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PubMed1996

Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows.

Animals↗
PubMed1995

Effect of coculture on subsequent survival and implantation of cryopreserved human embryos.

PURPOSE: This retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously match controls. RESULTS: During a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P < 0.05). CONCLUSION: Coculture of embryos prior to cryopreservation does not appear to improve cryosurvival; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation.

Animals↗
PubMed2005

Parents' conceptualization of their frozen embryos complicates the disposition decision.

OBJECTIVE: To ascertain what couples think about their embryos and how they approach making a decision about disposition in light of the fact that the disposition of unused frozen embryos has significant implications for medical research and embryo donation. DESIGN: Ethnographic qualitative interview study. SETTING: Academic research environment. PATIENT(S): Fifty-eight couples who had conceived using a donor oocyte and had at least one frozen embryo in storage. MAIN OUTCOME MEASURE(S): Tape-recorded interviews with 58 wives and 37 husbands were transcribed and analyzed for emergent themes. RESULT(S): With an average of 7.1 embryos per couple, after an average of 4.2 years of storage, 72% of couples with frozen embryos had not reached a disposition decision. Most couples had not anticipated or appreciated the consequences of having surplus embryos. Parents variously conceptualized frozen embryos as biologic tissue, living entities, "virtual" children having interests that must be considered and protected, siblings of their living children, genetic or psychological "insurance policies," and symbolic reminders of their past infertility. CONCLUSION(S): The disposition decision is not only a significant and frequently unresolved issue for couples with stored frozen embryos, but their deeply personal conceptualizations of their embryos contributes to their ambivalence, uncertainty, and difficulty in reaching a decision.

Adult↗
PubMed2000

Implantation rates after in vitro fertilization and transfer of a maximum of two embryos that have undergone three to five days of culture.

OBJECTIVE: To evaluate implantation and pregnancy rates in patients undergoing IVF after the transfer of a maximum of two embryos that had been cultured for 3-5 days. DESIGN: Prospective study. SETTING: An IVF laboratory at a tertiary referral university hospital. PATIENT(S): One thousand seven hundred eighty-seven couples who underwent their first IVF cycle between January 1995 and December 1997. INTERVENTION(S): In vitro fertilization and transfer of embryos after 3, 4, or 5 days of culture using a single medium without coculture. MAIN OUTCOME MEASURE(S): Implantation and pregnancy rates. RESULT(S): Overall implantation and pregnancy rates were not significantly different with different culture periods. Forty-one percent of all available embryos developed into blastocysts on day 5. The transfer of at least one good-quality blastocyst could be performed in 62% of patients. Blastocysts had an implantation rate of 26% per embryo, whereas the implantation rate of eight-cell embryos on day 3 was 18%. Implantation rates for retarded, normal, and advanced embryos were not significantly different with an extended culture period. CONCLUSION(S): Under the study conditions, the transfer of embryos after 5 days rather than 3 days of embryo culture did not change the overall implantation and pregnancy rates. The implantation potential of embryos available for transfer can be assessed better after an extended culture period. Five days of culture allows the transfer of a reduced number of embryos without decreasing overall pregnancy rates.

Adult↗
PubMed1994

Rescue of human embryos by micromanipulation.

This chapter evaluates clinical micromanipulation techniques aimed at rescuing abnormally developing zygotes and cleaved embryos. First, the possibility of reversing dispermic zygotes to a normal biparental diploid state was evaluated by extracting the distal pronucleus (that furthest from the polar body). The ratio of X:Y was determined in both groups of embryos by assessing a minimum of two blastomeres using duplex PCR or multiple colour FISH. The ratio of embryos containing only an X chromosome and those with X as well as Y chromosomes in the intact dispermic zygotes was 1.0:2.6, which is similar to the theoretical ratio of 1:3. This ratio was 1.0:1.5 in dispermic zygotes from which the distal pronuclei were removed. Although the ratio of X:Y was altered following removal of distal pronuclei, suggesting frequent targeting of male pronuclei, accidental removal of the female pronucleus could not be excluded. In a second set of mouse experiments, it was shown that the hatching process of embryos that develop with excessive amounts of degenerate material is adversely affected. It was shown that removal of such extracellular material by micromanipulation potentially reverted the hatching process. It was also indicated that immediate removal of the degenerate tissue was more beneficial than that following prolonged co-culture. Assisted hatching is probably the most frequently applied clinical embryo micromanipulation procedure. The outcome of assisted hatching is dependent largely on the mode by which the zona pellucida is breached, the size of the artificial gap and the thickness of the zona pellucida. Embryos with zonae thicker than 17 microns rarely implant. Zona drilling could be detrimental in embryos with thin zonae (< 12 microns). Superficial zona thinning has not enhanced implantation. These observations led to a routine procedure called selective assisted hatching, which involves measuring the zonae before zona drilling and replacement on day 3 of development. This appears to be most successful in older women and those with elevated basal FSH levels. Selective assisted hatching is routinely applied in consenting patients whose embryos have thick zonae, slow development or excessive fragmentation (> 20%). Zona drilling of all embryos, regardless of zona thickness, is being performed in patients aged over 40 years and in those with repeated failures or elevated basal FSH levels. Results in the first group of more than 900 patients indicate that nearly one-quarter of human embryos have the ability to implant.

