Kazutsugu Matsukawa

Freeze-dried somatic cells direct embryonic development after nuclear transfer.

The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies.

Pathway for the Movement of Water and Cryoprotectants in Bovine Oocytes and Embryos

The permeability of cells is important for cryopreservation. Previously, we showed in mice that the permeability to water and cryoprotectants of oocytes and embryos at early cleavage stages (early embryos) is low because these molecules move across the plasma membrane predominantly by simple diffusion through the lipid bilayer, whereas permeability of morulae and blastocysts is high because of a water channel, aquaporin 3 (AQP3). In this study, we examined the pathways for the movement of water and cryoprotectants in bovine oocytes/embryos and the role of AQP3 in the movement by determining permeability, first in intact bovine oocytes/embryos, then in bovine morulae with suppressed AQP3 expression, and finally in mouse oocytes expressing bovine AQP3. Results suggest that water moves through bovine oocytes and early embryos slowly by simple diffusion, as is the case in mice, although channel processes are also involved in the movement. On the other hand, water appears to move through morulae and blastocysts predominantly by facilitated diffusion via channels, as in mice. Like water, cryoprotectants appear to move through bovine oocytes/early embryos mostly by simple diffusion, but channel processes could also be involved in the movement of glycerol and ethylene glycol, unlike that in mice. In bovine morulae, although glycerol and ethylene glycol would move predominantly by facilitated diffusion, mostly through AQP3, as in mice, dimethylsulfoxide appears to move predominantly by simple diffusion, unlike in mice. These results indicate that permeability-related properties of bovine oocytes/embryos are similar to those of mouse oocytes/embryos, but species-specific differences do exist.

Bovine Nuclear Transfer Using Fresh Cumulus Cell Nuclei and In Vivo- or In Vitro-Matured Cytoplasts

We examined the effects of the source of recipient oocytes and timing of fusion and activation on the development competence of bovine nuclear transferred (NT) embryos derived from fresh cumulus cells isolated immediately after collection by ovum pickup (OPU). As recipient cytoplasts, we used in vivo-matured oocytes collected from hormone-treated heifers by OPU, or in vitro-matured oocytes from slaughterhouse-derived ovaries. NT embryos were chemically activated immediately (simultaneous fusion and activation, FA) or 2 h (delayed activation, DA) after fusion. When in vitro-matured oocytes were used as recipient cytoplasts, the development rate to the blastocyst stage of NT embryos produced by the DA method (23%) tended to be higher than those by the FA method (15%), but the difference was not significant. NT embryos derived from in vivo-matured cytoplasts have a high blastocyst yield (46%). Pregnancy rate at day 35 did not differ with the timing of fusion and activation (FA vs. DA; 50% vs. 44%) or oocyte source (in vivo- vs. in vitro-matured; 50% vs. 44%). Subsequently, the high fetal losses (88% of pregnancies) were observed with in vitro-matured cytoplasts, whereas no abortions were observed in NT fetuses from in vivo-matured cytoplasts. A total of three embryos derived from fresh cumulus cells developed to term. However, all three cloned calves were stillborn. These results indicate that improvement of development competence after NT is possible by using in vivo-matured oocytes as recipient cytoplasts in bovine NT.

Nitrous oxide emission mechanisms during intermittently aerated composting of cattle manure.

To investigate the mechanisms of nitrous oxide (N₂O) emission during intermittent aeration in the composting process, a laboratory scale experiment with continuous measurement of N₂O emission was conducted with cattle manure. A low oxygen mode (2.5% oxygen in the inlet for 1 day), anaerobic mode (0.13% oxygen for 0.25 day), and aerated mode (20.5% oxygen for 2 days) were sequentially set up three times after 22 days of continuous aeration to replicate intermittent aeration. The total N₂O emission was 0.26-0.35 mmol, 0.27-0.32 mmol, and 0.14-0.23 mmol during the low oxygen, anaerobic, and aerated modes, respectively. Denitrification was indicated as the main N₂O emission pathway in the anaerobic and low-oxygen modes, while nitrification was indicated as the main pathway in the aerated mode and under continuous aeration. Results from this study suggest that nitrification is an important pathway for N₂O emission as well as denitrification.

Fluorescent monitoring using microfluidics chip and development of syringe pump for automation of enucleation to automate cloning

We have developed a novel technique for fluorescent monitoring of a bovine oocyte nucleus for an automatic cloning device. Animal cells that have been chemically softened by cytochalasin and stained with Hoechst dye are aspirated into a thin microchannel of a microfluidic chip and stretched thin, allowing the nucleus of the expanded oocyte to be monitored. Half the volume of the oocyte is aspirated into the thin microchannel and a high-velocity fluid flow is generated in the wide microchannel to bisect the oocyte. Then, the half-oocytes are monitored to determine which contains the nucleus. To control flow velocity with high accuracy and rapid response, we also developed a syringe pump that is small, has no backlash, and has highly-accurate volume control and a good response for automatic cutting. In this report, we describe the monitoring method and construction of the syringe pump.

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.

