Effects of fusion/activation methods on development of embryos produced by nuclear transfer of porcine fetal fibroblast

The present studies were carried out to investigate the effects of intensity of dc pulse, number of dc pulse and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. In experiment 1, different fusion/activation intensity (two dc pulses of 0.4, 0.8, 1.2, 1.6 and 2.0 kV/cm for 30 micros, respectively) was carried out to investigate development of embryos. In experiment 2, the reconstructed oocytes were fused and activated with one, two or three dc pulses of 1.2 kV/cm for 30 micros. In experiment 3, reconstructed oocytes were equilibrated in TCM-199 medium for 0-6 h, respectively, and fused/activated with one dc pulse of 1.2 kV/cm for 30 micros. The reconstructed embryos were cultured in PZM-3 medium containing 0.3% BSA. When oocytes were fused with donor cell by two dc pulses of 0.4 kV/cm for 30 micros, the rates of cleavage and blastocyst formation were significantly lower (32.9% and 2.5%) than those of fused by 0.8 kV/cm (59.0% and 17.4%) or 1.2 kV/cm (63.3% and 18.4%), respectively. One dc pulse of 1.2 kV/cm for 30 micros was enough to fuse and activate embryos to develop to blastocyst (24.8%). Equilibration for 2-3 h in TCM-199 before fusion/activation was beneficial for improving the developmental ability of embryos produced by nuclear transfer (25.6-23.3% at blastocysts).     

Nuclear totipotency of cultured rabbit morulae to support full-term development following nuclear transfer.

The rabbit was used as a model for nuclear transfer. A critical step in nuclear transfer is oocyte activation, which was evaluated in this research. Optimal field strength of an electric stimulus for activation was examined. A significantly higher activation rate in all criteria tested was achieved when oocytes were activated electrically with a field strength of 2.4 kV/cm versus 1.2 or 1.8 kV/cm. Also, electrical stimulation with combined alternating current (AC) and direct current (DC) was superior to DC stimulation alone for activation. In another study involving 586 oocytes, exposure of oocytes to cytochalasin B for 1 h followed by activation with electrical stimulation significantly improved development of the oocytes to blastocyst stage compared to oocytes without cytochalasin B pre-exposure (38% vs 26%, p less than 0.05). Cytochalasin B exposure alone (control), however, had no effect on activation. Exposing oocytes to activation medium without electrical stimulation also activated some oocytes. In the nuclear transfer experiment, blastomeres from 8-cell embryos cultured for 20-24 h to the 32-64-cell stage were used as nuclear donor cells. Of 491 oocytes used, 459 (93%) survived the enucleation and fusion procedure, 370 (81%) fused, and 284 (77%) developed into 2-4-cell embryos. A total of 243 of these 2-4-cell embryos were transferred to 15 pseudopregnant recipients and produced 8 young (3%). Although the efficiency is low, this study demonstrated that rabbit morulae cultured for 20-24 h to the 32-64-cell stage as nuclear donors for transfer remain totipotent.     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚达11．7％、孵化囊胚率为6．7％,显著高于成纤维细胞组成的重构胚（P＜0．05）。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0或G1期,抽吸法／解剖法采集卵母细胞,体外培养33或44h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电泳冲结合6－DMAP激活处理,体外培养6天,结果表明,卵 母细胞采集方法、激活液中细胞松弛素（CB）并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,能在体外发育至囊胚。     

Improvement of an electrical activation protocol for porcine oocytes

Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-microsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56.7%,发育至桑椹胚达11.7%、孵化囊胚率为6.7%,显著高于成纤维细胞组成的重构胚(p<0.05)。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至GO或G1期,抽吸法/解剖法采集卵母细胞,体外培养33或44 h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电脉冲结合6-DMAP激活处理,体外培养6天,结果表明,卵母细胞采集方法、激活液中细胞松弛素(CB)并不影响重构胚的发育(以卵龄44h的卵母细胞为受体);而以电脉冲结合6-DMAP激活处理能提高重构胚发育能力(以卵龄33 h的卵母细胞为受体)(p<0.05)。本研究显示,以电脉冲结合6-DMAP激活卵丘细胞重构胚,能在体外发育至囊胚     

Developmental potential of porcine nuclear transfer embryos derived from transgenic fetal fibroblasts infected with the gene for the green fluorescent protein: comparison of different fusion/activation conditions.

