The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX- treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.     

Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% approximately 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 muM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% approximately 57.3%; 22.3% vs 7.4% approximately 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% approximately 46.2%; 18.0% vs 7.1% approximately 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8-74.4% vs 56.5-57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% approximately 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3-27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.     

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.     

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.     

Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.     

Effect of Different Parthenogenetic Activation Methods on the Developmental Competence of in vitro Matured Porcine Oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% ～ 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 μM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% ～ 57.3%; 22.3% vs 7.4% ～ 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% ～ 46.2%; 18.0% vs 7.1% ～ 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8–74.4% vs 56.5–57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% ～ 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3–27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.     

Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation. Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h, cleavage and blastocyst rates were 41.2% and 23.2%, respectively, which were lower than those of controls (77.5% and 42.0%, P < 0.05). Subsequently, we varied the ethanol concentration to increase the effectiveness of parthenogenetic activation. When either 5%, 6%, 7%, 8%, 9%, 10%, or 11% ethanol alone (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h) promoted optimal parthenogenetic activation.     

Development and calcium level changes in pre-implantation porcine nuclear transfer embryos activated with 6-DMAP after fusion

This study investigated the effect of treatment with 6-dimethylaminopurine (6-DMAP) following fusion on in vitro development of porcine nuclear transfer (NT) embryos. Frozen thawed ear skin cells were transferred into the perivitelline space of enucleated oocytes. Reconstructed oocytes were fused and activated with electric pulse in 0.3 M mannitol supplemented with either 0.1 or 1.0 mM CaCl(2). In each calcium concentration, activated oocytes were divided into three groups. Two groups of them were exposed to either ionomycin (I + 6-DMAP or 6-DMAP alone. In experiment 2, fused NT embryos in 0.3 M mannitol containing 1.0 mM CaCl(2) were exposed to 6-DMAP either immediately or 20 min after fusion/activation. For 0.1 mM CaCl(2), oocytes activated with either I + 6-DMAP or 6-DMAP alone showed a higher (P < 0.05) developmental rate to the blastocyst stage than those activated with an electric pulse alone (26.7 and 22.5 vs. 12.5%). For 1.0 mM CaCl(2), oocytes activated with either I + 6-DMAP or 6-DMAP alone showed significantly higher (P < 0.05) developmental rate to the blastocyst stage (35.6 and 28.3 vs. 19.8%). Developmental rate to the blastocyst stage was (P < 0.05) increased in NT embryos activated with 6-DMAP 20 min after fusion. 6-DMAP made a higher and wider Ca(2+) transient compared to that induced by electric pulses (Fig. 3). The fluctuation lasted during the time that oocytes were cultured in 6-DMAP. Regardless of Ca(2+) concentration in fusion medium, activation with 6-DMAP following electric pulses supported more development of porcine NT embryos. Activation of NT embryos with 6-DMAP after fusion in the presence of 1.0 mM CaCl(2) could support better developmental rate to the blastocyst stage.     

Effects of different activation protocols on preimplantation development, apoptosis and ploidy of bovine parthenogenetic embryos

