Advanced in vitro production of pig blastocysts obtained through determining the time for glucose supplementation

The objective was to determine the effect of glucose supplementation on development (to the blastocyst stage) of in vitro matured (IVM) porcine oocytes that were either in vitro fertilized (IVF) or electrically activated (EA). Embryos were incubated for 46 or 58 h post insemination (hpi) in an NCSU37-based medium containing 0.17 mM sodium pyruvate and 2.73 mM sodium lactate (IVC-PyrLac), and then transferred to an NCSU37-based medium containing 5.55 mM glucose (IVC-Glu) and cultured until Days 6 (Day 0 = day of EA or IVF). The proportions of oocytes that had formed full blastocysts by Day 6 following transfer to IVC-glu at 46 hpi was 23.5 and 41.2% in the IVF and EA groups respectively; these were lower (P<0.001) than the proportions of oocytes that formed full blastocysts after transfer at 58 hpi (60.3 and 78.7%). However, there was no significant difference in total cell number (at Day 6) between embryos transferred at 46 vs 58 hpi. We inferred that in vitro-derived pig embryos can efficiently use glucose as an energy source starting at approximately 58 hpi; exposure to glucose at that time enhanced development to the blastocyst stage as well as blastocyst quality.     

The effect of activation protocols on the development of cloned goat embryos

This study was conducted to compare the developmental competence of somatic nuclear transfer (NT) embryos, after either ionomycin or ethanol activation, in locally bred goats. Donor cells were prepared from the ear skin fibroblasts of a female goat. Cells, at passage 3-8, starved by culturing in 0.5% FCS for 4-8 d, were used for NT. Immature oocytes were obtained from FSH-stimulated goats and matured for 22 hr before enucleation and NT. After fusion, the reconstructed embryos were activated with either ionomycin or ethanol followed by culturing in 6-dimethylaminopurine (6-DMAP) and cytochalasin B (CB), for 3 hr. In experiment I, the fused NT embryos (n=63, ionomycin and n=68, ethanol treatments, respectively) were cultured in B2 with a Vero co-culture system and their developmental competence was evaluated through to Day 9. In experiment II, the NT embryos at the 2-4 cell stage on Day 2 derived from each treatment (ionomycin n=46, and ethanol n=37), were transferred into 10 synchronous recipients. There were no significant differences between the NT embryos derived from the ionomycin and ethanol groups, in fusion (86.3% versus 82.9%), cleavage (90.5% versus 82.4%) and for morula/blastocyst development rates (9.5% versus 5.9%). Sixty percent (3/5) of the recipients from ionomycin became pregnant by midterm (2.5 mts) while only 20% (1/5) from ethanol treatment was pregnant by Day 45. The results demonstrate that activation with either ionomycin or ethanol in combination with 6-DMAP-CB treatment does not affect the development of cloned goat embryos.     

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.     

Effect of recombinant bovine somatotropin (rbST) on cytoplasmic maturation of bovine oocytes and their developmental competence in vitro

The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca(2+)](is)) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2). The culture medium of the treatment groups was modified by supplementation of the control medium with 10 ng/ml rbST (CM 1A and CM 2A), 10(6)/ml granulosa cells (CM 1B and CM 2B), or 10 ng/ml rbST plus 10(6)/ml granulosa cells (CM 1C and CM 2C). No differences were observed in the percentages of oocytes reaching metaphase II between the groups. However, the proportion of blastocysts was highest in treatment groups CM 1C and CM 2C (P<0.05). The type of serum did not alter the positive effect of rbST on the developmental competence of embryos. The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 10 ng/ml rbST and granulosa cells (309.21 vs. 119.97 microA; P<0.01). In parallel, the concentration of [Ca(2+)](is) in oocytes, determined using fluorophore chlortetracycline, was significantly decreased (0.85 +/- 0.02 vs. 0.97 +/- 0.03 AU; P<0.05). Based on these results, we concluded that rbST, in interaction with granulosa cells stimulates the oxidative activity of ooplasmic mitochondria and decreases the content of [Ca(2+)](is) in oocytes. These facts support the hypothesis that somatotropin influences the developmental competence of bovine oocytes during maturation in vitro, and this effect can be modulated by granulosa cells.     

Effect of group culture and embryo-culture conditioned medium on development of bovine embryos

We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 mul/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P < 0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p < 0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.     

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.     

