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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., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

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]

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]

This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method. In experiment 2, the recovery rates, quality, and reactive oxygen species (ROS) concentrations of the spermatozoa selected using MIGLIS, washing, and Percoll DGC were compared. In experiment 3, the resultant in vitro fertilised embryos from spermatozoa selected using the three methods were evaluated for blastocyst formation rates and intracellular ROS concentrations at the 2–4 cell stage. The intracellular ROS concentrations were investigated using 2′, 7′-dichlorodihydrofluorescein diacetate staining. Using the MIGLIS device, significantly more spermatozoa were recovered at 2× dilution compared with the other dilution ratio, but the motility was not affected by the dilution ratio. On the selection of spermatozoa using the three methods, employing MIGLIS decreased the recovery rates. However, the MIGLIS method increased motility, viability, and acrosome integrity rates compared to those in spermatozoa from the other methods. The ROS concentration of spermatozoa in the MIGLIS method was significantly lower than that in the washing method. Nevertheless, blastocyst formation rates were similar among the three methods, but the ROS concentration of early-stage embryos produced using MIGLIS was significantly lower than those produced using Percoll DGC. In conclusion, the MIGLIS method has the potential to select functional, high-quality frozen-thawed bovine spermatozoa. • The MIGLIS method without centrifugation decreased the sperm recovery rates. • The MIGLIS method selected sperm with higher quality and lower ROS concentrations. • ROS concentrations of early-stage embryos produced using MIGLIS decreased. [ABSTRACT FROM AUTHOR]