Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p < 0.05) percentage of blastocyst development was observed in the NT embryos activated by calcium ionophore and 6-DMAP when compared with 6-DMAP alone (33% versus 17%). The results indicate that the somatic nuclei from buffalo can be reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.     

Development of embryos reconstructed by interspecies nuclear transfer of adult fibroblasts between buffalo (Bubalus bubalis) and cattle (Bos indicus)

The objective of this study was to explore the feasibility of employing adult fibroblasts as donor cells in interspecies nuclear transfer (NT) between buffaloes and cattle. Buffalo and bovine oocytes matured in vitro for 22 h were enucleated by micromanipulation using the Spindle View system. An ear fibroblast, pretreated with 0.1 microg/mL aphidicolin for 24 h, followed by culture for 2-9 days in Dulbecco's Modified Eagle's Media+0.5% fetal bovine serum, was introduced into the cytoplast by microinjection. Reconstructed oocytes were activated by exposure to 5 microM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h. When buffalo adult fibroblasts were used as donor cells, there were no differences (P < 0.75) in the cleavage rate (66.2% versus 64.0%) between bovine and buffalo recipient oocytes, but more embryos derived from bovine cytoplasts developed to blastocysts than from buffalo cytoplasts (13.3% versus 3.0%, P < 0.05). When bovine adult fibroblasts were used as donor nuclei, both cleavage rate (45.3%) and blastocyst yield (4.5%) of NT embryos derived from buffalo cytoplasts were lower than those of NT embryos derived from bovine cytoplasts (65.5 and 11.9%, P < 0.05). The proportion of parthenogenetic buffalo (29.1%) or bovine (35.6%) oocytes developing to blastocysts was higher than those of NT embryos (P < 0.01). Interspecies NT embryos were derived from the donor cells and 55.0-61.9% of them possessed a normal diploid karyotype. In conclusion, embryos reconstructed by interspecies NT of adult fibroblasts between buffaloes and cattle developed to blastocysts, but bovine cytoplasts may direct embryonic development more effectively than buffalo cytoplasts, regardless of donor cell species.     

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.     

Cloned endangered species takin (Budorcas taxicolor) by inter-species nuclear transfer and comparison of the blastocyst development with yak (Bos grunniens) and bovine

Interspecies cloning might be used as an effective method to conserve endangered species and to support the study of nuclear-cytoplasm interaction. In this study, we describe the development of takin-bovine embryos in vitro produced by fusing takin ear fibroblasts with enucleated bovine oocytes and examine the fate of mitochondrial DNA in these embryos. We also compare the blastocyst development of takin-bovine embryos with yak-bovine and bovine-bovine embryos and compare the cell numbers of the blastocyst. Our results indicate that: (1) takin-bovine cloned embryos can develop to the blastocyst stage in vitro (5%), (2) blastocyst mitochondria DNA are derived primarily from bovine oocytes in spite of a little takin donor cell mitochondrial DNA, (3) using the same cloned protocol, development efficiency is significantly different between bovine-bovine cloning, yak-bovine, and takin-bovine cloning (48 vs. 28% vs. 5%, P < 0.01), and (4) cell numbers in the blastocysts of the three species of embryos were not different. These results suggest that the bovine oocytes can reprogram the takin, yak, and bovine fibroblast nuclei. However, the development efficiency of intra-species cloning tends to be higher than inter-species cloning; the more close the species of the donor cell is to the recipient oocyte (yak versus takin), the greater the blastocyst development in vitro.     

in vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells

In the pig little information is available on cytoplasmic events during the reprogramming of oocytes reconstructed with somatic nuclei. The present study was conducted to determine the developmental potential of porcine cumulus cells (CC) and fetal fibroblasts (FF) after they were transferred into enucleated oocytes. Non-quiescent FF were fused to the enucleated oocytes using electrical pulse, whereas CC were directly injected into the oocytes. Transferred nuclei from both CC and FF underwent premature chromosome condensation (PCC), nuclear swelling and pronucleus formation. The remodeled oocytes developed to the mitotic and 2-cell stage at 18 to 24 h after nuclear transfer. The pattern of nuclear remodeling was similar regardless of the sources of karyoplasts or nuclear transfer methods. However, using FF, 24% of nuclear transferred embryos developed to the morula or blastocyst stage, whereas only 8% of those using CC developed to the morula or blastocyst stage. These results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of nuclear transferred embryos to the blastocyst stage.     