The objective of this study was to optimize the protocols for bovine oocytes activation through comparing the effectiveness of different treatments on the activation and subsequent development of oocytes and examining the effects of two combined activation treatments on the blastocyst apoptosis and ploidy. Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation, cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage. In Experiment 1, the matured oocytes were treated with single activation agents, including ionomycin (5 microM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5 microM for 5 min) or strontium (10mM for 5h). The pronuclear formation and cleavage rate were higher significantly in ionomycin (39.0 and 30.7%) and ethanol (41.5 and 28.1%) treatment alone compared to other treatments (9.7-25.2 and 11.3-23.7%, respectively, P<0.05). Very low blastocyst rates (3.9-5.3%) resulted which were not significantly different among treatments (P>0.05). For the combined activation treatment (Experiment 2), the same concentrations of ionomycin and ethanol as in Experiment 1 were used in combination with either 6-dimethylaminopurine (6-DMAP, 2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10 microg/ml for 3 h). The pronuclear formation, cleavage rate, blastocyst rate and cell number of blastocyst were higher significantly (P<0.05) in ionomycin+6-DMAP treatment (67.1, 69.2, 28.0 and 91.3%, respectively) and ethanol+CHX+CB treatment (68.9, 70.2, 25.5 and 89.3%, respectively) compared to other treatments (11.7-58.1, 10.2-47.1, 1.5-24.2 and 34.2-62.7%, respectively). In Experiment 3, the parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis using a terminal deoxynucleotidyl transferase mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The ethanol+CHX+CB treatment (7.0%) showed significantly lower blastocyst apoptosis index compared to ionomycin+6-DAMP treatment (9.1%, P<0.05). Furthermore, the chromosomal composition in the parthenotes embryos differed (P<0.05) among treatments. The percentage of haploid parthenotes was higher in ionomycin+6-DMAP treatment than ethanol+CHX+CB treatment. These results suggested that ethanol+CHX+CB treatment was more favorable protocol for parthenogenesis of bovine oocytes.     

Effects of chemical activation and season on birth efficiency of cloned pigs

The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs. Three different activation methods were used: (i) Electroporation (Ele); (ii) Ele followed by incubation with 6-dimethylaminopurine (6-DMAP); and (iii) Ele followed by a treatment with cycloheximide (CHX). In experiment 1, the rates of cleavage, developmental rates and cell number of porcine parthenogenetic (PA) embryos were investigated in the three treatment groups. In experiment 2, NT embryos produced by the three different activation treatments were compared for the rates of cleavage, development and cell number. Finally, the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared. The activated oocytes treated by combination activation generally showed a higher (P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele. The rates of cleavage and total cell number of parthenotes were not significantly different. Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly (P<0.05) higher rate than those treated with Ele, but the developmental capability was dramatically decreased in NT embryos. With the CHX activation method, the NT embryo blastocyst rate was substantially (P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods. The birth rate of cloned pigs increased in the CHX group, though the rate was not significantly different from Ele. The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study. Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter. However, no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons. The results obtained from PA and NT embryos, following different activation methods, were inconsistent, suggesting that activation mechanisms are dissimilar in PA and NT embryos. Although the chemical activation in our study leads to an elevation of the blastocyst rate, it does not improve the oocyte’s molecular programming and so does not significantly improve the efficiency of producing cloned pig births. Supported by National Key Basic Research and Development Program (China of Grant No. G200000161).     

Increasing glucose in KSOMaa basal medium on culture Day 2 improves in vitro development of cloned caprine blastocysts produced via intraspecies and interspecies somatic cell nuclear transfer

This study was conducted to evaluate the efficiency of potassium simplex optimization medium with amino acids (KSOMaa) as a basal culture medium for caprine intraspecies somatic cell nuclear transfer (SCNT) and caprine-bovine interspecies somatic cell nuclear transfer (iSCNT) embryos. The effect of increased glucose as an energy substrate for late stage development of cloned caprine embryos in vitro was also evaluated. Enucleated caprine and bovine in vitro matured oocytes at metaphase II were reconstructed with caprine ear skin fibroblast cells for the SCNT and iSCNT studies. The cloned caprine and parthenogenetic embryos were cultured in either KSOMaa with 0.2 mM glucose for 8 days (Treatment 1) or KSOMaa for 2 days followed by KSOMaa with additional glucose at a final concentration of 2.78 mM for the last 6 days (Treatment 2). There were no significant differences in the cleavage rates of SCNT (80.7%) and iSCNT (78.0%) embryos cultured in KSOMaa medium. Both Treatment 1 and Treatment 2 could support in vitro development of SCNT and iSCNT embryos to the blastocyst stage. However, the blastocyst development rate of SCNT embryos was significantly higher (P < 0.05) in Treatment 2 compared to Treatment 1. Increasing glucose for later stage embryo development (8-cell stage onwards) during in vitro culture (IVC) in Treatment 2 also improved both caprine SCNT and iSCNT embryo development to the hatched blastocyst stage. In conclusion, this study shows that cloned caprine embryos derived from SCNT and iSCNT could develop to the blastocyst stage in KSOMaa medium supplemented with additional glucose (2.78 mM, final concentration) and this medium also supported hatching of caprine cloned blastocysts.     

