Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer

Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage.     

Improving in vitro development of cloned bovine embryos with hybrid (Holstein-Chinese Yellow) recipient oocytes recovered by ovum pick up

In the present study, oocytes from F1 hybrid cattle, as well as their parental lines, were recovered by ovum pick up (OPU) and used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Four F1 hybrid (Holstein dam x Chinese Yellow sire), 10 Holstein and four Chinese Yellow cattle were subjected to OPU once weekly. There were no significant differences among breeds for number of recovered oocytes per session (overall average, 7.8+/-0.5; mean+/-S.E.M.), quality of the recovered oocytes, or oocyte maturation rate (72-73%). Matured oocytes were all used as recipient cytoplasm (without selection) and a single batch of cumulus cells collected from a Holstein cow were used as donor cells. Although reconstructed embryos initiated cleavage sooner when the recipient cytoplasm was from hybrid cattle versus the two parental breeds, the overall cleavage rate was indistinguishable among breeds. At Day 8, the blastocyst rate from the cleaved embryos (51% versus 37% and 27%), the total number of cells per blastocyst (135+/-4.1 versus 116+/-3.6 and 101+/-4.2), and the percentage of Grade-A (excellent quality) blastocysts (54% versus 42% and 29%) in the hybrid group were all higher than that of Holstein and Yellow groups. Furthermore, the proportion of blastocysts obtained at Day 7 (as a percentage of the total number of blastocysts) was greater in the hybrid group than in Holstein and Yellow groups (89% versus 71% and 63%). In conclusion, the use of F1 hybrid oocytes as recipient cytoplasm significantly improved in vitro development of cloned bovine embryos relative to oocytes derived from the parental lines.     

Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations

This study was conducted to improve a porcine somatic cell nuclear transfer (SCNT) technique by optimizing donor cell and recipient oocyte preparations. Adult and fetal fibroblasts, and cumulus and oviduct cells were used as donor cells, and in vivo- and in vitro-matured oocytes were employed as recipient oocytes. The percentages of fusion and development to the blastocyst stage, the ratio of blastocysts to 2-cell embryos, and cell number of blastocysts were monitored as experimental parameters. In Experiment 1, donor cells of four different types were transferred to enucleated oocytes matured in vitro, and more (P < 0.05) blastocysts were derived from SCNT of fetal fibroblasts than from that of other cells (15.9% versus 3.1-7.9%). For SCNT using fetal fibroblasts, increasing the number of subcultures up to 15 times did not improve developmental competence to the blastocyst stage (12.2-16.7%). In Experiment 2, fetal fibroblasts were transferred to enucleated oocytes that matured in vivo or in vitro. When parthenogenetic activation of both types of oocytes was conducted as a preliminary control treatment, a significant increase in blastocyst formation was found for in vivo-matured compared with in vitro-matured oocytes (36.4% versus 29.5%). However, no improvement was achieved in SCNT using in vivo-matured oocytes. In conclusion, the type of donor somatic cell is important for improving development after porcine SCNT, and fetal fibroblasts were the most effective among examined cells. A system with good reproducibility has been established using fetal fibroblasts as the donor karyoplast after subculturing 1-10 times, and using both in vivo and in vitro-matured oocytes as the recipient cytoplast.     

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.     

Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.     

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.     

Heterogeneous nuclear-transferred-embryos reconstructed with camel (Camelus bactrianus) skin fibroblasts and enucleated ovine oocytes and their development H-M

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and ovine oocytes as recipient cytoplasts for investigating the developmental potential of the reconstructed embryos. Serum-starved adult camel skin fibroblast cells were used as donor somatic cells. Ovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electrofusion. The fused oocytes were activated by 5mM/ml inomycin with 2mM/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168h. A total of 300 enucleated ovine oocytes were available for xenonuclear embryo reconstruction. The results showed that 71% of the nuclear transfer couplets were successfully fused, 55% of the fused oocytes cleaved within 48h after activation, 82% of the cleaved oocytes developed to 2-16-cell embryo stages and 18% of the cleaved nuclear transfer zygotes developed to the morula stage. This study demonstrated that the xenonuclear transfer camel embryos can undergo the first embryonic division and subsequent development to morula stage in vitro.     

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.     

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.     

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.     