Vitrification of fully grown and growing porcine oocytes using germinal vesicle transfer

The survival rate of vitrified germinal vesicle (GV) stage porcine oocytes is very low, and it is not known if the vitrification damages the nucleus, cytoplasm or both. We have evaluated the eventual GV or cytoplasmic damage in fully grown (FG) and growing vitrified oocytes. Fifty-five percent of nonvitrified FG cumulus-denuded oocytes reached the metaphase II (MII) stage in culture. When growing oocytes from preantral (PA) and early antral (EA) follicles were matured in vitro, almost all oocytes were arrested at the GV stage (GV stage: PA 88.9 and EA 79.5%, respectively). When fresh GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes and matured in vitro, some of them reached the MII stage (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). The maturation rate of vitrified FG oocytes was only 6.1% but increased dramatically when vitrified GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes (MII stage: VitFG/FG 43.9%, VitPA/FG 7.1% and VitEA/FG 26.3%, respectively). These results were not significantly different from those for the nonvitrified groups (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). We activated the reconstructed oocytes that received fresh or vitrified GVs (FG/FG, EA/FG, VitFG/FG and VitEA/FG) and examined their embryonic development. Cleaved embryos (nonvitrified groups 13.0-61.8%, vitrified groups 33.3-40.0%) and blastocysts (nonvitrified groups 0.0-18.2%, vitrified groups 0.0-2.9%) were obtained after activation. These results demonstrate that vitrified porcine GVs maintain maturational and developmental competence and that vitrification predominantly damages the cytoplasm.     

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.     

Effect of replacement of pyruvate/lactate in culture medium with glucose on preimplantation development of porcine embryos in vitro

The effect of glucose supplementation at different times in in vitro culture on the developmental competence of in vitro produced (IVP) porcine embryos was examined. In Experiment 1, when IVP embryos were cultured in modified NCSU-37 supplemented with pyruvate and lactate (IVC-pyr/lac) for 0 h, 24 h, 48 h, 72 h, 96 h, or 118 h and subsequently in modified NCSU-37 supplemented with glucose (IVC-glu) until Day 6 (Day 0=day of in vitro fertilization), the rates of blastocyst formation were significantly higher in embryos cultured in IVC-pyr/lac for 24 or 48 h (24.4% and 23.0%, respectively) than in embryos cultured in IVC-pyr/lac for the whole culture period (14.5%). However, there were no significant differences between embryos obtained after the energy source replacement and embryos cultured in IVC-glu for the whole culture period on the rates (15.2%-24.4%, and 16.8% respectively). Replacement of pyruvate/lactate with glucose at 58 h of culture in Experiment 2 significantly enhanced the rate (31.3%) compared to those after replacement at 48 h, 53 h and 63 h of culture (20.6%, 20.8%, and 21.1%, respectively). In conclusion, replacement of pyruvate/lactate with glucose as the energy substrate was optimal at 58 h of culture for the development of porcine embryos to the blastocyst stage.     

Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

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.     

The effect of nerve growth factor on nuclear progression of porcine oocytes during in vitro maturation and embryo development

The present study examined the effect of nerve growth factor (NGF) on in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development of porcine oocytes. Cumulus-oocyte complexes were cultured with or without 1.0 ng/ml NGF for 40 h. After IVF, they were cultured in vitro for 6 days. After 10 and 20 h of IVM, there was no difference in nuclear status between the NGF-treated and control oocytes. Significant differences were detected in nuclear progression of oocytes matured in the presence or absence of NGF at 30 h of culture. A higher proportion of NGF-treated oocytes were at M-II stage compared to the control. Nevertheless, at the end of the 40-h IVM period, there was no difference in the proportion of M-II stage oocytes between the NGF-treated and control groups. NGF in IVM medium did not influence the developmental competence of putative embryos. Most embryos remained at the 2- to 4-cell stage; however, a significant amount of embryos reached the morula stage both in the NGF and the control groups. These results suggest that NGF during IVM accelerates nuclear progression of porcine oocytes by enhancing the post-diakinetic events of meiosis.     

In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection

In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing.     

Bovine oocytes grown in serum-free medium acquire fertilization competence

We previously found that bovine oocytes 90-99 microm in diameter in early antral follicles grew to nearly their final size in serum-free medium, with some of the oocytes acquiring the nuclear competence to reach the second metaphase. In the present study, we examined the competence of the fertilization and pre-implantational development of the oocytes grown in serum-free medium. Bovine early antral follicles, 0.4-0.7 mm in diameter, were collected mechanically using fine forceps, embedded in collagen gels, and cultured in serum-free medium for 16 days. Grown oocytes which were enclosed by granulosa cells and did not show disintegrated ooplasm were recovered as normal oocytes, were transferred to the maturation medium, and then inseminated with spermatozoa. Ten to 12 h after insemination, 28% (41/145) of the oocytes were penetrated by spermatozoa. Of the penetrated oocytes, 18 (12%) formed a female and a male pronuclei, and 10 (7%) had a female pronucleus and an enlarged sperm head. Among the abnormally penetrated oocytes (13/41), 10 were penetrated by multiple spermatozoa and 3 were penetrated by a spermatozoon at the first metaphase stage. Of the 106 inseminated oocytes grown under serum-free conditions, 8 oocytes had cleaved and developed to the 2-cell stage 48 h after insemination, and 3-4-cell embryos and 5-8-cell embryos were observed after 72-96 h. However, no embryo developed to the blastocyst stage within 8 days. These results indicate that bovine oocytes grown in serum-free medium can be fertilized, but acquire insufficient embryonic development competence under the employed culture conditions.     

