Depth of Interbreed Difference in Postmortem Bovine Muscle Determined by CE-FT/MS and LC-FT/MS Metabolomics.

Japanese Brown (JBR) cattle have moderately marbled beef compared to the highly marbled beef of Japanese Black (JBL) cattle; however, their skeletal muscle properties remain poorly characterized. To unveil interbreed metabolic differences over the previous results, we explored the metabolome network changes before and after postmortem 7-day aging in the trapezius muscle of the two cattle breeds by employing a deep and high-coverage metabolomics approach. Using both capillary electrophoresis (CE) and ultra-high-performance liquid chromatography (UHPLC)-Fourier transform mass spectrometry (FT/MS), we detected 522 and 384 annotated peaks, respectively, across all muscle samples. The CE-based results showed that the cattle were clearly separated by breed and postmortem age in multivariate analyses. The metabolism related to glutathione, glycolysis, vitamin K, taurine, and arachidonic acid was enriched with differentially abundant metabolites in aged muscles, in addition to amino acid (AA) metabolisms. The LC-based results showed that the levels of bile-acid-related metabolites, such as tauroursodeoxycholic acid (TUDCA), were high in fresh JBR muscle and that acylcarnitines were enriched in aged JBR muscle, compared to JBL muscle. Postmortem aging resulted in an increase in fatty acids and a decrease in acylcarnitine in the muscles of both cattle breeds. In addition, metabolite set enrichment analysis revealed that JBR muscle was distinctive in metabolisms related to pyruvate, glycerolipid, cardiolipin, and mitochondrial energy production, whereas the metabolisms related to phosphatidylethanolamine, nucleotide triphosphate, and AAs were characteristic of JBL. This suggests that the interbreed differences in postmortem trapezius muscle are associated with carnitine/acylcarnitine transport, ß-oxidation, tricarboxylic acid cycle, and mitochondrial membrane stability, in addition to energy substrate and AA metabolisms. These interbreed differences may characterize beef quality traits such as the flavor intensity and oxidative stability.

Reviving vacuum-dried encapsulated ram spermatozoa via ICSI after 2 years of storage.

Introduction: Freeze-drying techniques give alternative preservation mammalian spermatozoa without liquid nitrogen. However, most of the work has been conducted in the laboratory mouse, while little information has been gathered on large animals that could also benefit from this kind of storage. Methods: This work adapted a technique known as vacuum-drying encapsulation (VDE), originally developed for nucleic acid conservation in anhydrous state, to ram spermatozoa, and compared it to canonical lyophilization (FD), testing long-term storage at room temperature (RT) and 4°C. Results and discussion: The results demonstrated better structural stability, namely lipid composition and DNA integrity, in VDE spermatozoa than FD ones, with outcomes at RT storage comparable to 4°C. Likewise, in VDE the embryonic development was higher than in FD samples (12.8% vs. 8.7%, p < 0.001, respectively). Our findings indicated that in large mammals, it is important to consider dehydration-related changes in sperm polyunsaturated fatty acids coupled with DNA alterations, given their crucial role in embryonic development.

Equilibrium vitrification of oocytes using low concentrations of cryoprotectants.

In order to make dry ice transportation of vitrified embryos practical, a near-equilibrium vitrification was developed using a cryoprotectant solution (EDFS10/10a), by which mouse embryos at various stages were vitrified in a near-equilibrium environment. EDFS10/10a consisted of 10% (v/v) ethylene glycol, 10% (v/v) Me2SO, 0.4 M sucrose and 24% (w/v) Ficoll PM70. This method exhibited the benefits of slow freezing and vitrification, with a low risk of osmotic injury. In this study, we investigated whether mouse oocytes are vitrifiable with EDFS10/10a in a highly dehydrated/concentrated state, and whether they can remain fertilizable and developing into embryos after vitrification. When mature mouse oocytes were vitrified in liquid nitrogen and after 4-28 days of storage at -80 °C, high survival rates were observed (88-99%). Vitrified and warmed oocytes were subjected to partial zona dissection and in vitro fertilized. The rate of 2-cell stage was 80-82%. Blastocyst formation rate was 55-70% which was similar to that of embryos derived from fresh oocytes. After the 2-cell embryos were transferred to recipient mice, the implantation and offspring rates did not differ significantly from those of embryos derived from fresh oocytes, indicating that vitrified oocytes retained the developmental ability. Therefore, it is possible to vitrify mouse oocytes in a near-equilibrium state using EDFS10/10a and conveniently transported using dry ice.

