Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.     

Carrying capacity and survival strategy for the Pacific bluefin tuna, Thunnus orientalis, in the Western Pacific

The carrying capacity for the Pacific bluefin tuna at each life stage is estimated and its survival strategy is examined numerically, using a new method to define the hypothetical capacity, the standard population, and the search volumes that are necessary and are feasible for the tuna. The carrying capacity for the adult is estimated at 1–2 × 10⁶ individuals, which corresponds with 5–10% of the hypothetical capacity and is comparable with the maximum levels of the southern and the Atlantic bluefin tuna populations. It is hypothesized semiquantitatively that the migration at each life stage and the remarkable decrement of growth at 120 days and about 40 cm occur as an evolutionary response to population excess over the carrying capacity. It is also hypothesized semiquantitatively that the early larvae have minimal food available in the Subtropical Water and develop the predatory morphology, high growth rate, and high mobility, however, at the expense of a high mortality as an evolutionary response to the tuna spawning in the Subtropical Water. This method may be an available tool to not only investigate the carrying capacity and survival strategy of a specific fish species, but also predict when and in how much abundance the fish species occurs in a specific area of its habitat.     

In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection

In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing.     

The Culture of the Rotifer Brachionus plicatilis with Chlorella vulgaris Containing Vitamin B12 in its Cells

The rotifer Brachionus plicatilis requires vitamin B₁₂. Freshwater Chlorella , which is produced by traditional culture, cannot support rotifer growth under bacteria-free conditions. However, Chlorella enriched with vitamin B₁₂ can support rotifer growth. To attain stable mass production of Brachionus , it is desirable to develop a food that can completely support growth of the rotifers. The authors cultured rotifers at experimental and mass culture scale with concentrated Chlorella vulgaris suspension enriched with vitamin B₁₂ in their cells.
Chlorella suspensions were prepared containing different amounts of vitamin B₁₂ in their cells, and rotifers were then cultured in 20 ml of the prepared suspensions. The highest rotifer yield was obtained from the group cultured with Chlorella containing more vitamin B₁₂ in their cells. The suitable content of vitamin B₁₂ in the concentrated Chlorella suspension commercially available for mass culture of the rotifer is considered to be 200 _μg per 100 g dry matter of Chlorella . The amount of vitamin B₁₂ necessary to produce one individual rotifer is calculated at 0.32 pg.
The authors conducted mass production of the rotifer with baker's yeast and refrigerated concentrated Chlorella containing vitamin B₁₂ Rotifer culture with vitamin B₁₂ was more stable and showed 1.3 times higher production than with normal Chlorella .     

Fundamental studies on the physiology of rotifers in mass culture—V. Dry Chlorella powder as a food for rotifers

The dietary value of dried, commercial Chlorella was compared with that of living marine Chlorella, and yeast, in relation to growth of the rotifer Brachionus plicatilis raised individually and by batch culture methods.A concentration of 50 μg/ml of dried Chlorella powder is near an optimal density for rotifer growth. The dried material in suspension is less effective for growth than living marine Chlorella, although it is much more effective than a suspension of yeast at the same density (50 μg/ml).In batch culture (12-l glass vessel), the rotifers grew from an initial inoculation of 13.2 individuals/ml to a density of 434 individuals/ml by the 16th day. About 10⁷ rotifers could be removed from one batch culture in five harvests in the 41-day experimental period.The results indicate that dried Chlorella powder is an effective food for the rotifer Brachionus plicatilis.     

Developmental ability of somatic cell nuclear transferred embryos aggregated at the 8-cell stage or 16- to 32-cell stage in cattle

Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.     

Constant transmission of mitochondrial DNA in intergeneric cloned embryos reconstructed from swamp buffalo fibroblasts and bovine ooplasm

Although interspecies/intergeneric somatic cell nuclear transfer (iSCNT) has been proposed as a tool to produce offspring of endangered species, conflict between donor nucleus and recipient cytoplasm in iSCNT embryos has been identified as an impediment to implementation for agricultural production. To investigate the nuclear–mitochondrial interactions on the developmental potential of iSCNT embryos, we analyzed the mtDNA copy numbers in iSCNT embryos reconstructed with water buffalo (swamp type) fibroblasts and bovine enucleated oocytes (buffalo iSCNT). As controls, SCNT embryos were derived from bovine fibroblasts (bovine SCNT). Buffalo iSCNT and bovine SCNT embryos showed similar rates of cleavage and development to the 8‐cell stage (P > 0.05). However, buffalo iSCNT embryos did not develop beyond the 16‐cell stage. Both bovine and buffalo mtDNA content in buffalo iSCNT embryos was stable throughout the nuclear transfer process, and arrested at the 8‐ to 16‐cell stage (P > 0.05). In bovine SCNT embryos that developed to the blastocyst stage, mtDNA copy number was increased (P < 0.05). In conclusion, both the donor cell and recipient cytoplast mtDNAs of buffalo iSCNT embryos were identified and maintained through the iSCNT process until the 8–16‐cell stage. In addition, the copy number of mtDNA per embryo was a useful monitor to investigate nuclear–mitochondrial interactions.