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes

Despite the numerous potential applications of oocyte cryopreservation, the poor success rate has limited its practical applications. In livestock, particularly in ovine, the oocytes have low developmental competence following vitrification/warming process. Considering the occurrence of osmotic and oxidative stresses during the vitrification/warming process, the application of antioxidants and osmolytes may improve the developmental competence of vitrified/warmed oocytes. In the present study, we aimed to evaluate the effects of the addition of ascorbic acid (AA) and N‐acetyl cysteine (NAC) as antioxidants and glycine as an organic osmolyte either to the vitrification/warming solutions (VWS) or to the IVM medium on the developmental competence of vitrified/warmed ovine germinal vesicle stage oocytes. The survival rate in the vitrified groups was significantly lower than fresh ones. In vitrified/warmed oocytes, there was no significant difference in survival rate between supplemented and non‐supplemented groups. The addition of AA and/or NAC to the VWS or IVM medium and adding glycine to the IVM medium reduced the proportion of apoptotic oocytes and fragmented embryos, which was reflected as an increase in the proportions of metaphase II stage oocytes and blastocyst production. The best result was achieved by supplementing the IVM medium with NAC. In our study condition, antioxidants and glycine could improve the developmental competence of vitrified/warmed ovine immature oocytes, especially when added during IVM.     

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.     

表儿茶素对小鼠卵母细胞mtDNA拷贝数和胚胎体外发育能力的影响

本研究旨在探究表儿茶素(epicatechin,EC)对小鼠体外成熟培养卵母细胞线粒体DNA(mtDNA)拷贝数及其随后孤雌激活胚胎发育能力的影响。小鼠卵丘-卵母细胞复合体(COCs)在添加不同浓度EC(0、5、10、15、20μmol/L)的成熟液中体外成熟培养16h后,采用实时荧光定量PCR的方法检测卵母细胞mtDNA拷贝数;同时,通过对卵母细胞进行孤雌激活处理,探讨其后续胚胎的体外发育能力。实时荧光定量PCR分析结果显示,添加EC各处理组的卵母细胞mtDNA拷贝数均有所增加,其中,10、15μmol/L组的mtDNA拷贝数均显著高于0μmol/L对照组(P0.05);但10μmol/L组mtDNA拷贝数更接近自然排卵周期合子的mtDNA含量(P0.05)。体外培养观察结果发现,成熟液中添加10μmol/L EC能提高卵母细胞第一极体排出率,与对照组相比差异不显著(P0.05),但能显著提高卵母细胞孤雌激活后胚胎的囊胚发育率(P0.05)。综上表明,小鼠卵母细胞体外成熟液中添加10μmol/L EC可提高卵母细胞的mtDNA拷贝数,有利于促进卵母细胞后续的发育能力。     

Efficiency of Two Enucleation Methods Connected to Handmade Cloning to Produce Transgenic Porcine Embryos

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41–42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 ± 2% vs 48.1 ± 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.     

Different thermotolerances in in vitro‐produced embryos derived from different maternal and paternal genetic backgrounds

The present study evaluated the effects of genetic backgrounds on the developmental competence and thermotolerance of bovine in vitro‐produced (IVP) embryos. First, Holstein (Hol) and Japanese Black (JB) oocytes were fertilized with sperm from Hol, JB and a thermotolerant breed (Brahman), and in vitro development was evaluated when the embryos were exposed to heat shock on Day 2 (Day 0 = day of fertilization). Sperm genetic backgrounds affected the developmental competence in controls (P < 0.05). Second, the effect of sperm pre‐incubation for 4 h on subsequent in vitro fertilization was assessed using different sperm genetic backgrounds. The pre‐incubation of sperm did not decrease the embryonic development regardless of the breed of the sperm. A milder heat shock (40.0°C) effect on parthenotes (Hol and JB) and IVP embryos were evaluated. JB parthenotes showed developmental arrest after Day 4, and the rate of development to the blastocyst stage decreased by heat shock, but not in Hol parthenotes. Heat shock decreased developmental competence after cleavage of IVP embryos regardless of genetic background. The thermotolerance of IVP embryos would be controlled by both maternal and paternal factors but genetic involvement was still unclear. Further evaluation is needed to reveal the genetic contribution to thermotolerance.     

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.