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Genetically modified animal models play an important role in elucidating pathogenesis and developing therapeutic strategies for human diseases. Pigs are considered one of the best animal models because their anatomy and physiology are similar to those of humans (Fan & Lai, 2013; Niemann & Lucas-Hahn, 2012). Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) are part of a genome engineering method based on the bacterial CRISPR immune system and have been developed and widely used for gene editing to produce genetically modified pigs (Wang, Du, et al., 2015; Yu et al., 2016). In these previous studies, modern techniques, such as somatic cell nuclear transfer (SCNT) and microinjection for the production of genetically modified pigs were used. Recently,

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. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.

Abstract. The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZENcontaining (0−1000 μg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 μg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 μg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 μg/l may not affect the fertility of the oocytes.Key words: Oocytes, Porcine, Sperm, Zearalenone (J. Reprod. Dev. 57: [547][548][549][550] 2011) earalenone (ZEN) is a nonsteroidal estrogen-like mycotoxin produced by Fusarium species on several grains. It is an estrogen receptor agonist; its distinct estrogenic and anabolic properties in several animal species exert detrimental effects on the reproductive system resulting in reproductive disorders in domestic animals, particularly in swine [1][2][3][4]. Although in vitro culture systems do not always provide accurate predictions of toxicity in animals, they can be used to assess risks and can help to define the mechanisms by which mycotoxins act on germ cells [5]. Several in vitro culture assays have been employed to determine the effect of ZEN and its metabolites on the reproductive organs of swine. Previous in vitro experiments revealed that exposure to these mycotoxins affects oocyte maturation, pronucleus formation and embryonic development [6,7], as well as viability, motility and acrosome reactions in sperm [8,9]. Nevertheless, the acute effects of exposure to ZEN during in vitro fertilization remain unknown. This study aimed to examine the effects of exposure to ZEN on porcine oocytes and sperm by using an in vitro maturation (IVM) and in vitro fertilization (IVF) systems to assess the state of nuclear DNA damage and fertilization.As shown in Table 1, exposure to 1000 μg/l of ZEN had a negative effect on the meiotic competence of porcine oocytes (P<0.05). However, there were no significant differences among the groups with respect to the percentages of oocytes showing degeneration and DNA damage. Exposure to 100 and 1000 μg/l of ZEN during maturation culture decreased the total rate of sperm penetration (P<0.05) compared with the control group, but did not influence the rates of normal or polyspermic fertilization of oocytes.As shown in Table 2, exposure to ZEN during IVF did not affect total rates of sperm penetration irrespective of the ZEN concentration. However, exposure to 1000 μg/l of...

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long-term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5-SM and 15-SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P< 0.05), and the 7.5-SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5-SM and 15-SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5-SM group showed similar rates of fertilization and blastocyst formation in the fresh non-stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5-SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.

Chlorogenic acid (CGA) and caffeic acid (CA) are potent antioxidants that are mostly found in coffee beans. This study aimed to investigate the effects of CGA and CA supplementation during semen freezing on the quality of frozen-thawed boar spermatozoa. The antioxidants CGA and CA were added to a semen extender to achieve final concentrations of 50, 100, 200 and 400 µM. Supplementation of 100 µM CGA and CA yielded a significantly higher percentage of sperm viability (increased by 8%-10%) and plasma membrane integrity (increased by 4%-6%) than the control groups without the antioxidants at 0 and 3 hr after thawing (p < 0.05). At a concentration of 100 µM, CGA and CA also yielded beneficial effects on total and progressive sperm motility. Increases of CGA and CA concentrations to more than 200 µM did not enhance any sperm quality parameters. When the sperm penetrability and oocyte development by spermatozoa frozen with CGA and CA were evaluated, CGA and CA supplementations had no positive effects on the percentages of total fertilization, monospermic fertilization, cleavage and blastocyst formation. In conclusion, the supplementation of 100 µM CGA and CA during sperm freezing improved certain sperm parameters including motility, viability and plasma membrane integrity.

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. Results First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.

Abstract. Zearalenone (ZEN) and its metabolites are important nonsteroidal estrogenic mycotoxins that cause reproductive disorders in domestic animals, especially pigs. We aimed to simultaneously detect ZEN and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in porcine follicular fluid (FF) by liquid chromatographytandem mass spectrometry. ZEN and α-ZOL, but not β-ZOL, were detected in all pooled FF samples collected from coexisting follicles (diameter ≥ 6 mm) within 10 ovaries. Furthermore, ZEN and α-ZOL were detected in samples pretreated with β-glucuronidase/arylsulfatase, but not in those left untreated, suggesting that the FF samples contained glucuronide-conjugated forms of the mycotoxins that may be less harmful to porcine oocytes due to glucuronidation affecting the receptor binding. Nonetheless, the effects of the glucuronide-conjugated forms should be studied, both in vitro and in vivo. earalenone (ZEN) is a nonsteroidal estrogenic mycotoxin that is produced by Fusarium species on several grains. Despite its low acute toxicity and carcinogenicity, ZEN and its metabolites exhibit distinct estrogenic and anabolic properties in several animal species because of their agonistic effect on the estrogenic receptor. Thus, they affect the reproductive system and play important roles in reproductive disorders in domestic animals, particularly swine [1][2][3][4].The presence of environmental pollutants with potential reproductive toxicity in the follicular fluid (FF) of livestock may be of particular importance because the oocyte completes maturation before ovulation within the FF [5]. Recently, we reported that the FF was naturally contaminated with ZEN and its metabolites and showed the in vitro effects of ZEN on oocyte maturation in cattle [6]. However, no reports are available on the contamination of porcine FF by ZEN and its metabolites.The objectives of this preliminary investigation were to determine the concentrations of ZEN and its metabolites, including α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL), in porcine FF by using liquid chromatography-negative ion electrospray tandem mass spectrometry with electrospray ionization (LC/MS/MS).As shown in Table 1, ZEN and α-ZOL were detected in all 10 FF samples supplemented with β-glucuronidase/arylsulfatase solution during preincubation, while the 10 FF samples that were not treated with β-glucuronidase/arylsulfatase solution did not contain ZEN or α-ZOL, even at trace levels. Additionally, β-ZOL was not detected even at trace levels, irrespective of β-glucuronidase/arylsulfatase treatment. Figure 1 shows representative LC/MS/MS chromatograms of the porcine FF samples contaminated with ZEN and α-ZOL. The mean (± SEM) concentrations of ZEN and α-ZOL were 38.9 ± 4.0 pg/ml (max and min: 54.8 and 15.2 pg/ml, respectively) and 17.6 ± 1.7 pg/ml (max and min: 26.4 and 10.0 pg/ ml, respectively), respectively.In the present study, we detected ZEN and α-ZOL but not β-ZOL in porcine FF only after the FF was treated with β-glucuronidase/arylsulfatase solution...