Effect of Ovary Storage on Development of Bovine Oocytes after Intracytoplasmic Sperm Injection, Parthenogenetic Activation, or Somatic Cell Nuclear Transfer

ABSTRACT The purpose of this study was to examine the effect of ovary storage on the development of bovine oocytes after intracytoplasmic sperm injection (ICSI), parthenogenetic activation, or somatic cell nuclear transfer (SCNT). Oocytes were obtained from ovaries stored in PBS for 2 to 6 h (control group) or 26 to 30 h (stored group) at 15°C. The maturation rate of the oocytes was significantly lower in the stored group (67%) than in the control group (78%). The degeneration rate of the oocytes was significantly higher in the stored group (24%) than in the control group (2%). ICSI and parthenogenetic oocytes from stored ovaries had a significantly decreased development to the blastocyst stage compared with the control (ICSI 8% vs. 24%, parthenogenetic activation 15% vs. 31%). However, the development rate to blastocysts of SCNT embryos derived from cumulus cells was not different between the two groups (38% vs. 38%). Also, the storage period of ovaries did not decrease the pregnancy rate of SCNT embryos, and cloned calves were produced in both groups with the same efficiency (21% vs. 21%). In summary, ovary storage at 15°C for 26 to 30 h reduced the maturation rate and in vitro development rate of bovine oocytes after ICSI or parthenogenetic activation, but did not decrease the blastocyst formation rate or survival rate after embryo transfer in SCNT.

Rapid Movement of Water and Cryoprotectants in Pig Expanded Blastocysts via Channel Processes: Its Relevance to Their Higher Tolerance to Cryopreservation

ABSTRACT Pig oocytes and embryos are highly sensitive to cryopreservation; however, tolerance to cryopreservation increases in embryos at the expanded blastocyst stage. This increased tolerance may be attributed to a decrease in cytoplasmic lipid droplets at this stage. We previously showed that an increase in the permeability of the plasma membrane in mouse oocytes to water and cryoprotectants, caused by the artificial expression of aquaporin 3, an aquaglyceroporin, enhanced tolerance to cryopreservation. In the present study, we investigated whether membrane permeability was also involved in the tolerance of pig embryos to cryopreservation. The permeability of oocytes and morulae to water and glycerol was low, whereas that of expanded blastocysts was high. Activation energy for permeability to water, glycerol, ethylene glycol, and dimethyl sulfoxide was markedly lower for expanded blastocysts than for oocytes. This suggests that water and these cryoprotectants move through expanded blastocysts predominantly by facilitated diffusion and through oocytes predominantly by simple diffusion. Aquaporin 3 mRNA was expressed in expanded blastocysts abundantly, but less so in oocytes. On the other hand, the permeability of expanded blastocysts to propylene glycol was as low as that of oocytes, and activation energy for its permeability was similar to that of oocytes, which suggests that propylene glycol moves through oocytes and embryos predominantly by simple diffusion. These results suggest that the higher tolerance of pig expanded blastocysts to cryopreservation is also related to high membrane permeability due to the expression of water/cryoprotectant channels, in addition to the decrease in cytoplasmic lipid droplets.

Adsorptive removal of sulfonamide antibiotics in livestock urine using the high-silica zeolite HSZ-385

The adsorptive removal of seven sulfonamide antibiotics using the high-silica zeolite HSZ-385 from distilled water, synthetic urine and real porcine urine was investigated. The pH greatly affected the adsorption efficiency, and the amounts of all sulfonamide antibiotics adsorbed on HSZ-385 decreased at alkaline conditions compared with that at neutral conditions. During storage, the pH and ammonium-ion concentration increased with urea hydrolysis for porcine urine. We clarified that the adsorption efficiency of sulfonamides in synthetic urine was equivalent to that in distilled water, suggesting that adsorption behavior was not affected by coexistent ions. HSZ-385 could adsorb sulfonamide antibiotics in real porcine urine even though the non-purgeable organic carbon concentration of porcine urine was 4-7 g/L and was two orders of magnitude higher than those of sulfonamides (10 mg/L each). Moreover, the adsorption of sulfonamides reached equilibrium within 15 min, suggesting that HSZ-385 is a promising adsorbent for removing sulfonamides from porcine urine.

Emission and control of N2O and composition of ash derived from cattle manure combustion using a pilot-scale fluidized bed incinerator

This study investigates the emission of nitrous oxide (N2O) and discusses the reduction of N2O emissions during the 24-h combustion of cattle manure using a pilot-scale fluidized bed incinerator under various experimental conditions. The results of these experiments were then validated against previously reported data. In addition, the characteristics of cattle manure incineration ash and their changes under different combustion conditions were estimated. In incineration experiments with composted cattle manure, N2O concentrations using multi-stage combustion were 75% lower than the concentrations resulting from normal combustion without additional auxiliary fuel, since N2O could be decomposed in the high-temperature zone formed by the inlet of the secondary combustion air. The N2O emission factor under normal combustion conditions (800°C) was 6.0% g-N2O-N/g-N. This result is similar to the values found in previous studies at the same temperature. The N2O emission factor was decreased to 1.6% g-N2O-N/g-N using a multi-stage combustion procedure. The current Japanese N2O emission factor of 0.1% g-N2O-N/g-N is an underestimate for some conditions and should be uniquely specified for each condition. Finally, cattle manure ash contains ample fertilizer elements, little Fe, Al and Zn, but abundant Cl. Therefore if Cl could be removed by some kind of pretreatment, cattle manure ash could be used as a favourable fertilizer.