Birth of viable female dogs produced by somatic cell nuclear transfer

Since the only viable cloned offspring born in dogs was a male, the purpose of the present study was to produce female puppies by somatic cell nuclear transfer (SCNT). Adult ear fibroblasts from a 2-month-old female Afghan hound were isolated and used as donor cells. In vivo-matured canine oocytes surgically collected (approximately 72h after ovulation) from the oviducts of 23 donors were used for SCNT. After removal of the cumulus cells, oocytes were enucleated, microinjected, fused with a donor cell, and activated. A total of 167 reconstructed SCNT embryos were surgically transferred (Day 0) into the oviducts of 12 recipient bitches (average 13.9 embryos/recipient, range 6-22) with spontaneous, synchronous estrous cycles. Three pregnancies were detected by ultrasonography on Day 23, maintained to term, and three healthy female puppies (520, 460, and 520g), were delivered by Caesarean section on Day 60. These puppies were phenotypically and genotypically identical to the cell donor. In conclusion, we have provided the first demonstration that female dogs can be produced by nuclear transfer of ear fibroblasts into enucleated canine oocytes.     

Heterogeneous nuclear transfer embryos reconstructed by bovine oocytes and camel (Camelus bactrianus) skin fibroblasts and their subsequent development

Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.     

Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries

For production of viable somatic cell nuclear transferred (SCNT) miniature pig embryos, in vitro condition for controlling the quality of recipient oocytes derived from domestic pig ovaries should be evaluated. In the present study, to get information on optimal in vitro maturation (IVM) condition of oocytes, we investigated the effect of IVM duration of recipient oocytes on subsequent development of SCNT miniature pig embryos, the maturation-promoting factor (MPF) activity in recipient oocytes before and after SCNT, and the occurrence of premature chromosome condensation (PCC) and spindle morphologies of donor nuclei following SCNT. The optimal window of the IVM period in terms of in vitro developmental ability of SCNT embryos was determined to be 36-40 h after the start of IVM. The use of recipient oocytes matured for 36 and 40 h resulted in a high level of MPF activity before and after SCNT, and increased the occurrence of PCC in transferred nuclei compared to the use of oocytes matured for 44 and 52 h. The proportion of abnormal spindle-like structures increased as the IVM period was prolonged. In addition, SCNT embryos constructed from recipient cytoplasts obtained after 40 h of maturation by using fetal fibroblasts of miniature pigs were transferred to surrogate miniature pigs, and developed to full term. These results suggest that recipient oocytes matured for 36 h and 40 h effectively induce PCC with a normal cytoskeletal structure because of a high level of MPF activity; furthermore, the 40-h IVM period improves in vitro development of SCNT embryos to the blastocyst stage, resulting in the production of viable cloned miniature pigs.     

Factors influencing chromosome condensation and development of cloned rat embryos

Factors influencing premature chromosome condensation (PCC) in transferred rat nuclei have been examined. Chromosome condensation of rat cumulus cell nuclei did not occur when the cell nuclei were injected into enucleated rat oocytes. By contrast, chromosome condensation did occur after transfer to enucleated mouse oocytes or intact rat oocytes. In the first serial NT experiment, rat somatic cell nuclei were injected into enucleated mouse oocytes, and the reconstructed oocytes were activated by strontium chloride. From these reconstructed embryos, karyoplasts containing pronucleus-like vesicles were transferred into pronuclear zygote-derived cytoplasts by a DC pulse. Transfer of a total of 340 serial NT zygotes into recipient females, including 206 two-cell embryos, resulted in only seven implantation sites. In the second serial NT experiment, rat somatic cell nuclei were injected into intact rat oocytes; the recipient metaphase-plate was then aspirated under UV light from the NT oocytes in which PCC of injected nuclei was observed. After activation of the NT oocytes, karyoplasts were introduced into zygote-derived cytoplasts. Transfer of a total of 115 serial NT zygotes, including 37 two-cell embryos, resulted in four implantation sites but no live offspring. These results establish a mean of inducing chromosome condensation in rat oocytes and demonstrate that reconstructed rat zygotes can be prepared by serial NT procedures. Developmental competence of these embryos remains to be clarified.     

Bovine nuclear transfer using fresh cumulus cell nuclei and in vivo- or in vitro-matured cytoplasts

We examined the effects of the source of recipient oocytes and timing of fusion and activation on the development competence of bovine nuclear transferred (NT) embryos derived from fresh cumulus cells isolated immediately after collection by ovum pickup (OPU). As recipient cytoplasts, we used in vivo-matured oocytes collected from hormone-treated heifers by OPU, or in vitro-matured oocytes from slaughterhouse-derived ovaries. NT embryos were chemically activated immediately (simultaneous fusion and activation, FA) or 2 h (delayed activation, DA) after fusion. When in vitro-matured oocytes were used as recipient cytoplasts, the development rate to the blastocyst stage of NT embryos produced by the DA method (23%) tended to be higher than those by the FA method (15%), but the difference was not significant. NT embryos derived from in vivo-matured cytoplasts have a high blastocyst yield (46%). Pregnancy rate at day 35 did not differ with the timing of fusion and activation (FA vs. DA; 50% vs. 44%) or oocyte source (in vivo- vs. in vitro-matured; 50% vs. 44%). Subsequently, the high fetal losses (88% of pregnancies) were observed with in vitro-matured cytoplasts, whereas no abortions were observed in NT fetuses from in vivo-matured cytoplasts. A total of three embryos derived from fresh cumulus cells developed to term. However, all three cloned calves were stillborn. These results indicate that improvement of development competence after NT is possible by using in vivo-matured oocytes as recipient cytoplasts in bovine NT.     

Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation

We conducted this study to examine whether serum starvation in culture contributes to better development of bovine reconstructed oocytes and to evaluate which serum-starved somatic cell is the most effective for cloned calf production. In Experiment 1, donor cells of four different types (cumulus cells, ear fibroblasts, oviduct cells and uterine cells) were either serum-starved or not before fusion with enucleated oocytes, and reconstructed oocytes were further cultured for 168 h. Regardless of serum starvation, cumulus cells or ear fibroblasts yielded higher (P < 0.05) rates of fusion than other cells (62.6-69.3 versus 33.3-38.7%). In the serum-starved group, the first cleavage after reconstruction was significantly increased in cumulus cells and ear fibroblasts, compared with oviduct cells (93.4-94.3 versus 78.8-86.0%), and oocytes reconstructed with either of these yielded more blastocysts than oocytes reconstructed with oviduct or uterine cells (40.6-43.8 versus 20.3-19.0%). We observed a similar pattern in the non-starved group, but we found a significant increase in blastocyst formation was found only in cumulus cells compared with other donor cells (42.6 versus 15.4-27.7%). Overall comparison showed that serum starvation increased the rates of cleavage and development to the blastocyst stage, but we found a statistical significance only in the cleavage rate (80.0 versus 89.5%). In Experiment 2, we transferred randomly selected 59 blastocysts that were developed from oocytes reconstructed with serum-starved cells to 44 synchronised recipients. Of those recipients, 23 became pregnant on Day 60 after transfer (52.3%) and 12 (27.3%) delivered cloned calves. The mean gestation length and birth weight was 275 +/- 8 days and 39.6 +/- 15.6 kg, respectively. Although there was no significant difference among donor cells, blastocysts that were derived from oocytes reconstructed with ear fibroblasts yielded the highest rates of pregnancy (50.0%) and delivery (27.3%). In conclusion, serum starvation is effective for improving preimplantation development of oocytes reconstructed with cumulus or ear fibroblast cells and it may positively influence on obtaining better pregnancy outcome.     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。     

Simplified cryopreservation of porcine cloned blastocysts

Recently, a non-invasive delipation (lipid removal) method combined with ultrarapid vitrification has been used successfully for in vitro produced (IVP) porcine embryos. In the present study, this method was combined with parthenogenesis and a recent form of somatic cell nuclear transfer (SCNT) - handmade cloning (HMC) - to establish a simplified and efficient cryopreservation system for porcine cloned embryos. In Experiment 1, zonae pellucidae of oocytes were partially digested with pronase, followed by centrifugation to polarize lipid particles. Ninety percent (173/192) oocytes were successfully delipated in this way. Parthenogenetic activation (PA) after complete removal of zona resulted in similar blastocyst rates in delipated vs. control oocytes (28+/-7% vs. 28+/-5%, respectively). Subsequent vitrification of produced blastocysts with the Cryotop technique resulted in higher survival rates in the delipated group compared to the control group (85+/-6% vs. 32+/-7%, respectively; P<0.01). In Experiment 2, delipated oocytes were used for HMC with normal oocytes as control. Partial zona digestion was further applied before enucleation both in delipated and control groups, to bisect oocyte successfully. Although the blastocyst rate of reconstructed embryos was similar between groups derived from delipated vs. control oocytes (21+/-6% and 23+/-6%, respectively), after vitrification higher survival rates were achieved in the delipated groups than in controls (79+/-6% vs. 32+/-8%, respectively). Our results prove that porcine embryos produced from delipated oocytes by PA or HMC can be cryopreserved effectively by ultrarapid vitrification. Further experiments are required to assess the in vivo developmental competence of the cloned-vitrified embryos.     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。