Animals↗
PubMed2001

Surrogacy in Australia: implantation rates have implications for embryo quality and uterine receptivity.

Since the passage, in November 1995, of the ACT Substitute Parents Agreement Act, The Canberra Fertility Centre has added a true gestational carrier pregnancy programme to its established infertility and IVF services. Embryos generated are transferred as frozen-thawed embryos to the carrier in an average of 2.2 embryos per transfer. Between 1 January 1996 and 31 December 1999 the results of 49 frozen embryo transfers to 25 gestational carriers were compared with 849 frozen embryo transfers on a routine IVF programme. In the carrier group, the embryo implantation rate of 13.8% per embryo transferred is double that of an exactly comparable group of patients undergoing routine frozen-thawed embryo transfer on the same IVF programme and considerably higher than those reported in large series of frozen-thawed embryo transfers. Exclusion from the carrier pregnancy programme of patients with incipient ovarian failure results in an implantation rate of 16.7%, a clinical pregnancy rate of 29.0% and a live birth rate of 19.4% per embryo transfer procedure.

Adult↗
PubMed2006

Metabolic regulation of in vitro-produced bovine embryos. II. Effects of phenazine ethosulfate, sodium azide and 2,4-dinitrophenol during post-compaction development on glucose metabolism and lipid accumulation.

The objective was to compare effects of three metabolic regulators on development of post-compaction bovine embryos. In-vitro-produced 8- to 16-cell embryos were allocated to treatments for 72 h in G2.2 medium as follows: 0.3 microm phenazine ethosulfate (PES); 27 microm sodium azide (NaN3); 30 microm 2,4-dinitrophenol (DNP); and control, no regulator. Treatments responded similarly for blastocyst rates and embryo quality responses (P > 0.1). The PES treatment resulted in higher glucose metabolism than the NaN3 treatment (18.5 v. 14.5 pmol per embryo per h, P < 0.05), and both did not differ from DNP or the control. The PES treatment tended to result in more flux of glucose through the pentose phosphate pathway (PPP) than the control (50.5 v. 21.5%, P < 0.11). The NaN3 treatment caused more glucose uptake than the PES treatment (38.9 v. 13.1 pmol per embryo per h, P < 0.01), but neither differed from the control or DNP treatment (P > 0.1). Glycolysis for the PES treatment was 187%, which was higher than any of the other groups (88-94%; P < 0.01). There were fewer medium + large lipid granules in the cytoplasm of PES-treated embryos than any other group, including the in vitro control (P < 0.01). However, in vivo control embryos had still fewer large and medium-sized lipid granules (P < 0.01) than the PES treatment. Developmental competence to Day 14 after embryo transfer was similar among treatments. The PES treatment increased glucose metabolism, tended to increase the PPP flux of glucose and clearly reduced accumulation of lipids in embryos produced in the chemically defined media used. Use of PES in culture media may be a promising approach to improving in vitro production of embryos.

2,4-Dinitrophenol↗
PubMed1998

Comparison of clinical outcome after cryopreservation of embryos obtained from intracytoplasmic sperm injection and in-vitro fertilization.

The impact of intracytoplasmic sperm injection (ICSI) on cryopreserved zygotes and embryos was evaluated by comparing embryo survival and implantation between embryos derived from ICSI and those derived from standard insemination procedures. The study included patients whose excess zygotes and embryos were cryopreserved between September 1993 and December 1994 and who subsequently underwent a frozen embryo transfer. Embryo survival, clinical pregnancy rates per transfer and pregnancy outcome were compared. Three hundred and thirty eight cryopreservation cycles, during which 1471 embryos were cryopreserved, were included in this study. Of those, 961 were derived from oocytes fertilized by insemination in vitro and 510 were derived from oocytes fertilized by ICSI. A total of 690 of the embryos (451 in the insemination group and 239 in the ICSI group) have since undergone a thaw cycle. The embryo survival rates were similar between the two groups (70.5 and 73.2%, insemination and ICSI respectively) and were not significantly affected by the stage at cryopreservation. There was no significant difference in pregnancy rates per transfer (31.8 and 32.3%), the preclinical pregnancy loss rate (16.7 and 23.8%), or the clinical miscarriage rate (16.7 and 23.8%) between the insemination and the ICSI groups respectively. It is concluded that ICSI does not have an adverse impact on the survival and successful implantation of cryopreserved and thawed embryos.

Cryopreservation↗
PubMed2000

Comparison between day-2 embryos obtained either from ICSI or resulting from short insemination IVF: influence of maternal age.

Short incubation time prevents deleterious effects of cumulus cell degeneration and excess spermatozoa in IVF embryos. We performed a short incubation (3 h) protocol in 328 IVF cycles, in order to compare the developmental potential of regular IVF embryos with those originating from 316 cycles entered our intracytoplasmic sperm injection (ICSI) programme over the same period. Embryo transfers were performed in all patients on day 2. The mean number of embryos transferred was 1.92 for the ICSI group and 1.73 for the IVF group (P < 0.007). This was related only to the wishes of patients. However, the policy of the centre is to transfer a low number of embryos in young patients in order to avoid multiple pregnancies. All spare embryos were permitted to grow to the blastocyst stage for freezing. Shortening incubation time did not decrease fertilization rates. In our overall population, no difference was observed in the implantation rates per embryo for IVF (19%) or for ICSI (20%). An age-related decrease in embryo production was observed for both groups of patients (P < 0.01 for ICSI and P < 0.001 for IVF). The age-related decrease in embryo implantation was only significant for the IVF group (P < 0.03 for patients <30 and >35 years of age). A significant overall decrease in blastocyst formation was observed for spare embryos after ICSI versus IVF (34.2 versus 43.8%; P < 0. 05). The significance of this observation is discussed.

Adult↗
PubMed2001

The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality.

BACKGROUND: Recent studies have shown that zygote morphology could be used for the assessment of human embryo quality. Pronuclear (PN) morphology is based on certain distinct features seen in zygotes 16-18 h after fertilization. In the present study PN stage morphology was assessed and combined with a single embryo transfer in order to investigate whether currently used zygote classifications are able to predict embryo quality and implantation rates. METHODS AND RESULTS: Zygotes were analysed according to two different classification systems. In the first, a total of 764 zygotes was analysed according to the degree of polarization of nucleolar precursor bodies (NPB). Zygotes with unpolarized PN (i.e. scattered localization of NPB) showed significantly slower (P < 0.005) cleavage rates (38.9%) than zygotes having at least one pronucleus polarized (57.3% and 54%). However, there was no difference in the pregnancy rate in 105 single embryo transfers between the groups. The appearance of a cytoplasmic halo was related to embryo morphology. Embryos derived from halo-positive zygotes had significantly better (P < 0.05) morphology (60.9%) compared to halo-negative derived embryos (52.2%), but in terms of pregnancy rates no difference was found. A total of 1520 zygotes was analysed according to a second classification system, which was based on the number and distribution of NPB. In the comparative analysis, none of the six different classes produced superior quality embryos or higher pregnancy rates in 144 single embryo transfers. CONCLUSIONS: Our results indicate that there are no significant differences in embryo quality or implantation/pregnancy rates between proposed zygote classes.

Cell Nucleolus↗
PubMed2001

The effect of pronuclear morphology on embryo quality parameters and blastocyst transfer outcome.

