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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...