Development of swamp buffalo (Bubalus bubalis) embryos after parthenogenetic activation and nuclear transfer using serum fed or starved fetal fibroblasts

The knowledge of oocyte activation and somatic cell nuclear transfer in the swamp buffalo (Buballus bubalis) is extremely rare. The objectives of this study were the following: (1) to investigate the various activation treatments on the parthenogenetic development of buffalo oocytes, (2) to examine the events of nuclear remodeling and in the in vitro development of cloned buffalo embryos reconstructed with serum fed or starved fetal fibroblasts, and (3) to investigate the in vivo development of cloned embryos derived from serum fed or starved cells after transfer into the recipients. The rates of cleavage and blastocyst development were found to be significantly higher (P < 0.05) when the oocytes were activated by the combination treatment of calcium ionophore (A23187) or ethanol followed by 6-DMAP than those activated by electrical pulses and 6-DMAP or other single treatments. Flow cytometric analysis revealed that the percentage in the G0/G1 phase in serum starved cells was significantly (P < 0.05) higher than that in serum fed cells (88.8 +/- 6.2 vs. 68.2 +/- 2.6). At 1 h post fusion (hpf), most of the transferred nuclei (71%) from serum fed cells did not change in size, and the nuclear envelope remained intact, whereas 29% underwent NEBD and PCC. When serum starved cells were used, 83% of the transferred nuclei underwent NEBD and PCC whereas 17% remained intact. The nuclear swelling and pronucleus (PN) formation were observed at 2-4 and 12 h post activation (hpa), respectively. The remodeled nuclei underwent mitotic division and developed to the 2-cell stage within 18-24 hpa. Fifty-five percent of oocytes reconstructed with serum fed cells were 2PN and 45% were 1PN, whereas 79% of the embryos reconstructed from starved cells were 1PN and 21% were 2PN. The percentage of blastocyst development of the embryos derived from starved cells was higher than that from the serum fed cells (35% vs. 21%, P < 0.05). Pregnancy was detected after the transfer of cloned blastocysts into the recipients but no recipients supported the development to term. The results of this work can be used to establish effective activation protocols for buffalo oocytes which can be used during nuclear transfer experiments.     

Genetic and environmental determinants of interferon-tau secretion by in vivo- and in vitro-derived bovine blastocysts

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-tau secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo's ability to develop to the blastocyst stage and of subsequent interferon-tau secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-tau. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-tau was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-tau by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.     

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.     

Production of cloned bovine embryos by somatic cell transfer into enucleated zona-free oocytes

Cloned bovine embryos were produced at the blastocyst stage. Prior to enucleation, oocytes were freed from the zona pellucida. Fibroblasts isolated from the bovine fetus were used as nuclear donors. Pairs of fetal fibroblasts and enucleated oocytes (cytoplasts) were glued in phytohemagglutinin solution under a binocular microscope. The subsequent electrofusion of 39 fetal fibroblast-cytoplast pairs yielded 36 reconstructed one-cell embryos (92.3%). After culturing in synthetic oviduct fluid for 7.5 days, seven cloned embryos developed to the blastocyst stage (19.4%) and six blastocysts were considered fit for transplantation. The applied technique of bovine embryo growth allowed 31.1% zona-free oocytes parthenogenetically activated by to reach the blastocyst stage.     

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.     

Cloned piglets born after nuclear transplantation of embryonic blastomeres into porcine oocytes matured in vivo

Nuclear transplantation in the pig is more difficult than in other domestic animals and only one embryonic nuclear transplantation (NT) pig has been born to date. In this study, reconstituted porcine embryos were produced by electrofusion of blastomeres from in vivo four-cell embryos to enucleated in vivo or in vitro matured (IVM) oocytes. Nuclear transfer using cumulus cells as nuclear donors was also conducted. When blastomeres were used as donors, the electrofusion rate was significantly higher in oocytes matured in vivo (91.5%) than in those matured in vitro (66.1%) (p < 0.01). After fusion, the NT embryos reconstituted from in vivo matured oocytes developed to blastocysts at a rate of 10.3% after culture in rabbit oviducts for up to 5 days, while only 5.9% of the NT embryos reconstructed from in vitro matured oocytes developed to blastocyst stage. Electrofusion rate of cumulus cell nuclei with enucleated IVM oocytes was lower (47.6%) and only 1.5% (2/136) of the reconstituted eggs developed in vitro to morula stage, and 1.9% developed to blastocysts when cultured in the ligated rabbit oviducts. Transfer of 94 embryos reconstructed by blastomere NT with in vivo matured oocytes to five synchronous recipients resulted in the birth of two cloned piglets. No piglet was born following transfer to two recipients of embryos (n = 39) derived from NT with in vitro matured oocytes. The results demonstrate that in vivo matured oocytes are better recipients than those matured in vitro for pig cloning.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic 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.     

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.     