BACKGROUND: Embryo quality may be accurately assessed as early as the pronuclear zygote phase, as shown in recent studies. However, it is not known whether good quality zygotes are destined to become good quality cleavage stage embryos and blastocysts. METHODS: In this retrospective study, 86 intracytoplasmic sperm injection-embryo transfer cycles were studied where each available embryo was scored from the zygote until the blastocyst stage. Embryonic normality parameters such as pronuclear pattern, early cleavage, cleavage stage embryo grade, the presence of embryos with > or =8 cells on day 3 and blastocyst quality were recorded. Embryo transfer was undertaken at the blastocyst stage and the outcome was studied according to the pronuclear pattern exhibited by the zygotes. RESULTS: Embryos that showed an ideal pronuclear pattern (0 PN pattern) cleaved earlier and faster and resulted in better quality cleavage stage embryos and blastocysts. The incidence of blastocyst formation was 72% in zygotes showing a 0 PN pattern, compared with 12.7% in zygotes with double pronuclear abnormality. Higher implantation and pregnancy rates were obtained when at least one blastocyst derived from a 0 PN pattern zygote was included in the set of embryos to be transferred. CONCLUSIONS: Our results indicate that the pronuclear pattern of the zygote is closely related to blastocyst formation and quality. Blastocysts derived from 0 PN zygotes have a higher potential for implantation.

Blastocyst↗
PubMed1996

Influence of "Solcoseryl" during culture on the sex-dependent repair of bovine demi-embryos.

The purpose of this experiment was to determine the effect of culture conditions on the development of split embryos after bisection and on the sex ratio of resultant bovine demi-embryos. Embryos that had developed into blastocysts on days 6 1/2 to 7 or on days 7 1/2 to 8 from oocytes matured and fertilized in vitro were bisected in BMOC-3 medium supplemented with 33% calf serum. The medium also contained 0%, 0.1% or 1.0% Solcoseryl, a deproteinized hemodialysate product from calf blood. The demi-embryos were first cultured for 4 hours in the same medium in which they had been bisected and then co-cultured with cumulus cells in TCM199 supplemented with 1% calf serum for an additional 20 hr. The rate of production of good to excellent quality demi-embryos obtained from days 6 1/2 to 7 blastocysts was higher than from those on days 7 1/2 to 8. The rate was also significantly improved when blastocysts were bisected in medium containing 0.1% or 1.0% Solcoseryl, compared to the medium without Solcoseryl. Male embryos seemed to recover more rapidly than female embryos, as assessed by morphological quality at 4 hr, although the quality of female embryos had improved by 24 hr. The percentage of males after culture was higher in the medium without Solcoseryl than in its presence. Thus, addition of Solcoseryl at either 0.1% or 1.0% to BMOC-3 medium seemed to improve the production efficiency of good quality demi-embryos, but did not influence the sex ratio. It appears as if female demi-embryos required more time than male embryos to be repaired after bisection.

Actihaemyl↗
PubMed1996

Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media.

Data from other laboratories have shown that speed of bovine blastocyst development is higher when Ménézo B2 is used for coculture compared to TCM199. It was our purpose to investigate whether this early blastocyst formation was also indicative of embryo quality by studying the allocation of inner cells in embryos generated by B2-coculture and by TCM199-coculture. For this purpose, a differential staining technique was used. General embryo development was similar for TCM199- and B2-embryos expressed as rate of cleavage at day 3 and morula-blastocyst formation at day 8 (P > 0.05), but significantly different when expressed as number of eight-cell stages at day 3 and expanded or hatched blastocysts at day 8 (P < 0.01). B2-embryos cultured until day 5, 6, and 7 post insemination, had total cell numbers of 24, 65, and 109 respectively, which was significantly higher than the cell number of TCM199-embryos cultured over the same time period (18, 41, and 71 respectively, P < 0.001). Morphological differentiation was significantly more advanced for B2-embryos at day 7 and 8 (P < 0.0001 and P < 0.001, respectively). First presumptive inner cells appeared in eight- to 16-cell stages at day 3. Because the determination of inner cells by differential staining is depending upon the presence of functional tight junctions, we concluded that the establishment of the tight junction seal in B2-embryos differed from that in TCM199-embryos: Inner cells appeared 0.56 cell cycle later in B2-embryos (P < 0.001) and a larger variation existed in the number of ICM-cells in B2-blastocysts (P < 0.001). The higher total cell number of B2-expanded blastocysts was mainly acquired by trophectoderm growth (P < 0.06). These data indicate that the apparent better quality of B2-embryos (faster cleavage, earlier blastocyst formation) is not reflected in a reliable number of inner cells of B2-blastocysts.