A comparison of two in vitro maturation media for use with adult porcine oocytes for adult somatic cell nuclear transfer

Two media used to mature adult porcine oocytes for somatic cell nuclear transfer were compared. In the first experiment, parthenogenetic embryos were produced using a maturation medium used by us previously to clone pigs (OMM199) and that described by Kühholzer et al. (2001) to transport oocytes overnight (BOMED). There was no difference in maturation rates between the two different media. However, BOMED medium increased the percentage of parthenogenetic embryos that developed to the blastocyst stage compared with OMM199 (49% vs. 29%, respectively). In a second experiment, BOMED medium increased the percentage of SCNT embryos that developed to the blastocyst stage compared with OMM199 (22% vs. 8%, respectively). The efficiency of our cloning protocol using adult oocytes matured in BOMED medium was then determined by transferring SCNT embryos reconstructed using adult fibroblasts to synchronized recipients. Primary cultures of adult fibroblasts were obtained from two adult male pigs and used for SCNT (passages 2-4). Between 82 and 146 fused couplets were transferred to seven recipients synchronized 1 day behind the embryos. Five recipients (71% pregnancy rate) subsequently farrowed a total of 23 piglets (4.4 average litter size). Overall efficiencies (liveborn/embryos transferred) were 3.2% for all transfers and 4.3% for animals that gave birth.     

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.     

Glucose and pyruvate metabolism of preimplantation goat blastocysts following in vitro fertilization and parthenogenetic activation

The energy metabolism of preimplantation embryos can be used to predict viability and postimplantation development. Although preimplantation development and mean blastocyst cell numbers of goat in vitro-fertilized (IVF) embryos and chemically activated parthenogenotes are comparable, mammalian parthenogenotes are not viable, with most dying shortly after implantation. The objective of this study was to compare glucose and pyruvate metabolism of IVF goat blastocysts with that of parthenogenetic blastocysts developing from chemically activated oocytes. Embryos derived from IVF and parthenogenotes produced by exposing oocytes to either ionomycin or ethanol followed by 6-dimethylaminopurine (6-DMAP) were cultured in G1.2/G2.2 sequential culture media. Metabolism was determined for individual blastocysts using [5-3H]glucose and [2-14C]pyruvate to determine glycolytic and Kreb's cycle activity, respectively. Data were analyzed by ANOVA. A significantly higher percentage of activated oocytes underwent cleavage and developed to the blastocyst stage compared to IVF oocytes (p < 0.05). There was no significant difference in glucose or pyruvate metabolism between IVF and parthenogenetically activated blastocysts. Mean glucose metabolism through glycolysis was 154.9 +/- 29.1, 130.3 +/- 17.1, and 129 +/- 16.5 pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Mean pyruvate metabolism through the Kreb's cycle was 28.1 +/- 8.0, 15.8 +/- 4.2, and 24.4 +/- 4.4 in pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Our results suggest that known differences in postimplantation development observed in IVF versus parthenogenetic embryos cannot be attributed to differences in pyruvate or glucose metabolism in the preimplantation blastocysts. Thus, these activation protocols result in embryos capable of appropriate regulation of key metabolic enzymes.     

