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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. In the first experiment, porcine semen was preserved with 0, 10, 25, 50 or 100 μM curcumin under UV‐C irradiation at 228 nm for 7 days at 15°C. The irradiation did not affect the motility and viability of preserved spermatozoa but decreased the percentage of plasma membrane integrity of spermatozoa. Curcumin supplementation at 25 μM significantly improved the plasma membrane and acrosome integrity of irradiated spermatozoa compared with spermatozoa preserved without curcumin (p <.05). In the second experiment, semen was preserved with or without 25 μM curcumin with UV‐C irradiation at 228 or 260 nm for 3 days at 15°C. Curcumin supplementation increased the percentages of total motility, sperm viability and plasma membrane integrity of preserved spermatozoa at both irradiation wavelengths (p <.05). All quality parameters of 260 nm irradiated spermatozoa decreased compared to those of the other groups, irrespective of curcumin supplementation. The collective findings indicate that porcine spermatozoa can retain their viability even after continuous long‐duration irradiation with 228 nm UV‐C. Curcumin supplementation improves the quality of UV‐C irradiated spermatozoa during semen preservation. [ABSTRACT FROM AUTHOR]

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. In Experiment 2, optimization of timing was performed at 10 or 29 h post‐in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry‐based technology needs further improvement, especially regarding editing efficiency and embryo development. [ABSTRACT FROM AUTHOR]

Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR‐Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene‐deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3‐galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene‐knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild‐type and gene‐deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene‐deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats‐CRISPR‐associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation‐ and lipofection‐mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)‐intact and ‐free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP‐intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9‐based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP‐intact or ‐free zygotes. [ABSTRACT FROM AUTHOR]