In vitro embryo developmental potentials are still suboptimal compared with in vivo potential due to the challenge of various unknown stressors that must be overcome by in vitro-cultured oocytes. To improve existing embryo developmental potentials, many chemicals have been treated in maturation media by dissolving in toxic substances such as dimethyl sulfoxide (DMSO) or other carrier molecule. The foremost effort of this study was to investigate the impact of the solvent tetrahydrofuran (THF) on the cytotoxicity of in vitro embryo production (IVP). The experiment was completed within 8 replicates. Statistical analyses were performed using SPSS version 22.0 (IBM/SPSS, Armonk, NY, USA), a one-way ANOVA followed by multiple pairwise comparisons (Tukey’s test), and Duncan’s multiple range post hoc test. The level of statistical significance was considered P < 0.05. Oocytes were cultured in vitro maturation media (IVM) followed by in vitro fertilization (IVF), in vitro culture media 1 (IVC1), and in vitro culture media 2 (IVC2). Composition of the media was as follows: IVM medium was TCM-199 supplemented with 10% (v/v) fetal bovine serum, 1 µg mL−1 oestradiol-17β, 10 µg mL−1 FSH, 0.6 mM cysteine, and 0.2 mM sodium pyruvate. The IVC1 medium consisted of CR1-aa supplemented with 44 µg mL−1 sodium pyruvate, 14.6 µg mL−1 glutamine, 10 IU mL−1 penicillin, 0.1 mg mL−1 streptomycin, 3 mg mL−1 BSA, and 310 µg mL−1 glutathione. The IVC2 medium was the same composition as IVC1 except that BSA was replaced with 10% (v/v) fetal bovine serum. The final concentration of the optimized (0.5 µM) THF in culture medium was 0.4%. When coculturing with 0.5 µM THF in the IVM stage, the cleavage rate (58.65 ± 1.90% v. 56.87 ± 1.68%) was not significantly different, but the blastocyst rate (35.21 ± 1.44% v. 28.34 ± 2.11%) was significantly higher compared with the control group. The TUNEL assay confirmed that apoptotic nuclei in THF group were significantly reduced compared with the control group (2.32 ± 0.14 v. 5.65 ± 0.12). The total cell number of trophectoderm (TE) in control and THF groups was 115.34 ± 0.98 and 132.13 ± 1.55, and that of the inner cell mass (ICM) was 29.67 ± 0.40 and 39.94 ± 0.44, respectively. However, the ICM:TE ratio in control and treated blastocysts was 1:3.34 and 1:3.9, which was not statistically significant. Immunocytochemistry analysis (using antibodies to IKBKB, NFkB, COX2, CASP9, and CASP3) demonstrated that THF supplementation significantly attenuated expression of these proteins. The quantitative recerse transcription PCR data established that relative mRNA expression level of the anti-apoptotic gene BCL2 was up-regulated, whereas that of COX2, iNOS, BAX, IKBKB, NFkB, CASP9, and CASP3 were significantly down-regulated in the THF treated group compared with the control. In conclusion, 0.5 µM THF supplement in the IVM media did not have injurious effects on in vitro-cultured bovine embryos.
This work was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets) and BK21plus.
Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity
