Effects of ergothioneine supplementation on meiotic competence and porcine oocyte development.

Background And Aim: The antioxidant effects of ergothioneine (EGT) on culture of porcine zygote are not well established. The study investigated the impact of EGT supplementation on meiotic competence and development of porcine oocytes.

Materials And Methods: The impact of EGT concentrations (0, 5, 10, 25, 50, and 100 μM) during maturation (IVM) on the progression of meiotic maturation, fertilization, and blastocyst formation in porcine oocytes was assessed.

Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h.

Quality and fertilizing ability of frozen-thawed porcine sperm separated using a migration sedimentation method.

We evaluated the quality and fertilizing ability of frozen-thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen-thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device.

Effects of centrifugation treatment before electroporation on gene editing in pig embryos.

Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation.

Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes.

The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates.

Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs.

The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency.

Effects of ergothioneine supplementation on the quality of liquid-preserved and frozen-thawed boar semen.

This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at 15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.

Bactericidal Efficacy and Mechanisms of Non-Electrolytic Slightly Acidic Hypochlorous Water on and .

Chilled pork is frequently contaminated with and . In this study, the bactericidal efficacy and mechanisms of non-electrolytic slightly acidic hypochlorous water (NE-SAHW) against two strains of these two species were evaluated. The results showed that the antibacterial efficacy of NE-SAHW was positively correlated with the concentration level of NE-SAHW and negatively correlated with the initial populations of the strains.

Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos.

The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization.

GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system.

Background: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models.

Effects of curcumin supplementation on quality of porcine spermatozoa irradiated with ultraviolet-C at 228 nm during liquid preservation.

It is important to prevent microbial contamination during liquid preservation of semen in pigs. We examined the effects of curcumin supplementation on the quality of porcine spermatozoa irradiated with ultraviolet-C (UV-C) at 228 nm. UV-C is used to disinfect gases and solid surfaces.

Pigs with an point mutation derived from zygotes electroporated with CRISPR/Cas9 and ssODN.

Just one amino acid at the carboxy-terminus of the B chain distinguishes human insulin from porcine insulin. By introducing a precise point mutation into the porcine () gene, we were able to generate genetically modified pigs that secreted human insulin; these pigs may be suitable donors for islet xenotransplantation. The electroporation of the CRISPR/Cas9 gene-editing system into zygotes is frequently used to establish genetically modified rodents, as it requires less time and no micromanipulation.

Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods.

Background And Aim: Mosaicism - the presence of both wild-type and mutant alleles - is a serious problem for zygotic gene modification through gene editing using the Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR/Cas9) system. Different delivery methods, such as microinjection (MI), electroporation (EP), and transfection (TF), can be used to transfer CRISPR/Cas9 components into porcine zygotes. This study aimed to develop a method that combines MI, EP, and TF to improve mutation efficiency mediated through the CRISPR/Cas9 system for a triple-gene knockout in pigs.

Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection.

Background And Aim: We previously developed the gene-editing by electroporation (EP) of Cas9 protein method, in which the CRISPR/Cas9 system was introduced into porcine fertilized (IVF) zygotes through EP to disrupt a target gene. This method should be further developed, and a combination of EP and MI methods should be evaluated in pigs. This study aimed to determine that a combination of microinjection (MI) and EP of CRISPR/Cas9 system could increase the rates of biallelic mutation for triple-gene knockout in porcine blastocysts.

Effects of the timing of electroporation during maturation on triple gene editing in porcine embryos using CRISPR/Cas9 system.

Mosaicism, including alleles comprising both wild-type and mutant, is a serious problem for gene modification by gene editing using electroporation. One-step generation of F0 pigs with completely desired gene modifications saves cost and time, but the major obstacles have been mosaic mutations. We hypothesized that the timing of electroporation prior to fertilization (IVF) can increase the rates of biallelic mutation for multiple gene knockout as the permeability of mature oocytes is greater than that of zygotes.

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.

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.

Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides.

This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2).

Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages.

Objective: Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos.

Curcumin supplementation in the maturation medium improves the maturation, fertilisation and developmental competence of porcine oocytes.

This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.