The effect of sucrose and trehalose on viability of one- and two-cell rabbit embryos

The effect of sucrose and trehalose on the viability of one- and two-cell rabbit embryos was investigated. A significant decrease in the viability of one- and two-cell embryos exposed for 30 min. at 20 degrees C was observed. At 38 degrees C none of the two-cell embryos in a sucrose solution survived after 30 min exposure, while approximately 50% of the embryos survived in a trehalose solution. The cleveage rate in culture of two-cell embryos exposed both to 2.0 M or 1.45 M trehalose was significantly lower in comparison with the control group. However the survival rate after transfer of two-cell embryos exposed to 1.45 M trehalose solution at 20 degrees C remained the same as that of the control group.     

Optimal equilibration conditions for practical vitrification of two-cell mouse embryos

The objective of the study reported here was to elucidate the optimal equilibration conditions for carrying out vitrification of two-cell mouse embryos, using a solution containing 2M dimethyl sulfoxide, 1M acetamide, and 3M propylene glycol (DAP213) as a cryoprotectant. Embryos were subjected to an equilibration process under 20 conditions of a combination of different temperatures (10 to 37 degrees C) and times (5 to 90 sec), and viability of the embryos was assessed by the rate of development into blastocysts and into live fetuses. As a result, these rates of development into blastocysts did not differ from those for unfrozen embryos. The rate of development of frozen-thawed embryos into live fetuses under conditions of 30 sec. at 20 degrees C, which was selected as having by highest operability, was 55.2%, comparable to the value (65.0%) for unfrozen embryos. Thus, the optimal equilibration condition for vitrification of two-cell mouse embryos, using DAP213 solution, was 30 sec at 20 degrees C, under which embryo viability was maximized, and this equilibration process was considered useful as a practical two-cell embryo freezing process in the vitrification method.     

Survival of bovine embryos stored at 4 degrees C

These experiments were designed to test the efficacy of storing bovine embryos at 4 degrees C. Of particular interest were the age of embryo at which maximum post-storage survival could be achieved and longevity at 4 degrees C. A greater proportion of day 8 blastocysts developed in vitro at 37 degrees C following refrigeration for 48 hr than did embryos collected 2, 4 or 6 days after estrus (P<0.01). Survival of blastocysts stored at 4 degrees C for 48 hr was similar to that of nonstored blastocysts. In a subsequent experiment, day 8 blastocysts were recovered nonsurgically and assigned to one of the following treatments: (a) immediate transfer; (b) culture at 37 degrees C; or (c) storage at 4 degrees C for 1, 2, 3 or 5 days. Post-storage viability was assessed by either development in culture at 37 degrees C or embryo survival following nonsurgical transfer to synchronized recipients. In vitro survival of nonstored embryos and embryos stored 1 day did not differ. Survival decreased after storage for 2 days (P<0.10) or longer (P<0.05). Similar results were observed for survival after transfer, but embryo viability decreased even more rapidly with increasing duration of storage. In vitro survival was approximately 50% for blastocysts stored for 3 and 5 days, but few pregnancies resulted from transfer of embryos stored for these periods. In another experiment survival after transfer of blastocysts stored at 4 degrees C for up to 2 days was similar to that of nonstored blastocysts.     

Cold culture of different stage mouse embryos in bicarbonated and bicarbonate-free media

Mouse embryos of different stages of development were cultured to expanded blastocysts following storage (1 to 8 d) at 4 degrees C in the presence or absence of HCO(3)(-). The effect of oxygen tension on the cold storage of one- and two-cell mouse embryos at 4 degrees C was evaluated by 37 degrees C culture and transfer to pseudopregnant recipients. Survival at 4 degrees C of early, one- to four-cell mouse embryos was improved with HCO(3)(-) in the medium. The presence of HCO(3)(-) was not of benefit for morulae or blastocyst survival following cold storage. Reducing the oxygen atmosphere from 20 to 5% O(2) improved survival of one-cell mouse embryos stored at 4 degrees C. The survival of two- and four-cell embryos, morulae and blastocysts at 4 degrees C was similar in 90% N(2), 5% CO(2) and 5% CO(2) in air, but it was significantly poorer in air alone. The collapse of morulae and blastocysts during cold storage up to 5 d was reduced with HCO(3)(-) in the storage medium. Blastocysts stored for 6 d at 4 degrees C failed to survive following immediate transfer to pseudopregnant recipients. Blastocyst survival was improved compared to controls (direct transfer of unstored blastocysts to recipients) when cultured for 36 h at 37 degrees C following 6 d of cold storage. This result suggests that cold-stored mouse blastocysts may require a metabolic period of readjustment to survive following transfer to synchronized recipients.     

