in vitro-produced bovine embryos.     

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.     

提高猪囊胚后期贴壁能力的优化培养体系

为了提高猪孤雌囊胚贴壁率,试验从饲养层及培养液两方面研究猪孤雌囊胚贴壁能力;用小鼠、猪和牛的胎儿成纤维细胞制作饲养层,分别添加DMEM、NCSU-23、DMEM/NCSU-23培养液,探讨猪孤雌囊胚在3种饲养层上的发育效果。结果表明,BEF饲养层能更好地促进猪孤雌囊胚贴壁生长,其囊胚贴壁率为33.67%,与MEF饲养层组的囊胚贴壁率(19.08%)之间差异显著(P0.05),与PEF饲养层组之间囊胚贴壁率差异不显著(P0.05),MEF饲养层组和PEF饲养层组之间囊胚贴壁率差异不显著(P0.05);在BEF牛胎儿成纤维细胞饲养层组,用猪胚胎培养液NCSU-23培养猪孤雌囊胚后,囊胚贴壁率(22.53%)显著高于DMEM培养液组(10.41%)和DMEM/NCSU-23培养液半量混合组(12.05%)(P0.05),DMEM培养液组和DMEM/NCSU-23培养液半量混合组之间差异不显著(P0.05)。牛胎儿成纤维细胞饲养层和猪胚胎培养液NCSU-23能更好地促进猪孤雌囊胚后期贴壁。     

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.     

Predictive value of the area of expanded cumulus mass on development of porcine oocytes matured and fertilized in vitro

The objective of the present study was to investigate the correlation between the degree of cumulus expansion and in vitro development of porcine cumulus-oocytes complexes (COCs) matured and fertilized in vitro. The COCs were matured in the maturation medium (IVMM) supplemented with 15% or 5% of porcine follicular fluid (PFF) from small, medium and large follicles (<2 mm, 2-5 mm and >5 mm, respectively). COCs cultured in IVMM with PFF for 48 h displayed less expansion than those cultured in IVMM alone (P<0.05), irrespective of follicle size. After culture for 24 h in IVMM with PFF and for another 24 h in IVMM alone, the degree of cumulus expansion was more prominent than culture in the presence of PFF for the entire 48 h period (P<0.05), but the percentages of oocytes with PB I showed no significant difference between the control and experimental groups (P>0.05). After in vitro fertilization, the oocytes failed to develop to the morula/blastocyst stages except for those matured in IVMM supplemented with 15% or 5% PFF obtained from >5 mm follicles for the first 24 h and followed by in IVMM alone for the second 24 h (12.5% and 11.1% of the embryos developed to morulae and blastocysts, respectively). The expanded cumulus areas of COCs were significantly positively correlated with their in vitro development (p=0.0058, 0.0001 and 0.0348 for the percentages of embryos developed to 2-4 cell, beyond 4 cell and morula and blastocyst stages, respectively). In conclusion, PFF had an inhibiting effect on cumulus expansion, and the inhibitory effect decreased progressively with the increase in size of follicles from which PFF was obtained, and the action of PFF on cumulus expansion was affected by the PFF culture time. The areas of the expanded cumulus mass may be used as a parameter to predict development of porcine oocytes matured and fertilized in vitro.     

Effect of mSOF and G1.1/G2.2 Media on the Developmental Competence of SCNT‐Derived Bovine Embryos

The objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell–cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p < 0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p < 0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p < 0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.     

咖啡因和Scriptaid对绵羊核移植重构胚发育的影响

为提高绵羊体细胞核移植效率,以绵羊卵丘细胞为核供体,在融合后的核质互作期间加入咖啡因,在激活后对重构胚进行培养的过程中,加入组蛋白去乙酰化酶抑制剂Scriptaid。结果表明:咖啡因浓度以2.5 mmol/L或5 mmol/L为宜,尽管卵裂率、桑椹胚率以及囊胚发育率与对照组差异不显著(P>0.05),但囊胚细胞数显著高于对照组(P<0.05);以0.2μmol/L Scriptaid处理组囊胚发育率最高,达24.31%,显著高于对照组和0.8μmol/L Scriptaid处理组(P<0.05),但与0.4μmol/L Scriptaid处理组相比差异不显著(P>0.05);囊胚细胞总数以0.2μmol/L Scriptaid处理组最高,显著高于0.8μmol/L Scriptaid处理组(P<0.05),但是与对照组和0.4μmol/L Scriptaid处理组差异不显著(P>0.05)。以上结果表明,咖啡因处理对绵羊核移植重构胚的囊胚发育率无显著影响,但可以显著提高囊胚细胞总数;Scriptaid可以显著提高绵羊核移植重构胚的囊胚发育率。     

Oviduct epithelial cell co-culture of early porcine embryos.

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144-168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4- and 8-16-cell embryos formed blastocysts in culture at a rate of 80-90%.     

Follicular fluid and cumulus cells synergistically improve mouse early embryo development in vitro

The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used independently, to improve preimplantation embryo quality in culture. This study was undertaken to evaluate the influence of a cumulus cell monolayer in human follicular fluid on mouse early embryo development in vitro. One-cell embryos were obtained from NMRI mice after superovulation with eCG and hCG. Cumulus cells were prepared from mouse egg-cumulus mass. These cells were separated from red blood cells using a Percoll gradient. Follicular fluid was collected from patients undergoing an IVF program during oocyte pick-up. The cumulus cell monolayer was prepared in follicular fluid (FC) and Ham's F10 (HC). Mouse one-cell embryos were cultured in FC, HC, Ham's F10 (HF) and follicular fluid (FF) for 120 h. Only 10.5% of embryos passed the two-cell block in HF. However, the proportions of embryos passing the two-cell block were 23.1%, 21.4% and 68.5% in FF, HC, and FC treatments, respectively; which were significantly different from HF (p<0.05). The differences between FC and the two other treatments were also significant (p<0.001). In FC, 33.7% of one-cell embryos continued to grow to the blastocyst stage whereas only 2.1% and 1.9% of one-cell embryos in FF and HC reached this stage and no embryos developed to blastocyst in HF. The proportion of blastocysts in FC was significantly higher than all other treatments (p<0.001). It can be concluded that follicular fluid and cumulus cells in monolayer form synergistically improve the early embryo culture condition.     

影响小鼠早期胚胎发育的几个重要因素

以Ｗｈｉｔｅｎ氏液为基本培养液,研究了卵丘细胞、谷氨酰胺及不同浓度的葡萄糖对ＨＣＧ注射后１７～１８ｈ、２５～２７ｈ和３６～３８ｈ昆明小鼠胚胎体外发育的影响。结果表明,不论葡萄糖或谷氨酰胺存在与否,卵丘细胞与受精卵的共同培养对胚胎克服体外发育阻断无明显效果。葡萄糖能抑制１－细胞胚胎发育,促进４－细胞以后的胚胎发育。谷氨酰胺是小鼠早期胚胎发育的良好能源物质,可使９６％（３０８／３２１）的体外发育到２－细胞的胚胎克服阻断而继续发育到４－细胞。谷氨酰胺与葡萄糖联合使用,则可使小鼠受精卵的囊胚发育率达５７．５％（２２９／３９８）。ＨＣＧ注射后３６～３８ｈ的小鼠胚胎能利用葡萄糖。     

