Mosaic Mutation on Tumour Development in Pigs: A Case Study.

Pigs rarely develop cancer; however, tumour protein p53 ()-modified pigs may have an increased incidence of cancer. In this study, two pigs with mosaic mutations induced by gene editing were compared to determine the role of the wild-type sequence in tumorigenesis and to speculate how amino acid changes in TP53 sequences are related to tumorigenesis. The pig without tumours had a wild-type sequence and a 1-bp deletion in the sequence that resulted in a premature stop codon.

Viability and developmental potential of porcine blastocysts preserved for short term in a chemically defined medium at ambient temperature.

This study developed an efficient method for liquid storage of in vitro-derived porcine blastocysts at ambient temperature for 24 hr. We evaluated the effects of new chemically defined media (cell wash and preservation solution, Cellstor -W [Cell-W] and cell suspension and preservation solution, Cellstor -S [Cell-S]) for short-term storage. In the first experiment, in vitro-derived blastocyst were stored at 25ºC for 24 hr in Cell-W solution, Cell-S solution and pig embryo culture (PBM) medium.

Generation of mutant pigs by lipofection-mediated genome editing in embryos.

The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques.

Effects of individual or in-combination antioxidant supplementation during in vitro maturation culture on the developmental competence and quality of porcine embryos.

The oocyte maturation process requires a high supply of energy, which generates reactive oxygen species (ROS), adversely affecting oocyte and embryo development. Balancing ROS by antioxidant supplementation is essential for maintaining oocyte maturation and embryonic quality in vitro. This study aimed to evaluate the impact of four antioxidants: β-mercaptoethanol (β-ME), chlorogenic acid (CGA), curcumin and sericin, when applied individually or in combinations, during oocyte maturation on development of porcine oocytes.

Zona pellucida treatment before CRISPR/Cas9-mediated genome editing of porcine zygotes.

Background: Increasing the permeability of the zona pellucida (ZP) of oocytes before CRISPR/Cas9 electroporation may improve the efficiency of gene editing; however, the effects of this approach on subsequent developmental processes are unclear. In this study, the effects of ZP treatment before electroporation on embryonic development and gene editing in porcine embryos were evaluated.

Methods: The ZP of zygotes was weakened or removed by exposure to 0.

Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events.

Objective: Lipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection.

Improvement of the in vitro fertilization and embryo development using frozen-thawed spermatozoa of microminipigs.

This study aimed to compare the quality and the penetration ability of frozen-thawed spermatozoa from three microminipigs and Large White boars and to evaluate the effects of caffeine and heparin as well as the sperm-oocyte co-incubation length on the fertilization and embryonic development in vitro. Results showed that the fertilization rates of spermatozoa from three microminipig boars were significantly lower than those of a Large White boar. In the post-thaw spermatozoa from one of three microminipig boars, the sperm quality, penetration ability, and the oocyte development after in vitro fertilization were significantly lower than those of the spermatozoa from other boars.

Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos.

Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection.

Enrichment of bovine X-sperm using microfluidic dielectrophoretic chip: A proof-of- concept study.

The microfluidic dielectrophoretic (MF-DEP) chip is a new, economical and readily-available technology that might be used to enrich X-sperm for increasing female offspring in dairy farms. In this study, we sought to develop an MF-DEP chip to enrich X bovine sperm. The MF-DEP chip was composed of an electrode attached to a glass slide and a microchannel made from polydimethylsiloxane.

Development of IFN-Stimulated Gene Expression from Embryogenesis through Adulthood, with and without Constitutive MDA5 Pathway Activation.

Pathogen-associated molecular patterns (e.g., dsRNA) activate expression of IFN-stimulated genes (ISGs), which protect hosts from infection.

Cilostamide and forskolin maintain gap junction function of incubated dog follicles.

Disruption of the communication between the oocyte and granulosa cells is one of the major causes of poor development of in vitro grown ovarian follicles and oocytes. The present study investigated the effect of two cAMP modulators, cilostamide and forskolin, on in vitro growth of isolated dog secondary follicles and enclosed oocytes, communication between the gamete and surrounding granulosa cells, expression of GJA1 and GDF9, as well as cAMP level. Secondary follicles were incubated with cilostamide or forskolin alone or a combination of 20 μM cilostamide +1 μM forskolin, and the diameter of the incubated follicles and enclosed oocytes assessed every 72 h.