The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42-44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3-5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 microsec (group A), or one pulse of 1.6 kV/cm for 30 microsec followed in 30 min by one pulse of 1.2 kV/cm for 30 microsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean +/- SEM, 68.4% +/- 3.9%) was higher (P < 0.05) than in group B (59.4% +/- 2.3%). The rates of cleavage (50.1% +/- 4.6% to 62.8% +/- 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% +/- 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% +/- 1.7% and 4.9% +/- 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% +/- 4.2%) was higher (P < 0.05) than in group C (58.9% +/- 2.7%). The cleavage rates were not different among control and treatment groups (58.1% +/- 8.1% to 73.6% +/- 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% +/- 1.7%) was lower (P < 0.05) than in controls and group C (11.4% +/- 2.0% to 16.4% +/- 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% +/- 1.4%) was higher (P < 0.05) than in the group without CB (70.9% +/- 0.2%). The cleavage rate of the control group (85.5% +/- 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% +/- 2.7% and 63.9% +/- 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% +/- 2.5% to 19.1% +/- 6.0%) and the mean cell number of blastocysts (29.4 +/- 5.2 to 45.7 +/- 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.     

In vitro developmental competence of domestic cat embryos after somatic cloning: a preliminary report

This work was undertaken in order to study the developmental competence of nuclear transfer feline embryos with regard to the recipient-cytoplast's age and type of somatic cells used as donor nuclei. Oocytes were recovered by mincing the ovaries in HEPES-buffered TCM-199. Selected cumulus-oocyte complexes (COCs) with compact cumulus cell mass and a dark, homogenous ooplasm were cultured for maturation in the modified medium TC-199 for 24, 35, and 43 h, and after enucleation were used as a source of recipient cytoplasts for exogenous somatic nuclei. Two experiments were carried out. In Experiment 1, the source of recipient cytoplasts was oocytes matured in vitro for 24 h (Group 1), 35 h (Group 2), and 43 h (Group 3), while the source of donor nuclei was cycling fetal fibroblasts. Somatic cell-cytoplast complexes (SC-CCs) were fused electrically by double DC pulses of 2.0 kV/cm for 15 micros. The reconstructed embryos were cultured in B2 medium for 72 h after NT, then co-cultured with BRL cells in the same medium supplemented with 10% FBS at 38.5 degrees C under 5% CO2 in air. In Groups 1, 2, and 3, the fusion rates were 71.4 (25/35), 74.6 (47/63), and 57.5% (46/80), respectively. The cleavage rates in Groups 1, 2, and 3 were 80.0 (20/25), 55.3 (26/47), and 60.8% (28/46), respectively. The development to morula and blastocyst stages was higher in Groups 1 and 2 compared to Group 3 (morula stage 14/25 (56.0%), 16/47 (34.0%), and 13/46 (28.2%); blastocyst stage 2/20 (8.0%), 4/47, (8.5%), and 0/46, respectively). In Experiment 2, the oocytes matured for 24-35 h were used as a source of recipient cytoplasts and cycling fetal fibroblasts and cumulus cells derived from mature COCs were used as a source of donor nuclei. The fusion rates were 115/193 (59.6%) versus 65/143 (45.4%) for fetal fibroblasts and cumulus cells, respectively. The cleavage rate was 72/115 (62.6%) versus 48/65 (73.8%), and the development to blastocyst stage 6/115 (5.2%) versus 5/65 (7.7%), for fetal fibroblast and cumulus cells, respectively. In conclusion, a prolonged maturation period of cat oocytes decreases developmental competence of reconstructed embryos, especially the ability to reach the blastocyst stage. The in vitro development of reconstructed embryos with either nuclei of fetal fibroblasts or cumulus cells was at approximately the same level.     