The effect of donor cell serum starvation and oocyte activation compounds on the development of somatic cell cloned embryos

Two experiments, one comparing nuclear transfer (NT) embryo activation compounds, the other donor cell treatments, were conducted with a goal of identifying factors that improve the in vitro development of cloned bovine embryos. In experiment 1, 539 NT embryos were produced by combining serum starved bovine fetal fibroblasts with enucleated in vitro matured oocytes, activated with ionomycin, then randomly allocated to be incubated for 4 hours in either Butyrolactone-I (BL-I) or 6-dimethylaminopurine (DMAP). There was no significant difference in development to blastocyst or compact morula of fused embryos at Day 6.5 between BL-I and DMAP activated embryos (22.4% vs. 20.2%; p = 0.18). Karyotyping of 20 blastocysts and compact morula from each group determined that 65% of BL1 and 63% of DMAP embryos were diploid with the remainder mixoploid (2n + 4n). In Experiment 2, the development of 389 NT embryos reconstructed from either serum starved or serum fed fetal fibroblasts was assessed. More Day 7 blastocysts and compact morula developed in the serum starved group (34.5% vs. 18.8%; p = 0.008). To verify the viability of BL-I activated embryos, 10 blastocytes from experiment 2 were transferred into 4 recipient cows. Two morphologically normal fetuses, genetically identical to the original fetal cell line, were surgically recovered at day 45 of gestation. In summary, serum starvation of bovine fetal fibroblasts prior to NT significantly improved development to blastocyst. Additionally, we have shown that BL-I is a novel alternative compound for use in combination with ionomycin to activate NT embryos.     

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.     

Pregnancies established from water buffalo (Bubalus bubalis) blastocysts derived from in vitro matured, in vitro fertilized oocytes and co-cultured with cumulus and oviductal cells

Buffalo ovaries were collected immediately after slaughter and were transported to laboratory in sterile saline at 37 degrees C. Follicular oocytes with the cumulus mass aspirated from 2 to 6 mm in diameter follicles were cultured in TCM-199 medium supplemented with 10% buffalo estrus serum (BES) in 5% CO(2) at 38.5 degrees C. After 20 to 24 h of incubation, the oocytes were inseminated with precapacitated frozen thawed spermatozoa for 6 h. The fertilization rate was 78.15% of the matured oocytes. Over an in vitro culture period of 3 to 9 d, 4.02% of the inseminated oocytes developed to the morula stage when cultured with cumulus cells alone and 17.83% when cumulus cells plus oviductal epithelial cells were used. The percentage of developed blastocysts was very low (0.57%) when the oocytes were co-cultured with cumulus cells from the original oocytes. However, 8% of the inseminated oocytes that were denuded 3 d after insemination developed to the blastocyst stage when they were co-cultured with cumulus and oviductal epithelial cells. Sixteen early/expanded blastocysts were transferred non-surgically to 16 recipients. Four of the 16 recipients became pregnant, of which 2 delivered normal buffalo male calves.     

Cell synchronization for the purposes of nuclear transfer in the bovine

We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1. Cells synchronized in G0, M, and G1 phase were transferred to enucleated bovine MII oocytes by direct injection using the Piezo-Drill microinjector. NT oocytes were artificially activated following injection. Cells at the M phase were also transferred to enucleated oocytes after artificial activation. Cells induced into quiescence by serum starvation and unsynchronized donor cells produced the highest rates of development to the morula/blastocyst stage (20% and 18%, respectively). Development to blastocyst was significantly higher in parthenogenetic controls compared to NT embryos. The transfer of M phase nuclei to MII cytoplasts was not associated with high development to the blastocyst stage. Nevertheless, determining the viability of these embryos requires transfer to recipient animals and assessment of in vivo development.     

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.     

Vitrification of bovine oocytes and its application to intergeneric somatic cell nucleus transfer

We determined the efficacy of a microdrop vitrification procedure for cryopreservation of bovine oocytes, using vitrified oocytes as cytoplasts for intraspecies and intergeneric somatic cell nucleus transfer (NT). In vitro matured bovine MII oocytes were vitrified in microdrops with a vitrification solution containing 35% ethylene glycol, 5% polyvinyl pyrrolidone, and 0.4 M trehalose. After warming, approximately 80% of the vitrified oocytes were morphologically normal, and their enucleation rate was similar to that of fresh oocytes. The NT embryos constructed with bovine cumulus cells and the vitrified oocytes developed similar to blastocysts constructed with fresh oocytes, although the cell number of NT blastocysts originating from vitrified oocytes was lower than that of the fresh control. In a second experiment, we examined the development of NT embryos constructed with vitrified bovine oocytes and bovine fibroblasts (intraspecies NT embryos) or swamp buffalo fibroblasts (intergeneric NT embryos). There were no differences between the intraspecies and intergeneric NT embryos in fusion, cleavage and development to blastocysts, except for lower cell numbers in the intergeneric NT blastocysts. In conclusion, the efficacy of this microdrop vitrification procedure and the production of swamp buffalo NT blastocysts using vitrified bovine oocytes was demonstrated.