Somatic Cell Nuclear Transfer Using Freeze-Dried Protaminized Donor Nuclei.

Somatic cell nuclear transfer (SCNT) is the only nuclear reprogramming method that allows rewinding an adult nucleus into a totipotent state. As such, it offers excellent opportunities for the multiplication of elite genotypes or endangered animals, whose number have shrunk to below the threshold of safe existence. Disappointingly, SCNT efficiency is still low. Hence, it would be wise to store somatic cells from threatened animals in biobanks. We were the first to show that freeze-dried cells allow generating blastocysts upon SCNT. Only a few papers have been published on the topic since then, and viable offspring have not been produced. On the other hand, lyophilization of mammalian spermatozoa has made considerable progress, partially due to the physical stability that protamines provide to the genome. In our previous work, we have demonstrated that a somatic cell could be made more amenable to the oocyte reprogramming by the exogenous expression of human Protamine 1. Given that the protamine also provides natural protection against dehydration stress, we have combined the cell protaminization and lyophilization protocols. This chapter comprehensively describes the protocol for somatic cell protaminization, lyophilization, and its application in SCNT. We are confident that our protocol will be relevant for establishing somatic cells stocks amenable to reprogramming at low cost.

Metabolomic profiling of postmortem aged muscle in Japanese Brown beef cattle revealed an interbreed difference from Japanese Black beef.

OBJECTIVE: Japanese Brown (JBR) cattle, especially the Kochi (Tosa) pedigree (JBRT), is a local breed of moderately marbled beef. Despite the increasing demand, the interbreed differences in muscle metabolites from the highly marbled Japanese Black (JBL) beef remain poorly understood. We aimed to determine flavor-related metabolites and postmortem metabolisms characteristic to JBRT beef in comparison with JBL beef. METHODS: Lean portions of the longissimus thoracis (loin) muscle from four JBRT cattle were collected at 0, 1, and 14 d postmortem. The muscle metabolomic profiles were analyzed using capillary electrophoresis time-of-flight mass spectrometry. The difference in postmortem metabolisms and aged muscle metabolites were analyzed by statistical and bioinformatic analyses between JBRT (n = 12) and JBL cattle (n = 6). RESULTS: A total of 240 metabolite annotations were obtained from the detected signals of the JBRT muscle samples. Principal component analysis separated the beef samples into three different aging point groups. According to metabolite set enrichment analysis, postmortem metabolic changes were associated with the metabolism of pyrimidine, nicotinate and nicotinamide, purine, pyruvate, thiamine, amino sugar, and fatty acid; citric acid cycle; and pentose phosphate pathway as well as various amino acids and mitochondrial fatty acid metabolism. The aged JBRT beef showed higher ultimate pH and lower lactate content than aged JBL beef, suggesting the lower glycolytic activity in postmortem JBRT muscle. JBRT beef was distinguished from JBL beef by significantly different compounds, including choline, amino acids, uridine monophosphate, inosine 5'-monophosphate, fructose 1,6-diphosphate, and betaine, suggesting interbreed differences in the accumulation of nucleotide monophosphate, glutathione metabolism, and phospholipid metabolism. CONCLUSION: Glycolysis, purine metabolism, fatty acid catabolism, and protein degradation were the most common pathways in beef during postmortem aging. The differentially expressed metabolites and the relevant metabolisms in JBRT beef may contribute to the development of a characteristic flavor.

Effects of Trichostatin A on the Timing of the First Cleavage and In Vitro Developmental Potential of Bovine Somatic Cell Nuclear Transfer Embryos.