猪孤雌胚胎干细胞建系的研究

以猪孤雌激活囊胚为材料,囊胚透明带消化后采用全胚培养,培养液中添加不同培养成分或因子（如FGF2,LIF,2i等）,以及选择不同的初始培养液体积来筛选猪胚胎干细胞（embryonic stem cell,ES细胞）建系的优化培养体系。囊胚内细胞团形成的细胞集落采用胰酶消化传代。结果显示：透明带消化后,囊胚贴壁率显著升高（19．4％VS．8．8％）（P〈0．05）;初始培养液体积比平常培养液体积（0．30mL／孔,24孔培养板）减半条件下,能显著提高其贴壁率（91．7％VS 20．0％）（P〈0．01）,而且获得了可传至7代的类ES细胞系2株,碱性磷酸酶染色成阳性;当用2i因子（CHIR99021和PD03025901）去替代培养液中的FGF2,囊胚贴壁率（29．400VS53．3％）和原代集落形成率（20．0％VS 87．5％）反而显著下降（P〈0．01）。这表明培养液添加了FGF2和LIF（不舍2i因子）,用24孔板培养,最初培养体积为0．15mL,透明带消化的培养体系比较适合猪孤雌激活胚的ES细胞建系。     

Developmental Competence of Frozen-thawed Blastocysts from Fair-quality Bovine Embryos Cultured with β-Mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.     

Developmental competence of frozen-thawed blastocysts from fair-quality bovine embryos cultured with beta-mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.     

Vascular endothelial growth factor (VEGF) promotes the early development of bovine embryo in the presence of cumulus cells

Three experiments were conducted to determine the effect of Vascular Endothelial Growth Factor (VEGF) on bovine embryonic development in vitro. Human recombinant VEGF(165) was employed at 5 ng/ml in modified synthetic oviduct fluid. In Exp. 1, bovine cumulus oocyte complexes were matured with or without VEGF for 22 hr, inseminated without VEGF for 6 hr, then cultured with or without VEGF for 42 hr. The cleavage rate and the development rate to 4- to 8-cell were higher (P<0.05) in groups with VEGF during in vitro maturation (IVM, 71.4% and 59.6%), in vitro culture (IVC, 70.3% and 62.3%), and both IVM and IVC (75.9% and 67.8%) than in the group cultured thoroughly without VEGF (49.9% and 38.4%, respectively). In Exp. 2, 4- to 8-cell embryos produced in vitro without VEGF were removed from cumulus cells at 48 hr post-insemination (Pi) and cultured with or without VEGF for 144 hr. The development rates to blastocyst at 96 hr (D6), 120 hr (D7) and 144 hr (D8) were similar (P>0.05) in both groups. In Exp. 3, cumulus cells were removed from presumptive embryos produced by IVM and IVF without VEGF at 10 hr Pi. Denuded embryos were cultured with or without VEGF for 38 hr or 182 hr. The cleavage rate and the development rates to 4- to 8-cell at 48 hr Pi and to blastocyst on D6, D7 and D8 were similar (P>0.05) in all groups. These results suggest that VEGF has a beneficial effect on the initial development of bovine embryo through surrounding cumulus cells.     

卵丘细胞对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响

为探讨表皮生长因子(epidermal growth factor,EGF)的添加浓度及脱卵丘细胞时间对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响.试验通过在体外成熟液中添加不同浓度(0、10、15、20、30、40 ng/mL)的EGF来研究其对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响;在培养开始后的不同时间(18、24、38、44 h)进行脱卵丘细胞处理来研究不同时间脱卵丘处理对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响.结果表明,成熟培养基中添加10 ng/mL EGF能显著提高卵母细胞的卵裂率和囊胚率(P <0.05).共培组和独培组卵母细胞培养18 h后脱卵丘细胞成熟率均低于44 h,但差异不显著(P >0.05);共培组卵母细胞培养18 h后脱卵丘细胞的卵裂率和囊胚率显著高于培养44 h(P <0.05);独培组卵母细胞培养18 h后脱卵丘细胞的卵裂率与44 h无显著差异(P >0.05),但囊胚率显著高于培养44 h后脱卵丘细胞(P <0.05).添加10 ng/mL EGF对猪卵母细胞体外成熟及孤雌胚胎体外发育较好;卵母细胞培养18 h后脱卵丘细胞可提高孤雌胚胎早期发育能力.     

Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini-pig (Sus Scrofa)

To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.