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 μg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX- blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.     

Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.     

Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation

The purpose of our work was to establish an efficient protocol for activation of porcine cytoplast-fibroblast constructs produced by the handmade cloning technique. Firstly, we investigated a combined electrical and chemical activation protocol for parthenogenetic development of in vitro matured zona-free oocytes. Oocytes were activated by one 80 micros pulse and subsequently cultured in cytochalasin B and cycloheximide. Developmental rates of blastocysts from activated oocytes were 49+/-1 and 40+/-2%, when using one 80 micros pulse of 0.85 or 1.25 kV/cm, respectively. The activation procedure was further confirmed by a simultaneous re-fusion and activation of bisected oocytes, resulting in a blastocyst rate of 41+/-8%. Secondly, the activation protocol was applied in the handmade cloning technique. In vitro matured zona-free porcine oocytes were bisected and halves containing no chromatin, i.e. the cytoplasts, were selected. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused to one fibroblast by one 80 micros pulse of 1.25 kV/cm. After 1h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously by one 80 micros pulse of 0.85 kV/cm, and subsequently cultured in cytochalasin B and cycloheximide. The development of reconstructed embryos to the blastocyst stage was in average 21+/-4%, and total blastocyst cell counts were in average 48+/-3. Thus, the combined electrical and chemical activation procedure resulted in efficient blastocyst development in the handmade cloning technique.     

猪体细胞核移植重构胚的体外发育(英文)

以卵丘细胞为核供体细胞组成重构胚 ,卵裂率达到 5 6.7% ,发育至桑椹胚率达到1 1 .7% ,囊胚率为 6.7% ,显著高于成纤维细胞重构胚 (P <0 .0 5 )。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0 G1 期 ,抽吸法 解剖法采集卵母细胞 ,体外培养 3 3～ 44h ,将卵丘细胞放至去核卵母细胞的卵周隙中 ,重构胚以钙离子载体A2 3 81 7或电脉冲结合 6 DMAP激活处理 ,体外培养 6d。研究表明 ,卵母细胞采集方法、激活液中细胞松弛素 (CB)、激活程序并不影响重构胚的发育 (以卵龄 44h的卵母细胞为受体 ) ;而以电脉冲结合 6 DMAP激活处理能提高重构胚发育能力 (以卵龄 3 3h的卵母细胞为受体 ) (P <0 .0 5 )。本研究显示 ,以电脉冲结合 6 DMAP激活卵丘细胞重构胚 ,体外能发育至囊胚     

Brief exposure to cycloheximide prior to electrical activation improves in vitro blastocyst development of porcine parthenogenetic and reconstructed embryos

To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10 microg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100 micros) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30 micros) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX+Ele); electrical activation followed by exposure to cycloheximide for 6h (Ele+CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6h (CHX+Ele+CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for > or =30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX+Ele, Ele+CHX and CHX+Ele+CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX+Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.     

Somatic cell nuclear transfer in domestic cat oocytes treated with IGF-I for in vitro maturation

Oocyte maturation and somatic cell nuclear transfer (NT) studies conducted in the domestic cat can provide valuable insights that are relevant to the conservation of endangered species of felids. The present investigation focuses on the in vitro maturation (IVM) of domestic cat oocytes stimulated by insulin-like growth factor-I (IGF-I) and their possible use as recipient cytoplasts for somatic cell NT. In Experiment I, the effects of IGF-I on cat oocyte IVM were monitored. Cumulus-oocyte complexes (COCs) were recovered in TALP-HEPES medium following ovarian follicular aspiration and were classified under a stereomicroscope into four grades using criteria based on cumulus cell investment and the uniformity of ooplasm. The COCs were either cultured in Dulbecco's modified Eagle medium (DMEM) alone as a control group or supplemented with 100 ng/ml IGF-I. After culturing for 32-34 h, oocytes were denuded and maturation rate was evaluated by observing the extrusion of the first polar body and staining with aceto-orcein. The percentages of maturation of Grades 1 and 2 oocytes were significantly increased (P<0.05) in IGF-I supplemented medium compared with medium alone (85.8 versus 65.5 and 70.3 versus 51.8, respectively) whereas the maturation rates of Grades 3 and 4 oocytes were not different. The IVM of Grade 1 oocytes was significantly higher (P<0.05) than for all other grades in both control and experimental groups. In Experiment II, the in vitro development of cat NT embryos using cumulus cells, fetal or adult fibroblasts as donor nuclei was investigated. The IVM oocytes in medium containing IGF-I were enucleated and fused with cumulus cells, fetal or adult fibroblasts between passages 2 and 4 of culture. Reconstructed embryos were cultured and monitored every 24h for progression of development through Day 9. There was no significant difference in the percentage of fusion of NT embryos using different donor nuclei whereas the cleavage rates of NT embryos reconstructed with fetal fibroblasts and cumulus cells were significantly higher (P<0.05) than those reconstructed with adult fibroblasts (72.5 and 70.7% versus 54.8%, respectively). Development of NT embryos reconstructed with adult fibroblast to the morula stage was significantly lower (P<0.05) compared with cumulus cell or fetal fibroblast donor cells (25.8% versus 37.9 or 47.5%, respectively). However, no difference was observed in development to the blastocyst stage. These results demonstrated that IGF-I promoted the IVM of domestic cat oocytes. The enucleated IVM oocytes could be used as recipient cytoplasm for fetal and adult somatic cell nuclei resulting in the production of cloned cat embryos.     

Oocyte activation procedures and influence of serum on porcine oocyte maturation and subsequent parthenogenetic and nuclear transfer embryo development

The viability of SCNT embryos is poor, with an extremely low cloned piglet production rate. In the present work, we studied the effect of three activation protocols based on ionomycin treatment (5 microM ionomycin for 5 min and incubated in 2 mM 6-DMAP for 3.5 h) or electric stimuli (two square wave electrical DC pulses of 1.2 kV/cm for 30 micros) combined or not with 6-DMAP on parthenogenetic embryo development. Oocytes activated by ionomycin plus 6-DMAP showed lower cleavage (47.2 vs. 78.5-81.5; p < 0.05) and blastocyst rates (11.3 vs. 29.2-32.1; p < 0.05) than those activated by electrical and electrical plus 6-DMAP treatments. Also, we studied the effect of addition of serum to maturation medium (0% vs. 10%) on nuclear maturation and further parthenogenetic and SCNT embryo development. We observed in the parthenogenetic embryos that cleavage rates in the serum-free group were significantly higher than in the serum-supplemented group (81.8 vs. 69.6% respectively; p < 0.05), although these differences were not detected in blastocyst rates or blastocyst nuclei numbers. Regarding SCNT embryos, no significant differences were observed in cleavage or blastocyst rates between different experimental groups of SCNT embryos. In conclusion, electrical pulse followed or not by 6-DMAP was found to be an efficient procedure to artificially activate MII porcine oocytes. Moreover, the addition of serum to oocyte maturation media did not seem to improve parthenogenetic or SCNT porcine embryo development.     

猪卵母细胞体外成熟的发育潜力及核移植重构胚构建方法的研究

为了提高猪体细胞核移植重构胚发育潜力,本研究对体外成熟28 h、32 h、36 h、40 h、44 h、48 h、52 h和56 h的猪卵母细胞分别进行去核构建重构胚.研究结果表明,成熟44 h的卵母细胞核移植后有较高的融合率(58.99%)、卵裂率(67.52%)和囊胚率(22.78%),而成熟48 h的卵母细胞则分别为56.51%、65.73%和15.96%;且卵龄为44 h的卵母细胞核移植后分裂率与囊胚率显著高于卵龄为40 h、36 h、32 h、28 h的卵母细胞的分裂率与囊胚率(P＜0.05).卵龄为48 h的卵母细胞融合率高于卵龄为52 h卵母细胞的融合率(P＜0.05).同时我们还探讨了不同去核方法(盲吸法、Hochest33342染色法和Spindle-view system)对猪体细胞核移植重构胚发育能力的影响.研究结果发现,盲吸法、Hoechest33342染色法和Spindle-view system法的去核率分别达到76.33%,100.00%和98.40%.Hoechest染色法去核率显著高于盲吸法的去核率(P＞0.05),而与Spindle-view法去核率没有差异(P＞0.05).三种方法在融合率和囊胚率方面差异不显著(P＞0.05),但Hoechest染色法的分裂率较低,差异显著(P＜0.05).进一步的研究表明,细胞质内注射进行核移植构建重构胚的分裂率和囊胚率分别为68.13%和6.44%;透明带下注射法则为60.37%和8.08%,两者差异不显著(P＜0.05);两者均可运用于猪体细胞的核移植,这为建立有效的猪体细胞核移植体系提供了参考.     

