Purpose This study was designed to investigate whether artificial shrinkage, induced by a laser pulse or hyperosmotic sucrose solutions, improves in vitro survival and/or implantation of vitrified-warmed human expanded blastocysts. Methods Before Cryotop vitrification, the blastocoelic cavity was collapsed either by a laser pulse or sucrose solutions. Non-treated blastocysts were used as control. Post-warm blastocyst survival and implantation after transfer were examined. Implantation rate outcome was retrospectively analyzed by morphological grading and developmental kinetics of post-warm blastocysts. Results Survival rates in the three groups were high. Implantation rates in the laser-pulse group (59.7%) were comparable with those in the sucrose group (49.3%), and were significantly higher than those in the control group (34.2%). The proportion of blastocysts showing fast development tended to be higher when the blastocysts underwent artificial shrinkage treatment before vitrification. There was no clear correlation between morphology of post-warm blastocysts and implantation rate. Conclusion Artificial shrinkage treatment before vitrification is associated with an increased probability of fast-developing embryos, resulting in higher implantation rates.

Abstract. For developmental competence of porcine embryos in vitro, it is important to improve the culture environment. The present study was performed to evaluate four different culture systems for in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI); drop, well and two sizes of the well of the well (WOW) systems (500 and 1,000 µm in diameter). The cleavage rate on Day 2 and the mean cell number in blastocysts on Day 6 were not significantly different among the four treatments. However, the 1,000 µm WOW (24.6%) resulted in a significantly higher (P<0.05) blastocyst rate than those in the other culture systems (12.9, 14.8, and 7.1% for drop, well, and 500 µm WOW, respectively). The present study indicates that the microenvironment created by the 1,000 µm diameter WOW improves blastocyst production of in vitro matured porcine oocytes after ICSI, and that the effectiveness of the WOW system is dependent on the size (diameter) of the WOW. Key words: In vitro culture, Microenvironment, Porcine embryos, WOW (J. Reprod. Dev. 51: [533][534][535][536][537] 2005) n vitro production (IVP) for porcine oocytes has advanced remarkably during the past decades. T h e i n v i t r o c u l t u r e ( I V C ) s y s t e m f o r preimplantation embryos is an important step for IVP. Recently, it has been noted that the in vitro production techniques for pigs can be applied to o t h e r r e p r o d u c t i v e t e c h n o l o g i e s , s u c h a s production of alternative organs and specific proteins for humans through the modification of genes [1]. However, the proportions of blastocyst development and pregnancy rates of in vitroproduced embryos after transfer are usually lower than that of in vivo produced counterparts [1].In general, embryonic development in vitro is affected by a number of factors, such as co-culture w i t h s o m a t i c c e l l s [ 2 ] , s u p p l e m e n t w i t h antioxidants [3] and growth factors [4], and oxygen tension [5]. Moreover, another important factor is embryo density during culture [6,7], which influences the interaction of embryonic factors in the microenvironment. In vivo, embryonic factors are secreted by the embryo itself and appropriate c o n c e n t r a t i o n s a r e m a i n t a i n e d i n t h e microenvironment surrounding the embryo. The embryonic factors enhance the development of embryos through the autocrine-paracrine action. For improving the developmental competence of embryos in vitro, it is important to provide a culture environment similar to the in vivo conditions. Vajta et al. [8] developed the well of the well (WOW) system as a new culture system. The WOW created at the bottom of a four-well dish p r o v i d e s a m o r e c o n s t a n t a n d s u i t a b l e microenvironment for embryos. The open condition of the WOW may also provide an appropriate method of nutrition and dilution of

SummaryGerminal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.

The objective of this study was to investigate the developmental ability of the whale embryos with interspecies somatic cell nuclear transfer (SCNT) in comparison with bovine or porcine SCNT embryos and parthenotes. There were no significant differences among the proportions of pseud-pronucleus (PPN) formation of whale SCNT oocytes, intraspecies SCNT oocytes and parethenotes. Furthermore, no significant difference was found in the cleavage rates of whale SCNT embryos between 6-dimethylaminopurin (DMAP) and cycloheximide (CHX) as secondary activation treatments. In effects of different donor cell types (viable or non-viable cells) on whale SCNT embryos, there was no significant difference in the cleavage rates of whale SCNT embryos between the two cell types. Two-to four-cell stages of whale SCNT embryos were obtained, but none of the embryos reached the blastocyst stage. The cleaved whale embryos were confirmed to have whale genomic DNA. From the results, it was shown that both bovine and porcine oocyte cytoplasms have the potential to form PPN and to produce cleaved whale interspecies SCNT embryos, regardless of the survivability of donor cells.

Abstract. The present study was conducted to investigate whether a portable CO2 incubator was effective for in vitro maturation (IVM) of bovine, porcine and minke whale oocytes, and the effect of maturation media supplemented with different hormones; porcine follicle stimulating hormone (pFSH), estradiol-17β (E2), or pregnant mare's serum gonadotropin (PMSG): human chorionic gonadotropin (hCG) for minke whale immature oocytes was also examined. In vitro maturation rates of bovine and porcine oocytes cultured in the portable CO2 incubator were not significantly different from the standard CO2 incubator. In minke whale IVM culture using the portable incubator, the maximum expansion of cumulus mass was observed by pFSH/E2 and PMSG/hCG at the end of IVM culture. Moreover, the IVM culture period was shortened to 28-30 h from 96-120 h previously reported. The proportion of matured oocytes cultured in the medium supplemented with pFSH/E2 (26.7%) was significantly higher (P<0.05) than that with PMSG/hCG (6.9%). The present study indicates that a portable CO2 incubator is a useful device for minke whale IVM culture on a research base ship, and the addition of pFSH/E2 into an IVM medium enhanced cumulus expansion and the proportion of minke whale matured oocytes.

This study was performed to establish an individual bovine oocyte-IVP system using a chemically defined simple medium (mSOFaa containing 1 mg/ml polyvinyl alcohol: PVA) and to investigate the effects of epidermal growth factor (EGF) during oocyte maturation on in vitro maturation, fertilization and embryonic development. Cumulus–oocyte complexes were collected from bovine ovaries and were matured in mSOFaa containing PVA (control medium) supplemented with 0, 1, 10 or 50 ng/ml of EGF. Two further groups (TCM199 and mSOFaa, supplemented with 10% fetal calf serum were also included. In this study, mSOFaa containing PVA were used as a basic medium for fertilization and embryo development in vitro. Experiments were conducted in both group- and individual-IVP systems. In the group-IVP system, the proportion of matured oocytes (MII) in the control medium (62.7%±5.0%) was significantly (p<0.05) lower than in all other treatments, and in the individual-IVP system, the addition of 1 ng/ml EGF significantly (p<0.05) increased the maturation rate (1 ng/ml EGF vs control: 76.2%±5.4% vs 57.1%±14.4%). The addition of EGF did not affect the proportions of penetrated and normally fertilized oocytes in either individual- or group-culture systems. In the group-IVP system, no significant difference among treatments was found in the rate of blastocyst formation, whereas in the individual-IVP system the control medium supplemented with 10 ng/ml EGF resulted in a significantly (p<0.05) higher the rate of blastocyst formation (20.0±5.2%) than that in the control medium (6.2%±3.5%). These results indicate that bovine oocytes can successfully develop to blastocysts in an individual-IVP system using a single chemically defined medium, and that the group-IVP system also resulted in a similar level of blastocyst formation to that in a standard multiple-media system in our laboratory. The effect of EGF during oocyte maturation medium differed depending on whether embryos were cultured individually or in groups.

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Abstract. The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prebuertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.