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 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.

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.

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) MeSO, 0.4 M sucrose and 24% (w/v) Ficoll PM70.

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.

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.

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

This study examined the relationship between the timing of the first cleavage and 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.

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.

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.

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).

Timing of the First Cleavage and 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 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).

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).

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.

Emission and control of NO and composition of ash derived from cattle manure combustion using a pilot-scale fluidized bed incinerator.

This study investigates the emission of nitrous oxide (NO) and discusses the reduction of NO 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.

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.

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.

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.

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.

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.

Emission of greenhouse gases from controlled incineration of cattle manure.

Greenhouse gas emission is a potential limiting factor in livestock farming development. While incineration is one approach to minimize livestock manure, there are concerns about significant levels of nitrogen and organic compounds in manure as potential sources of greenhouse gas emissions (N2O and CH4). In this study, the effects of various incineration conditions, such as the furnace temperature and air ratio on N2O and CH4 formation behaviour, of cattle manure (as a representative livestock manure) were investigated in a pilot rotary kiln furnace.

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.

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.

Influence of intergeneric/interspecies mitochondrial injection; parthenogenetic development of bovine oocytes after injection of mitochondria derived from somatic cells.

Interspecies/intergeneric mitochondrial heteroplasmy can occur in interspecies/intergeneric hybrid embryos or following nuclear transfer. In the present study, intergeneric buffalo (Bubalus bubalis) mitochondria (WB-mt) or interspecies murine (Mus spretus) mitochondria (M-mt) were injected into bovine (Bos taurus) oocytes, and the subsequent embryonic development was characterized. Fibroblast mitochondria (WB-mt or M-mt) were microinjected into in vitro matured bovine oocytes followed by oocyte activation by a combination of electrical stimulation and 6-dimethylaminopurine treatment.

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).

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.

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.