This study examined the relationship between the timing of the first cleavage and in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos treated with trichostatin A (TSA). SCNT embryos were visually assessed at 22, 26, and 48 hours after activation. Each embryo with two or more distinct blastomeres was transferred into a microwell and cultured until day 7. Irrespective of TSA treatment, approximately half of the cleaved embryos were observed at 22 hours, and a significantly higher blastocyst formation rate was shown in the SCNT embryos cleaved at 22 hours than those cleaved at ≥26 hours. The blastocyst formation rate of TSA-treated embryos cleaved at 22 hours (80%) was slightly higher than that of the control embryos (70%). In addition, interferon-τ (IFN-τ) expression was significantly lower in control SCNT embryos and late-cleaving (>26 hours) TSA-treated embryos than in in vitro fertilized (IVF) embryos. However, a significant difference was not observed between TSA-treated SCNT embryos cleaved at 22 and 26 hours, and IVF embryos. These results suggest that TSA treatment has no influence on the timing of the first cleavage of SCNT embryos; however, it slightly improves the blastocyst formation rate and the expression level of IFN-τ in early-cleaving embryos.

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.

Equilibrium vitrification of mouse embryos using low concentrations of cryoprotectants.

Previously, we developed a method for vitrification of mouse embryos in a near-equilibrium state using EFS35c, PB1 medium containing 35% (v/v) ethylene glycol, and 0.98 M sucrose. This method has advantages in both slow freezing and vitrification. However, since the vitrification solution in this method contains high concentrations of cryoprotectants and thus has high osmolality, the solution would injure oocytes and embryos with high sensitivity to chemical toxicity and high osmolality. In this study, we examined whether embryos could be vitrified in a near-equilibrium state using a solution containing low concentrations of cryoprotectants and thus with low osmolality. To investigate whether embryos were vitrified in a near-equilibrium state, 2-cell mouse embryos were vitrified with EDFS10/10a, PB1 medium containing 10% (v/v) ethylene glycol, 10% (v/v) DMSO, and 0.4 M sucrose, in liquid nitrogen, stored at -80 °C for 4-28 days, and warmed in water at 25 °C. The viability of the embryos was evaluated by the appearance of embryos after warming and developmental ability. When embryos were vitrified in liquid nitrogen using EDFS10/10a, the survival and developmental ability into blastocysts after storage at -80 °C for 7 days were high, indicating that embryos were vitrified in a near-equilibrium state. A high proportion of embryos vitrified with EDFS10/10a developed to term after transportation with dry ice, re-cooling in liquid nitrogen, and transfer to recipients. Therefore, new equilibrium vitrification developed in this study may be useful for oocytes and embryos that are highly sensitive to the toxicity of cryoprotectants and high osmolality.

Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature.

Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to - 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA). TGA revealed more than 90% of water subtraction in both groups. A minor DNA damage, Double-Strand Break (DSB) in particular, characterized by a lower degree of abnormal chromatin structure (Alpha-T), was detected in the SF-group, comparing to the LN-one. In accordance with the structural and DNA integrity data, spermatozoa from SF-group had the best embryonic development rates, comparing to LN-group: cleaved embryos [42/100 (42%) versus 19/75 (25.3%), P < 0.05, SL and LN respectively] and blastocyst formation [7/100 (7%) versus 2/75 (2.7%), P < 0.05, SF and LN respectively]. This data represents a significant technological advancement for the development of lyophilization as a valuable and cheaper alternative to deep-freezing in LN for ram semen.

Timing of the First Cleavage and In Vitro Developmental Potential of Bovine Somatic Cell Nuclear Transfer Embryos Activated by Different Protocols.

The objective of this study was to examine the relationship between the timing of the first cleavage and in vitro development of somatic cell nuclear transfer (SCNT) embryos produced by different activation protocols. SCNT embryos were activated with calcium ionophore A23187 and further treated with 6-dimethylaminopurine (DMAP group), cycloheximide (CHX group), or anisomycin (ANI group). The proportion of SCNT embryos that cleaved within 18 hours after activation was significantly higher in the DMAP group (20%) than that in the CHX and ANI groups (3% and 2%, respectively). More than 70% of the cleaved embryos were observed within 24 hours in the DMAP and CHX groups, and within 26 hours in the ANI group. The blastocyst formation rate of SCNT embryos decreased gradually as the time from activation to the first cleavage increased in the DMAP group. The blastocyst formation rate of SCNT embryos cleaved at 22 hours (>20 to ≤22 hours) in the CHX group or within 26 hours in the ANI group was significantly higher than that of SCNT embryos cleaved more than 26 hours in each group. These results indicate that the activation protocol affects the timing of the first cleavage and subsequent in vitro development potential of bovine SCNT embryos and that late-cleaving embryos have a low developmental potential irrespective of the activation protocol.