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle

Ooplasmic factors drive nuclear organization after fertilization and are also important for re-programming in nuclear transfer procedures, in which artificial activation is essential for reconstructed embryos to progress in development. The present research evaluated the effect of pronuclear transfer (PT) between zygotes parthenogenetically activated with ionomycin followed by strontium (S) or 6-DMAP (D) on early embryonic development. PT was performed in the same zygote to obtain embryos in control groups (S-PT and D-PT) and between zygotes activated with S and D to achieve embryos with differentially activated cytoplasm (C) and nucleus (N) (SCDN and DCSN). PT procedure did not affect cleavage and blastocyst rates, respectively, in PT control groups compared to non-manipulated control (S-PT: 73.6% and 7.3% compared with S-Control: 77.9% and 7.8%; and D-PT: 73.3% and 31.7% compared with D-Control: 83.1% and 41.5%). Cleavage, eight-cell, and blastocyst rates, respectively, were similar between SCDN (76.5%, 36.4%, and 6.8%) and DCSN (69.5%, 25.0%, and 4.9%) embryos. Developmental rates in SCDN were similar to S-PT, but inferior to D-PT. Developmental arrest up to eight-cell stage was greater in SCDN and DCSN than in S-PT and D-PT. In conclusion, karyoplast exchange between parthenogenetic zygotes activated with strontium and 6-DMAP can lead to nuclear–cytoplasmic incompatibilities and affect embryonic development to the eight-cell and blastocyst stages.     

In vitro development of porcine transgenic nuclear-transferred embryos derived from newborn Guangxi Bama mini-pig kidney fibroblasts

Porcine transgenic cloning has potential applications for improving production traits and for biomedical research purposes. To produce a transgenic clone, kidney fibroblasts from a newborn Guangxi Bama mini-pig were isolated, cultured, and then transfected with red and green fluorescent protein genes using lipofectamine for nuclear transfer. The results of the present study show that the kidney fibroblasts exhibited excellent proliferative capacity and clone-like morphology, and were adequate for generation of somatic cell nuclear transfer (SCNT)-derived embryos, which was confirmed by their cleavage activity and blastocyst formation rate of 70.3% and 7.9%, respectively. Cells transfected with red fluorescent protein genes could be passed more than 35 times. Transgenic embryos cloned with fluorescent or blind enucleation methods were not significantly different with respect to cleavage rates (92.5% vs. 86.8%, p?>?0.05) and blastocyst-morula rates (26.9% vs. 34.0%, p?>?0.05), but were significantly different with respect to blastocyst rates (3.0% vs. 13.2%, p?p?>?0.05), blastocyst (14.1%, 16.1% vs. 23.1%, p?>?0.05) and morula/blastocyst rates (43.5%, 47.0% vs. 57.6%, p?>?0.05) were not significantly different between the groups of transgenic cloned embryos, cloned embryos, and parthenogenetic embryos. This indicates that long-time screening by G418 caused no significant damage to kidney fibroblasts. Thus, kidney fibroblasts represent a promising new source for transgenic SCNT, and this work lays the foundation for the production of genetically transformed cloned Guangxi Bama mini-pigs.     

Developmental rate and ploidy of embryos produced by nuclear transfer with different activation treatments in cattle

Bovine oocyte activation is one of the essential elements that determine the success of nuclear transfer and the subsequent development of cloned embryos. Three methods for oocyte activation, including 5 microM ionomycin (5 min, Group 1) alone, ionomycin+1.9 mM 6-dimethylaminopurine (DMAP, 3h, Group 2), and ionomycin+10 microg/ml cycloheximide (CHX, 3h, Group 3) were compared for the development of embryos produced by somatic nuclear transfer (SCNT) to parthenotes and IVF counterparts. At 19-h post-activation/insemination (hpa/hpi), 27.5% of oocytes in Group 2 cleaved and this rate was greater (P<0.05) than other groups (Group 1, 2.1%; Group 3, 3.0%). None of the oocytes in the IVF control group cleaved at 19-22 hpi. At 24 hpa, the rates of cleavage of oocytes in Group 2 (52.1%) were greater (P<0.05) than those in Groups 1 and 3 (7 and 38.3%, respectively). Only six oocytes (3.3%) in the IVF control group cleaved at 24 hpi. The overall cleavage rates of oocytes in Group 2 (85.5%) at 48 hpa were greater (P<0.05) than other treatments, but it did not show any difference when compared with the IVF control group (75.0%). The development rate to two-cell stage embryos of Group 2 was consistently greater at all observation points followed by Groups 3 and 1. Similar results were obtained in SCNT embryos, but the rates of cleavage at 48 hpi and blastocyst development in Group 2 (68.4 and 16.3%, respectively) did not differ from Group 3 (63.0 and 13.1%, respectively). The chromosomal composition in the parthenotes and SCNT embryos differed (P<0.05) among treatments. In Groups 1 and 3, greater percentages of haploid parthenotes (86 and 71%, respectively) were observed. In contrast, 84% of parthenotes in Group 2 had abnormal ploidy (44% polyploid and 40% mixoploid). In the case of SCNT embryos, Groups 1 and 3 had greater percentages of diploid chromosomal sets (77 and 70%, respectively), whereas 54% in Group 2 were polyploid or mixoploid. These results indicate that DMAP treatment after ionomycin greatly increases the developmental rates of parthenotes, but did not differ in blastocyst development compare with CHX treatment. However, DMAP treatment increased the time-dependent cleavage rate to two-cell stage embryos. Further, it greatly enhanced the incidence of chromosomal abnormalities in parthenotes and SCNT embryos. Hence, it is concluded that CHX combined with ionomycin is more desirable than DMAP for oocyte activation during nuclear transfer in cattle.     

Effect of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos

In order to develop a culture system and recipient cytoplasm that could improve the developmental competence of somatic cell nuclear transfer (SCNT) embryos for successful cloning of pigs, we evaluated the effect of donor oocytes and in vitro maturation (IVM) media on maturation of oocytes and developmental competence of SCNT embryos. In Experiment 1, oocytes derived from sows or gilts were matured in two IVM media (TCM-199 versus NCSU-23) and maturation of oocytes was evaluated by the status of chromatin configuration, the diameter of matured oocytes, the thickness of the zona pellucida, and the size of the perivitelline space (PVS). Sow oocytes matured in TCM-199 (S-TCM group) and NCSU-23 (S-NCSU group) showed significantly higher (P<0.05) maturation rates (S-TCM and S-NSCU, 86+/-4 and 82+/-4%, respectively) when evaluated by metaphase-II status than the gilt oocytes matured in TCM-199 (G-TCM group, 71+/-3%) and in NCSU-23 (G-NCSU-23 group, 71+/-3%). Oocyte diameter, the thickness of the zona pellucida, and the perivitelline space of sow oocytes (S-TCM and S-NCSU) were larger than those of gilt oocytes (G-TCM and G-NCSU) after IVM (P<0.05). In Experiment 2, SCNT was performed, using in vitro-matured oocytes from each group as recipient cytoplasm and porcine fetal fibroblasts as karyoplasts. The reconstructed embryos were electrically fused and activated, and cleavage and blastocyst formation were monitored under a stereomicroscope. The total cell number of flattened blastocysts stained with 5 microM bisbenzimide on day 7 were counted. In addition, in vitro matured non-enucleated oocytes were also electrically activated (parthenogenetic activation) and pronuclear formation was monitored. No difference in pronuclear formation rate after parthenogenetic activation and fusion rate after SCNT was observed among experimental groups. A significantly higher cleavage rate (P<0.05) was observed in S-TCM (69+/-4%) when compared with only G-NCSU (58+/-4%), but not with G-TCM (60+/-4%) or S-NCSU (68+/-4%). The rate of blastocyst formation was significantly higher (P<0.05) in sow oocytes (24% in S-TCM and S-NCSU), when compared to that observed in G-TCM (15%), and G-NCSU (14%). When the same source of oocytes was used, there was no significant difference in rate of blastocyst formation in the two culture media. Total cell number of blastocysts were not significantly different among experimental groups. In conclusion, the present study clearly demonstrated that sow oocytes have a greater developmental competence than gilt oocytes, regardless of the maturation medium examined.