Cold storage of mouse embryos of different stages of development

Experiments were designed to evaluate the survival rates of preimplantation mouse embryos of different stages of development in cold culture at 4 degrees C. Several developmental stages, from one-cell to the blastocyst, were stored at 4 degrees C from 1 to 8 d. Viability following cold culture was determined by blastocyst expansion during culture in Whitten's medium at 37 degrees C. Blastocyst formation of nonstored controls ranged from 93 to 100% for all developmental stages tested. Only 3% of one-cell embryos survived 1 d and none survived 2 days at 4 degrees C. Survival improved using two-cell embryos, with 84, 69 and 15% forming expanded blastocysts following storage for 1, 2 and 3 d, respectively. Eighty five and 38% of eight-cell embryos formed expanded blastocysts following cold storage for 3 and 4 d, respectively. Survival rates for cold stored morulae and blastocysts remained above 75% for 6 d but decreased significantly to 30 and 36%, respectively, when stored for 8 d. A large percentage of blastocysts were observed to collapse when placed in cold storage from 1 to 8 d but almost all expanded when placed in culture at 37 degrees C. This study showed that one-cell embryos were particularly sensitive to cold storage compared to later-stage mouse embryos. Cold storage survival increased with increasing age of the embryo; morula and blastocyst survival rate was similar.     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.     

Synchronous division of mouse two-cell embryos with nocodazole in vitro.

Mouse two-cell embryos were cultured in a medium supplemented with nocodazole or colcemid for 12.5-14.5 h in vitro, and development after elimination of these drugs was examined. All embryos cultured with nocodazole stopped at the metaphase of the second cell cycle. When nocodazole was removed, almost all embryos divided to the normal four-cell stage within 1 h and then developed into blastocysts (98%). The proportion of embryos that developed into young after transfer to recipients was not significantly different from the control (35 versus 36%), but the developmental ability of the embryos treated with colcemid was reduced, especially after transfer to recipients.     

The effect of partial dehydration on the developmental capacity of mouse embryos stored in the supercooled state

The effect of partial dehydration on the ability of mouse blastocysts to withstand storage at subzero temperatures without freezing was studied. The embryos were equilibrated with a supercooling medium developed at the Centre for Food and Animal Research, containing 3% (Medium A) or 6% (Medium B) methanol and propanediol, and then with the same medium, A or B, containing 0–0.5 mol sucrose. The embryos were placed in 0.25 ml straws, cooled to −5°C or −10°C and stored for up to 3 days. After storage, the embryos were cultured for 48 h in M16 and their ability to develop into expanded blastocysts was used to gauge their survival in supercooled storage.

The maximal beneficial effect of partial dehydration occurred in media supplemented with 0.3–0.5 mol sucrose: the proportions of dehydrated embryos surviving 24 h storage at −5°C and −10°C were 84–85% and 91–100%, respectively, compared with only 58% and 52% of non-dehydrated, supercooled embryos. The corresponding figures for dehydrated embryos after 48 or 72 h storage at −5°C were 86–92% and 38–58% compared with 13% and 4% of non-dehydrated embryos. Similarly, 75–85% and 47–55% of partially dehydrated embryos survived storage for 48 h or 72 h, respectively, at −10°C, compared with 5% and 0% of non-hydrated embryos. Thus, reducing the water content of early mouse blastocysts improved their ability to withstand subzero storage.     

Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.     

Viable spermatozoa can be recovered from refrigerated mice up to 7 days after death

To develop a model for utilizing germ cells collected from dead animals, male mice were euthanized and refrigerated for various periods, and the viability of the epididymal spermatozoa was examined by in vitro fertilization, embryo culture, and embryo transfer. Higher proportions of fresh oocytes were fertilized when males had been stored at 4-6 or 8-10 degrees C than at 0 degrees C. By partially dissecting the zona of freshly ovulated oocytes, spermatozoa from ICR male mice could fertilize oocytes (21% fertilization rate) after being stored for 5 days at 4-6 degrees C, and spermatozoa from BDF1 male mice could fertilize oocytes (39%) after being stored for 7 days at 4-6 degrees C. The resulting two-cell embryos had the ability to develop into expanded blastocysts in culture (81-100%) and into live young after transfer (34-47%). With further refinement of this system, it should be applicable not only for rescuing valuable genetic variants in laboratory animals or livestock animals but also for wild species in the future.     

Effects of beta-mercaptoethanol and cycloheximide on survival and DNA damage of bovine embryos stored at 4 degrees C for 72 h

The objectives of this study were to determine the effects of cycloheximide (CHX) and beta-mercaptoethanol (beta-ME) during storage of in vitro-produced (IVP) bovine blastocysts for 72 h at 4 degrees C on their survival, hatching capacity and DNA damage. In Experiment 1, when blastocysts were stored in a medium supplemented with 25, 50 or 100 microg/mL of CHX, or 25, 50 or 100 microM of beta-ME, the blastocysts stored with 25 microg/mL of CHX had a significantly higher survival rate than that of the blastocysts stored without CHX (79.5% versus 54.2%). In contrast, beta-ME had no apparent effects on the survival and hatching capacity of stored embryos. In Experiment 2, to investigate synergistic effects of CHX and beta-ME during storage of blastocysts on their developmental parameters and DNA damage, they were stored in the medium with CHX (25 microg/mL) and beta-ME (50 microM). The combination of CHX and beta-ME had no significant effects on the survival of blastocysts. The proportion (6.8%) of DNA-fragmented cells in the blastocysts stored with CHX was similar to that (5.4%) in the non-stored blastocysts (positive control) and significantly lower than that (9.7%) in the blastocysts stored without CHX and beta-ME (negative control). However, there were no significant differences among the proportions of dead cells of blastocysts in the storage groups. Therefore, the supplementation of CHX in the storage medium had a beneficial effect on the proportions of survival and DNA-fragmented cells in the stored embryos, whereas the beta-ME alone or in combination with CHX had no positive effects on either of these proportions.     

Effect of dimethyl sulfoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique

Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.5 M dimethyl sulfoxide (Me2SO), and 0, 4, 8, 16, or 32 mg/ml bovine serum albumin (BSA). In the absence of BSA, significantly more embryos were lost or damaged during freezing and thawing. Increasing the BSA concentration from 4 to 32 mg/ml had no significant effect on subsequent embryo viability in vivo or in vitro. Blastocyst formation in vitro was greater than 90% in embryos exposed to the cryoprotective solutions only. Although development to blastocysts was not significantly different to nonfrozen controls in most groups frozen in 3 and 4.5 M Me2SO (up to 92% blastocysts), it was significantly reduced when embryos were frozen in 1.5 M Me2SO (up to 65% blastocysts). The development to fetuses of embryos frozen in 3 M Me2SO (64 to 74% fetuses) was not significantly different from nonfrozen controls (68 to 79% fetuses) or embryos frozen by a conventional slow cooling method (70%). Frozen thawed two-cell embryos developed into normal adults which were able to reproduce normally. We conclude that this freezing method can efficiently cryopreserve early cleavage stage mouse embryos.     

Nuclear transplantation of mouse fetal germ cells into enucleated two-cell embryos

Mouse fetal germ cells were fused with enucleated blastomeres of two-cell embryos. Donor germ cells were obtained from fetuses of albino CD-1 strain or pigmented F(1) (C57BL x CBA) female mice mated with the same strain males at 11.5 to 16.5 days post coitum. Recipient two-cell embryos, which were of a different strain from the donors, were obtained at 37 to 42 hours (Group 1), 42 to 47 hours (Group 2), and 47 to 52 hours (Group 3) after treatment with human chorionic gonadotropin (hCG). After removing the nucleus from one two-cell blastomere, a single germ cell was fused with the enucleated blastomere using the Sendai virus; the second blastomere was left intact. The reconstituted embryos were cultured for 3 days in vitro, to examine their developmental capacity. The fused blastomeres in Groups 1 and 2 did not divide, but a few transplanted blastomeres in Group 3 divided several times, and some of them developed into normal blastocysts. Most embryos developed into blastocysts from one blastomere, with an undivided blastomere remaining. Embryos developing into normal blastocysts or blastocysts with small blastomeres were transferred to the oviducts of Day-1 or the uteri of Day-3 pregnant albino CD-1 mice. None of the young showed any contribution of the germ cells, judging by the eye and coat colors and by the germ cells in the germ line following mating with albino mice. Possible reasons for failure of pluripotency of the germ cells are discussed here.     

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.     

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.     

Vitrification of two-cell rat embryos derived from immature hypothyroid rdw rats by in vitro fertilization in ethylene glycol-based solutions

Two-cell embryos derived from immature rdw rats by in vitro fertilization (IVF) were vitrified in ethylene glycol-based solutions. Embryos exposed to EFS20 before being vitrified in EFS40 exhibited improved viability in vitro. All embryos exposed to EFS20 for 1-3 min before vitrification in EFS40 were morphologically normal. However, 2-3 min of exposure to EFS20 increased the number of embryos that developed beyond the four-cell stage. More embryos exposed to EFS20 for 2-3 min developed to morulae (63-64%) and blastocysts (34-38%) than those exposed for 1 min (35 and 10%, respectively). After transfer, 33% of embryos exposed to EFS20 for 3 min and vitrified in EFS40 developed to term compared to 29% of fresh embryos. Fifteen (47%) of live young were homozygous rdw and all of the others were heterozygous rats. The present study demonstrated that vitrification in EFS solution can be routinely used to cryopreserve rat two-cell IVF-embryos with no loss of viability.     

Heat shock induces apoptosis related gene expression and apoptosis in porcine parthenotes developing in vitro

Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. However, the molecular aspects involved in the thermoprotection of embryos, against heat and cold stress it is not clear. The aim of this study was to determine the effects of heat and cold shock on the viability and development of porcine diploid parthenotes developing in vitro. Exposure of two-cell stage embryos to 41 degrees C did not affect further cleavage. However, prolonged heat shock, greater than 12h, reduced the percentage of blastocysts that developed from two-cell stage parthenotes, as well as the total number of nuclei in the blastocysts that formed. Furthermore, the degree of apoptosis was increased (P<0.05) in these blastocyst stage parthenotes. In contrast, exposure of two-cell parthenotes to cold (30 degrees C) for 24h did not affect the cleavage rates, development to blastocyst, nor the total cell numbers per blastocyst. Real time PCR revealed that quantitative expression of the Bcl-xL gene was not different, but amounts of HSP 70.2, Bak, and Caspase 3mRNA were significantly increased in the heat shocked embryos, as compared with untreated controls. These results suggest that porcine embryos are more tolerant to cold shock than to heat shock. Heat stress seems to induce apoptosis related gene expression in porcine parthenotes developing in vitro, which results in diminished parthenote viability.     

Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa

This study was undertaken to try to reduce the number of animals required to maintain mouse strains by banking of embryos or spermatozoa. The principal objective was to cryopreserve ejaculated mouse spermatozoa, using a method recently developed for epididymal spermatozoa. Within 30 min after mating, ejaculated spermatozoa were flushed from the uterus of mated females; shortly afterwards, epididymal spermatozoa were also collected from the same males that had mated with the females. The average values for spermatozoal motility and viability of ejaculated specimens of nine males were 43 and 46%, respectively, and for epididymal specimens, the corresponding values were 60 and 52%. In experiment 1, ejaculated or epididymal spermatozoa were incubated with oocytes for 0.5 to 4 h. As evidenced by development into two-cell embryos within 24 h, kinetics of fertilization of the two spermatozoa types were similar. In experiment 2, ejaculated and epididymal spermatozoa of three males were separately cryopreserved in medium containing raffinose, glycerol, and egg yolk. Samples were cooled and seeded at -4 degrees C, cooled to -70 degrees C at 20 degrees C/min, and then were placed into liquid nitrogen for storage. When cryopreserved epididymal or ejaculated spermatozoa were thawed at > 1,000 degrees C/min and used for in vitro fertilization, > 60% of oocytes cleaved, and approximately 95% of cleaved embryos developed into morulae or blastocysts. When embryos produced with cryopreserved spermatozoa were transferred into recipients, 18 and 22 live pups were obtained from 62 and 54 embryos resulting from ejaculated or epididymal spermatozoa, respectively. This study documented the feasibility of cryopreserving ejaculated spermatozoa as an effective alternative to preserving germ plasm from genetically valuable mice.     

In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.