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.     

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.     

The effect of electrical field strength on activation and development of cloned caprine embryos

The activation procedure used in nuclear transfer (NT) is one of the critical factors affecting the efficiency of animal cloning. The purpose of this study was to compare the effect of two electrical field strengths (EFS) for activation on the developmental competence of caprine NT embryos reconstructed from ear skin fibroblasts of adult Alpine does. The NT embryos were obtained by transfer of the quiescent fibroblasts at the fourth passage into the enucleated metaphase II (M II) oocytes. Four to five hours after electrical fusion, the NT-embryos were activated by EFS either at 1.67 or at 2.33 kV/cm and immediately incubated in 6-DMAP (2 mM) for 4 h. The cleavage rate of the NT-embryos activated with 2.33 kV/cm was greater than that activated with 1.67 kV/cm after in vitro culture for 18 h (65.6% versus 19.6%, p < 0.001). No pregnancy was found in 14 recipient does after transferring 51 NT embryos at 1-2 cell stages activated with 1.67 kV/cm. In contrast, two of the seven recipients were pregnant and gave birth to three kids after transferring 61 NT embryos at 1-2 cell stages activated by 2.33 kV/cm. The birth weights of three cloned kids were within the normal range of Alpine goats. However, one kid died 1h after birth while the remaining two are still healthy. DNA analysis by polymerase chain reaction (single-strand conformation polymorphism, SSCP) confirmed that the three kids were genetically identical to the nuclear donor.     

Establishment of a porcine cell line from in vitro-produced blastocysts and transfer of the cells into enucleated oocytes

The present study was conducted to establish a porcine cell line from blastocysts produced in vitro and to examine the developmental ability of nuclear transfer embryos reconstituted with the cells and enucleated mature oocytes. When hatched blastocysts were cultured in Dulbecco's modified Eagle's medium with supplements, no colonies of embryo-derived cells were observed. In contrast, 56% of embryos that were attached to feeder layers of STO cells formed colonies in NCSU-23 with supplements. When the colonies were subcultured in the absence of feeder cells, a cell line with an epithelial-like cell morphology was obtained. This cell morphology was stable up to at least passage 30. Although no fused embryos were observed when a pulse of 100 V/mm was applied, the fusion rate increased significantly at 150 V/mm (28%) and 200 V/mm (64%). At 200 V/mm, 39% of fused embryos cleaved, but no embryos developed beyond the 3-cell stage. When cocultured with electro-activated oocytes, percentages of reconstructed embryos cleaved (65%) and developed to the 4-cell stage (23%) were significantly higher than percentages for those (cleavage: 38%; 4-cell stage: 3%) in the absence of activated oocytes. At 7 days after culture, one reconstructed embryo successfully developed to the blastocyst stage in the presence of activated oocytes. When green fluorescent protein-expressing cells and enucleated oocytes were fused and the fused embryos were cultured with electro-activated oocytes, 3 of 102 reconstructed embryos developed to the blastocyst stage. All of the blastocysts were positive for fluorescent green under ultraviolet light. The results of the present study indicate that a porcine cell line can be established from the hatched blastocyst and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cells into enucleated oocytes have the ability to develop to the blastocyst stage in vitro.     

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.