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. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen-thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen-thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize in vitro-matured oocytes and cultured in vitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high-quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation.

Development of porcine embryos cultured in media irradiated with ultraviolet-C.

Sterilization of the culture medium using ultraviolet (UV)-C reduces the potential adverse effects of microorganisms and allows for long-term use. In the present study, we investigated the effects of a medium directly irradiated with UV-C prior to in vitro culture on the development and quality of porcine in vitro-fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM-5] and porcine blastocyst medium [PBM]) were irradiated with UV-C at 228 and 260 nm for 1 and 3 days, respectively. Next, the culture media were irradiated with UV-C at 228 nm for 3, 7, or 14 days. After in vitro fertilization, the embryos were cultured in the UV-C-irradiated media for 7 days. Free amino acid levels in culture media irradiated with 228 and 260 nm UV-C for 3 days were analysed. The blastocyst formation rate of embryos cultured in media irradiated with 260 nm UV-C for 3 days was significantly lower than that of embryos cultured in non-irradiated control media. However, 228 nm UV-C irradiation for up to 14 days did not affect blastocyst formation rates and quality in the resulting blastocysts. Moreover, 260 nm UV-C irradiation significantly increased the taurine concentration in both culture media and decreased methionine concentration in the PBM. In conclusion, UV-C irradiation at 228 nm before in vitro culture had no detrimental effects on embryonic development. However, 260 nm UV-C irradiation decreased embryo development and altered the composition of free amino acids in the medium.

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 < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0, 50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods.

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. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs.

Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes.

Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs.

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. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.

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. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency.