Effects of voltage strength during electropolation on the development and quality of in vitro-produced porcine embryos
Electroporation conditions and embryonic development
Nishio, Katsutoshi Tokushima University
Nguyen, Thanh-Van Tokushima University
Kunihara, Toshiki Tokushima University
Nii, Masahiro Tokushima Prefectural Livestock Research Institute
in vitro fertilization
This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration, and pulse number fixed at 30 V/mm, 1 msec, and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm – 40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < 0.05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < 0.05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.
Reproduction in Domestic Animals
This is the peer reviewed version of the following article: Nishio, K, Tanihara, F, Nguyen, T-V, et al. Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos. Reprod Dom Anim. 2018; 53: 313– 318., which has been published in final form at https://doi.org/10.1111/rda.13106. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
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