Animals↗
PubMed2000

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism.

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.

Animals↗
PubMed1995

Mouse half embryos: viability and allocation of cells in the blastocyst.

We have investigated the developmental capacity of mouse embryos in which one blastomere was destroyed by lysis at the 2-cell stage. The allocation of cells to the trophectoderm and inner cell mass (ICM) was documented by differential cell counts on single embryos after 2 days under different culture conditions. Viability and further developmental potential were tested by embryo transfer to foster mothers. The conditions used were: (1) in vitro culture in modified BMOC-2 medium, (2) in vivo oviduct transfer to immature (prepuberal) females, and (3) in vivo oviduct transfer to pseudopregnant females. Half embryos almost always fared less well for all parameters of development than control embryos developing under the same conditions. Lower total cell numbers in half embryos were accounted for by decreases in both ICM and trophectoderm with a disproportionate decrease in ICM in smaller embryos. In both half and control embryos, the growth conditions affected the rate of morphological development, the total cell number, and embryo viability. Unlike the effect of halving embryos, the growth condition effects on total cell number can be accounted for primarily by differences in ICM cell number, with trophectoderm cell number remaining constant. These results provide new information on the ability of the mouse embryo to differentially regulate ICM and trophectoderm cell number under different conditions, and confirm our previous work showing the advantage of short-term development in vivo over short-term in vitro culture (Papaioannou and Ebert [1986] J. Reprod. Fertil. 76:603-608).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Development of porcine embryos from the one-cell stage to blastocyst in mouse oviducts maintained in organ culture.

Successful development of porcine embryos from the one-cell stage to the blastocyst stage has been accomplished using mouse oviducts in organ culture. One-cell embryos were transferred to mouse oviducts maintained in organ culture and were cultured for 6 days. Control embryos from each donor pig were cultured in a modified Krebs-Ringer bicarbonate medium. Thus control and experimental embryos obtained from the same individual pig could be directly compared. At the end of the culture period, all embryos were scored for the stage of development attained and stained to allow the cell number of each embryo to be counted. In medium alone, only 35.7% of the one-cell embryos reached the morula or blastocyst stage, whereas 78.1% of the one-cell embryos transferred to mouse oviducts reached the morula or blastocyst stage. Of those embryos reaching the morula or blastocyst stage, cell numbers were similar for the two treatments (medium alone vs. oviduct culture). The procedure described for mouse oviduct organ culture provides a simple method for culturing early-stage pig embryos to the morula or blastocyst stage prior to embryo transfer.

Animals↗
PubMed1991

Expression of an antigen specific for trunk lateral cells in quarter embryos of the ascidian, Halocynthia roretzi.

Cell-lineage analysis has demonstrated that a pair of the right and left A7.6 cells of a 64-cell embryo of the ascidian Halocynthia roretzi, descendants of A4.1 cells of an 8-cell embryo, give rise to trunk lateral cells (TLCs). In this study, in order to investigate cellular mechanisms involved in the specification of TLCs, we have examined the expression of a TLC-specific antigen in cleavage-arrested embryos and in quarter partial embryos. Although cleavage arrest of embryos by treatment with cytochalasin B at early stages, prior to and including the 16-cell stage, inhibited expression of the TLC-specific antigen, embryos arrested at the 32-cell stage and at later stages developed the antigen. The only blastomeres exhibiting expression of the antigen were the presumptive TLCs, as predicted by cell-lineage assignments. When the developmental potential of quarter embryos that originated from four isolated blastomere-pairs (a4.2, b4.2, A4.1, and B4.1 pairs) of an 8-cell embryo was examined, the A4.1 quarter embryos, which are developmentally fated to give rise to TLCs, rarely showed evidence of expression of the antigen. Expression of the antigen was not observed in a4.2 and b4.2 quarter embryos, which are not associated with the TLC fate. By contrast, expression of the antigen was detected in about a half of the B4.1 quarter embryos which are also not associated with the TLC fate. These results are discussed with reference to the relationship between TLCs and mesenchyme cells.

Animals↗
PubMed2005

Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro.

The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.

Animals↗