Effect of anti-apoptotic drug Z-VAD-FMK on in vitro viability of dog follicles.

It is recognized that ovarian follicular atresia is associated with apoptosis, and the most important effector of cell death is caspase-3. The aim of this study was to investigate the influence of anti-apoptotic drug Z-VAD-FMK on in vitro follicle growth in the domestic dog. Ovaries were obtained from peri-pubertal and adult domestic dogs, and cortical fragments recovered and incubated on 1.

Insulin promotes preantral follicle growth and antrum formation through temporal expression of genes regulating steroidogenesis and water transport in the cat.

The aims of the present study were to determine the effects of insulin, invitro, on: (1) the viability and growth of domestic cat ovarian follicles; (2) mRNA expression of genes regulating steroidogenesis (cytochrome P450 family 17 subfamily, A polypeptide 1 (Cyp17a1), cytochrome P450 family 19 subfamily, A polypeptide 1 (Cyp19a1) and steroidogenic acute regulatory protein (Star)) and water transport (aquaporins (AQPs) Aqp1, Aqp3, Aqp7, Aqp9); and (3) steroid production (17β-oestradiol (E2), progesterone (P4), androstenedione (A4)). Cat secondary follicles were isolated from ovarian cortices and cultured in 0 (Control), 1 or 10µgmL-1 insulin for 14 days (Day 0=culture onset). Follicle and oocyte viability (based on neutral red staining), diameter and antrum formation were assessed every 72h and at the end of incubation (Day 14).

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos.

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7.

Spermatozoa isolated from cat testes retain their structural integrity as well as a developmental potential after refrigeration for up to 7 days.

The objective of this study was to compare the efficiency of preservation media for isolated feline testicular spermatozoa as well as the concentrations of bovine serum albumin (BSA) on: (1) the membrane (sperm membrane integrity (SMI)) and DNA integrity of spermatozoa; and (2) the developmental potential of spermatozoa after intracytoplasmic sperm injection (ICSI). Isolated cat spermatozoa were stored in HEPES-M199 medium (HM) or Dulbecco's phosphate-buffered saline (DPBS) at 4°C for up to 7 days. Results indicated that HM maintained a better SMI than DPBS throughout the storage periods (P > 0.

Selective TGF-β1/ALK inhibitor improves neuronal differentiation of mouse embryonic stem cells.

The transforming growth factor-β1 (TGF-β1), a polypeptide member of the TGF-β superfamily, has myriad cellular functions, including cell fate differentiation. We hypothesized that suppression of TGF-β1 signaling would improve the efficacy of neuronal differentiation during embryoid body (EB) development. In this study, mouse embryonic stem cells (ESCs) were allowed to differentiate into their neuronal lineage, both with, and without the TGF-β1 inhibitor (A83-01).

Insulin-like growth factor-1 (IGF-1) enhances developmental competence of cat embryos cultured singly by modulating the expression of its receptor (IGF-1R) and reducing developmental block.

Objective: The aim of this study is to determine the effects of insulin-like growth factor-1 (IGF-1) and the mRNA expression of IGF-1 receptor (IGF-1R) during the in vitro development of cat embryos cultured in groups versus singly.

Methods: Cumulus-oocyte complexes (COCs) were matured and fertilized in vitro with frozen-thawed semen. Cleaved embryos (48h post-fertilization) were randomly assigned to one of the following treatments: 1) group embryo culture without IGF-1 (10 embryos per 50μl droplet), 2) single-embryo culture without IGF-1, and 3) to 6) single-embryo culture (50μl droplet per embryo) supplemented with different concentrations of IGF-1 (5, 25, 50 and 100ng/ml, respectively).

Characterization and in vitro culture of putative spermatogonial stem cells derived from feline testicular tissue.

Spermatogonial stem cells (SSCs) function to regulate the balance of self-renewal and differentiation of male gametes. SSCs have been successfully isolated and cultured in vitro in several species, but not in feline. Therefore, in this study, we aimed to culture and characterize feline SSCs.