Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

Development of cloned pig embryos by nuclear transfer following different activation treatments

This study compared the effects of activation treatments on the development and ploidy of nuclear transferred (NT) pig embryos. After in vitro maturation of oocytes collected from the slaughterhouse, oocytes were enucleated and reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 kV/cm, 20 musec). Oocytes were pulsed thrice electrically with 1.4 kV/cm for 20 musec and NT eggs were divided into three treatment groups: Group 1 (no further treatment), Group 2 (10 mug/mL cycloheximide [CHX], 3 hr), and Group 3 (1.9 mM 6-dimethylaminopurine [DMAP], 3 hr). All the eggs were cultured in sets of 30 in 60 muL drops of NCSU-23 supplemented with 4 mg/mL fatty acid free BSA, and compared for the rates of development and ploidy. The rates of cleavage, development, and total cell number of parthenotes in Group 3 were significantly (P < 0.05) higher than those in Groups 1 and 2. Cleavage rates of NT embryos in Group 1 were significantly (P < 0.05) lower than those in Groups 2 and 3 (73% vs. 81% and 82%, respectively). Development into blastocyst stage and total cell number of NT embryos in Group 3 were significantly (P < 0.05) higher than those in Groups 1 and 2. Although the embryos in Group 3 had higher development, approximately 58% of NT embryos evaluated were abnormal ploidy (6% haploidy, 9% polyploidy, and 42% mixoploid). The results suggested that although DMAP enhanced development and higher cell number, due attention should be paid to abnormal ploidy in pig NT embryos.     

In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.     

Effects of glucose, glutamine, ethylenediaminetetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro.

Analysis over the first 48 h of development in vitro from the one-cell stage to the early four-cell stage indicated that (i) ethylenediaminetetraacetic acid (EDTA) exerts the major beneficial effect on culture to the blastocyst stage of F1 and MF1 embryos, (ii) glutamine assists development of MF1, but not F1, embryos to the blastocyst stage and probably functions as part of a metabolic response to oxidative damage to mitochondria and (iii) exposure to glucose at some time during early cleavage is essential for full development to blastocysts. None of the culture conditions examined affected significantly the increase in concentration of reactive oxygen species in late two-cell embryos in vitro, although F1 embryos in vitro often had lower peroxide concentrations than MF1 embryos. A decline in oxygen tension from 20 to 50% had no consistent effect on culture to the blastocyst stage or production of reactive oxygen species. Aminooxyacetate, an inhibitor of transaminase activity, prevented non-blocking embryos from developing beyond G2 of the second cell cycle. It is concluded that the chelation of transitional metals provides the most effective method of overcoming the block to development in vitro.     

In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep

Development to the blastocyst stage and survival following embryo transfer were assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The rates of maturation, fertilization and cleavage in vitro did not differ significantly between oocytes from prepubertal and adult sheep. The proportion of cleaved zygotes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal than for those from adult sheep (15.4% and 34.1% respectively). There were no differences in the pregnancy rate and number of lambs born following transfer of blastocyst stage embryos derived from prepubertal and adult sheep to adult recipients. These data show that embryos derived from prepubertal lamb oocytes have reduced developmental potential in vitro but, of those which do reach the blastocyst stage, they have equal capacity to develop to term as embryos derived from adult sheep.     

Growth factors effects on preimplantation development of mouse embryos exposed to tumor necrosis factor alpha

The success rates of assisted reproduction techniques are still unsatisfactory. Relatively few in vitro cultured embryos reach the blastocyst stage. The purpose of the study was to evaluate the protective potential of epidermal growth factor (EGF), insulin-like growth factors 1 and 2 (IGF-I, IGF-II) and stem cell factor (SCF) on in vitro development of pre-implantation mouse embryos exposed to tumor necrosis factor alpha (TNFalpha). C3B6F1 female mice were superovulated with 5 IU of pregnant mare serum gonadotropin (PMSG) and 48 h later with 5IU of equine chorionic gonadotropin (eCG). Following the second injection females were mated with DBA males. Two cell embryos were flushed out from the fallopian tubes 40 h after eCG administration. After retrieval, the embryos were divided into control and experimental media and incubated in groups of ten for 96 h (37 degrees C, 5%CO(2), in droplets of 50 microl under mineral oil). In the first part of experiment, the embryo development was tested in media containing EGF, IGF-I, IGF-II, SCF, TNF-alpha (1 to 1000 ng/ml). In the second part of the study, the development of embryos was examined in medium containing 100 ng/ml TNFalpha and one of following factors: IGF-I, IGF-II; EGF or SCF (100 ng/ml). During the culture embryos were examined at 24 hours intervals to assess the embryo development. Blastocyst rate was determined following 96 hours of culture. Evaluation of total blastocyst cell number (TB) and inner cell mass (ICM) was also performed. TNFalpha significantly reduced (p<0.05) the blastocyst rates as well as TB and ICM. The examined growth factors improved the development of embryos exposed to TNFalpha. Thus, in this study, the protective action of IGF-I and II, EGF and SCF against the detrimental influence of TNFalpha was demonstrated.     

In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa

Embryo development during in vitro culture of polyspermic porcine oocytes was investigated in the present study. After in vitro fertilization (IVF) of in vitro matured oocytes, putative zygotes were centrifuged to visualize pronuclei. Two pronuclear (2PN) and poly-pronuclear (PPN) zygotes were selected and cultured in vitro. Their development to the blastocyst stage and total cell numbers, dead cell rates and ploidy at the blastocyst stage and morphology of resultant embryos after first cleavage were compared. A cleavage rate of PPN embryos was lower than that of 2PN (61.3% and 82.2%, respectively), however, the ability of cleaved embryos to develop to the blastocyst stage did not differ between the PPN and the 2PN groups (22.4% and 32.9%, respectively). Also there was no difference in total cell numbers and rates of dead cells between PPN and 2PN blastocysts. The majority of blastocysts in 2PN group were found to be diploid. In contrast, blastocysts in PPN group showed heterogeneous status in their ploidy including polyploidy and mixoploidy, whereas a remarkable proportion (31.3%) of them was found to be diploid. After the first cleavage (at 36 h after IVF), there was no difference in the number of nuclei/embryo between the two groups, nevertheless embryos in PPN group had significantly higher numbers of blastomeres than that of embryos in 2PN group, mainly due to an increased frequency of anuclear blastomeres. The present results indicate that correction of embryo ploidy in polyspermic embryos can occur during IVC. Nevertheless the frequency of partial fragmentation in polyspermic embryos is increased.     

Effect of activation time on the nuclear remodeling and in vitro development of nuclear transfer embryos derived from bovine somatic cells

This study was conducted to investigate the effect of recipient activation time on the chromatin structure and development of bovine nuclear transfer embryos. Serum-starved skin cells were electrofused to enucleated oocytes, activated 1-5 hr after fusion, and cultured in vitro. Some fused eggs were fixed at each time point after fusion without activation, or 3 or 7 hr after activation. Some nocodazole treated zygotes were fixed to analyze their chromosome constitutions. The proportion of eggs with a morphologically normal premature chromosome condensation (PCC) state increased 1-2 hr after fusion. Whereas eggs with elongated chromosome plate increased as activation time was prolonged to 3 hr, and 5 hr after fusion, 58.1% of eggs showed more than two scattered chromosome sets. The proportion of eggs with a single chromatin mass (40.6-56.7%) significantly increased when eggs were activated within 2.5 hr after fusion (P < 0.05). Only 23.3% of reconstituted embryos activated 5 hr after fusion formed one pronucleus-like structure (PN), whereas, 64.5-78.3% of embryos activated 1-2.5 hr after fusion formed one PN. The proportion of embryos with normal chromosome constitutions decreased as activation time was prolonged. Development rates to the blastocyst stage were higher in eggs activated within 2 hr after fusion (17.3-21.7%) compared to those of others (0-8.6%, P < 0.05). The result of the present study suggests that activation time can affect the chromatin structure and in vitro development of bovine nuclear transfer embryos.     

The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.     

SP600125孵育影响鲫骨骼发育基因表达

前期研究表明ATP竞争性小分子抑制剂SP600125在体外可以高效诱导鱼类细胞多倍化。研究首次尝试用SP600125直接孵育鲫受精卵以获得多倍体鱼的可能性。结果显示SP600125处理受精卵虽然能够引起部分胚胎倍性发生改变, 但也显著影响胚胎发育并导致胚胎高死亡率, 同时伴随着畸形鱼苗的发生, 其中主要表现为脊椎弯曲和尾缺失等骨骼发育异常。SP600125处理导致骨骼畸形在SP600125孵育鲫鱼苗试验中也同样获得验证。荧光定量PCR检测结果表明, 在体(受精卵和鱼苗)或离体(培养的尾鳍细胞)状态下, SP600125孵育会引起smad6、stat1、 lef1、bmp2和twist1等骨骼发育相关基因mRNA水平显著上调。相关结果为进一步调整SP600125诱导的鱼类倍性操作策略和方法提供了重要理论参考。     

Bovine embryos contain a higher proportion of polyploid cells in the trophectoderm than in the embryonic disc

The frequency of polyploid cells in the embryonic disc (ED) and in the trophectoderm (TE) was assessed in 50 in vitro produced bovine embryos fixed at days 7-8 post insemination (pi) and in 20 in vitro produced embryos that were transferred to uteri of recipients at day 7 and then recovered and fixed at day 12 pi. Separation of TE and ED cells was obtained by microdissection and the frequency of polyploid cells was determined by interphase cytogenetic analysis using fluorescence in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes. The results show that 96% of day 7 embryos contain polyploid cells in the TE, whereas only 58% contain polyploid cells in the ED. In day 12 embryos 85% of TE and 40% of ED preparations contain polyploid cells. Statistical analysis revealed that the frequency of polyploid cells was significantly higher in the TE than in the ED in embryos containing less than 25% polyploid cells (n = 65). The few embryos (n = 5), which contained more than 25% polyploid cells, did not show this difference. Further, it was revealed that the level of polyploidy on day 7-8 was significantly higher than on day 12, both in the TE (two-fold) and in the ED (seven-fold).     

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.     

Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells.

Control ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty-one (38%) reached morula/blastocyst stage. Twenty-one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty-one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.     

Effects of exogenous putrescine on murine preimplantation development in vitro.

After first demonstrating that murine embryos take up putrescine from the medium in which they are cultured in vitro, fertilized eggs were placed in culture and maintained for 4 days in medium supplemented with varying amounts of putrescine. Their development was monitored each day. While embryos that were cultured in putrescine-supplemented medium developed at the same rate as control embryos, a significantly higher percentage of the putrescine-treated embryos attained the blastocyst stage as compared to the control group.     

Allele‐specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre‐implantation bovine embryo development by characterizing the allele‐specific expression pattern of the X chromosome‐linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4‐, 8‐ to 16‐cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT‐PCR‐RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele‐specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4‐, 8‐ to 16‐cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele‐specific expression of an X‐linked gene that is subject to XCI in in vitro bovine embryos from the 4‐cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. 77: 615–621, 2010. © 2010 Wiley‐Liss, Inc.     

Successful in vitro and in vivo development of in vitro fertilized two- to four-cell cat embryos following cryopreservation, culture and transfer

In vitro and in vivo survival of in vitro-derived 2- to 4-cell cat embryos following cryopreservation was examined. Prefreeze 1- vs 2-step cryoprotectant exposure (Experiment 1) and warming method (Experiment 2) on zona pellucida damage and development in vitro were compared. To determine viability in vivo, frozen/thawed embryos were cultured in vitro to the morula/early blastocyst stage and transferred to synchronous recipients (Experiment 3). At 24 to 26 h after IVF, embryos were cryopreserved in 1.4 M propanediol (Pr) + 0.125 M sucrose (Su) by cooling at 0.3 degrees C/min from -6 degrees C to -30 degrees C and storing in liquid nitrogen. Autologous embryos were cultured in vitro for 7 d. After warming for 5 sec in air and 10 sec at 37 degrees C in water (Experiments 1 to 3), or at room temperature air (22 degrees C; Experiment 2), the cryoprotectant was removed and embryos were cultured in vitro for 6 d (Experiments 1 and 2). Development was assessed after staining by counting cell numbers/embryo and determining the percentages at the 2- to 4-cell (nonsurvivor), pre (5 to 15), early (16 to 32), mid (33 to 50), late (>50) morula or blastocyst stages. Post-thaw development to late morula/blastocyst after 1-step exposure (68%, 15 min Pr + Su) was higher (P< 0.05) than that after 2-step exposure (36%, 15 min Pr and 15 min Pr + Su). Both warming methods produced similar percentages of embryos with damaged zonae (13 to 15%) and equivalent development to morula/blastocyst (64 to 69%). Development in vitro to early morula/blastocyst of frozen embryos with intact zonae was similar to that of nonfrozen embryos. Following cryopreservation, most 2- to 4-cell cat embryos retained their capability for in vitro development to morula/blastocyst, and in vivo viability was demonstrated by the birth of 3 live kittens to 2 of 4 recipients following the transfer of 58 embryos.     

Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro

Factors influencing the developmental potential of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n = 1053) were recovered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova). However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-free, growth factor-supplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively). Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes.     

Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium

Development of outbred CF1 mouse zygotes in vitro was studied in a chemically defined, protein-free medium both with and without amino acids. The addition of amino acids to protein-free potassium simplex optimized medium (KSOM) had little effect on the proportion of embryos that developed at least to the zona-enclosed blastocyst stage. In contrast, amino acids stimulated very significantly, in a dilution-dependent way, the proportion of blastocysts that at least partially or completely hatched. Amino acids also stimulated cell proliferation in both the trophectoderm and inner cell mass (ICM) cells, at rates that favored proliferation of cells in the ICM; had no effect on the incidence of cell death (oncosis or apoptosis); and improved development of the basement membranes, which form on the blastocoelic surface of the trophectoderm and between the primitive endoderm and the primitive ectoderm. Thus, KSOM, supplemented with amino acids but containing no protein supplements, supports development of a newly fertilized ovum to the late blastocyst stage, in which its normal, three-dimensional structure is preserved and in which the ICM has been partitioned into the primitive ectoderm and primitive endoderm.     

Environmental variables influencing in vitro development of hamster 2-cell embryos to the blastocyst stage

This study is a systematic analysis of environmental variables influencing development of 2-cell hamster embryos to the blastocyst stage in vitro. Experiments were done using a chemically defined (protein-free) culture medium (HECM-2). Physicochemical variables examined were temperature, the concentrations of CO2, HCO3-, Ca2+, Mg2+, K+, and O2, the presence of a silicone oil overlay, and osmotic pressure. The optimal temperature for development in vitro was 37.5 degrees C; lower temperatures were inhibitory. There was no significant effect on blastocyst development of alterations in the concentrations of NaHCO3, CaCl2, MgCl2, and KCl, or in the ratios of Ca2+:Mg2+ and Na+:K+, over the ranges tested. Development to the blastocyst stage was significantly stimulated by increased CO2 concentrations (7.5% and 10%), by reduced O2 concentrations (10% and 5%), and by the presence of silicone oil overlying the culture medium. Reduction of blastocyst development in the absence of an oil overlay was not caused by increased osmotic pressure. Cleavage stage embryos were not affected by osmolalities ranging from 250 to 350 mOsmols, but blastocyst development was inhibited at greater than or equal to 325 mOsmols. Under optimized conditions (37.5 degrees C, 10% CO2, 25 mM HCO3-, 2.0 mM Ca2+, 0.5 mM Mg2+, 3.0 mM K+, 10% O2, 250-300 mOsmols, with silicone oil overlay), 51-57% of 2-cell hamster embryos developed to the blastocyst stage. This represents a significant improvement over previous "standard" culture conditions, which supported development of 26% blastocysts from 2-cell hamster embryos. The culture system described here provides an adequate baseline response to support detailed investigations into the regulation of embryo development in the hamster.