A negative feedback loop between nuclear factor 90 (NF90) and an anti-oncogenic microRNA, miR-7.

Alterations in microRNAs (miRNAs) levels deeply correlate with tumorigenesis. However, the molecular mechanism for the regulation of the miRNA production in tumors is not fully understood. We previously reported that downregulation of miR-7, which is an anti-oncogenic miRNA, was caused by overexpression of the nuclear factor 90 (NF90)-nuclear factor 45 (NF45) complex through the binding of double-stranded (ds) RNA-binding proteins to primary miR-7, resulting in promotion of tumorigenesis (Higuchi et al 2016). During this study, we found that the level of NF90 protein was dramatically decreased by overexpression of miR-7. Interestingly, the miR-7-mediated reduction in NF90 family proteins was only observed in NF90 protein, but not in NF110 protein, which is a longer form of the NF90 gene. Luciferase reporter analysis indicated that the overexpression of miR-7 significantly repressed the luciferase activity in the coding region of NF90 mRNA harboring a predicted target sequence of miR-7. The luciferase activity of the reporter vector, which has a mutated miR-7 target site in the coding region, was the same in the control and miR-7 overexpressed cells. Furthermore, the translation of TARGET-tagged NF90 mRNA without the 3'UTR of the NF90 mRNA was inhibited by the overexpression of miR-7. These results imply that miR-7 suppresses NF90 at the protein level through the binding of miR-7 to the complementary site of the seed sequence in the coding region of the NF90 mRNA. We further confirmed increased endogenous NF90 protein levels in SK-N-SH cells transfected with antisense oligonucleotides targeting miR-7, indicating that miR-7-mediated translational repression of NF90 is a physiological event. Taken together with our previous findings (Higuchi et al 2016), it suggests that the level of NF90 is increased by a negative feedback loop between NF90 and miR-7 in tumor tissues under physiological conditions.

Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange.

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

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.

Relationship between respiratory quotient, nitrification, and nitrous oxide emissions in a forced aerated composting process.

We assessed the relationship between respiratory quotient (RQ) and nitrification and nitrous oxide (N2O) emission in forced aerated composting using lab-scale reactors. Relatively high RQ values from degradation of readily degradable organics initially occurred. RQ then stabilized at slightly lower values, then decreased. Continuous emission of N2O was observed during the RQ decrease. Correlation between nitrification and N2O emission shows that the latter was triggered by nitrification. Mass balances demonstrated that the O2 consumption of nitrification (∼24.8mmol) was slightly higher than that of CO2 emission (∼20.0mmol), indicating that the RQ decrease was caused by the occurrence of nitrification. Results indicate that RQ is a useful index, which not only reflects the bioavailability of organics but also predicts the occurrence of nitrification and N2O emission in forced aerated composting.

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.

Rapid movement of water and cryoprotectants in pig expanded blastocysts via channel processes: its relevance to their higher tolerance to cryopreservation.

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.

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.

A trial to cryopreserve immature medaka (Oryzias latipes) oocytes after enhancing their permeability by exogenous expression of aquaporin 3.

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.

Differentiation potential and GFP labeling of sheep bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow-derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34- and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre-clinical sheep model to test the efficiency and safety of cell replacement therapy.

Equilibrium vitrification of mouse embryos at various developmental stages.

Previously, we developed a new method by which 2-cell mouse embryos can be vitrified in liquid nitrogen in a near-equilibrium state, and then kept at -80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight-cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol-based solutions, named EFSc because of their composition of ethylene glycol (30-40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at -80°C. When 8-cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at -80°C, the survival rate was high even after 4 days in storage and remained high after re-cooling in liquid nitrogen. On the other hand, the survival of vitrified-expanded blastocysts kept at -80°C was low. Therefore, 8-cell embryos and morulae can be vitrified in a near-equilibrium state using the same method as for 2-cell embryos. A high proportion of C57BL/6J embryos at the 2-cell, 8-cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re-cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near-equilibrium vitrification method, which is effective for 2-cell mouse embryos, is also effective for embryos at the 8-cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice.