This study evaluated a modified plastic straw method for vitrification of in vitro-produced (IVP) bovine blastocysts. A modified straw was used that has a depressed area on its inner surface to which embryos attach. The IVP blastocysts were randomly assigned into 3 groups: (1) attachment of embryos to the inner surface of a plastic straw (aV), (2) attachment of embryos to the inner surface of a modified plastic straw (maV), and (3) non-vitrified (control). The recovery rates of blastocysts were not significantly different between the aV and maV groups (95.8 v. 94.3%; P > 0.05). The survival of post-thaw blastocysts did not significantly differ between the aV and maV groups (86.4 v. 88.2%; P > 0.05). The total cell number of blastocysts was significantly higher in the control group than in the aV and maV groups (142 ± 21.8 v. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not different between the aV and maV groups. The mRNA levels of the pro-apoptosis-related genes Bax and caspase-3 were significantly higher in the aV and maV groups than in the control group. By contrast, the mRNA levels of Bcl-2 and Mcl-1, which are anti-apoptotic genes, and of MnSOD and Prdx5, which are antioxidant-related genes, were significantly lower in the aV and maV groups than in the control group (P < 0.05). In conclusion, the aV and maV methods can be used to vitrify IVP bovine blastocysts, and embryos are more easily loaded using the maV method than using the aV method. This work was partly supported by the Next-Generation BioGreen 21 Program (grant no. PJ009587022013), IPET (grant no. 110020-5 and 112020-3), and a scholarship from the BK21 Program, Republic of Korea.
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.
Serum has widely been used as a main supplement to embryo in vitro culture media as it contains embryotrophic factors. Charcoal:dextran treatment of fetal bovine serum (FBS) removes lipophilic chemicals and certain steroid hormones and growth factors. The objective of this study was to investigate the effects of charcoal:dextran-stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium (SOF-BE1 medium supplemented with 10% of serum) on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The experiment was conducted in 6 replicates (350 oocytes per group). The differences in embryo development, integrated optical intensity, and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P < 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% v. 36.85 ± 0.89%, respectively). The total number of cells per Day 8 blastocyst was not significantly different (P > 0.05) between the CDS FBS group (208.40 ± 14.77) and the HI FBS group (195.11 ± 19.15). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly increased mitochondrial activity, as identified by MitoTracker Green, and reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P < 0.05) higher after 24 h in the CDS FBS than in the HI FBS group (85.33 ± 4.84% v. 68.67 ± 1.20%). Quantitative reverse transcription PCR showed that the mRNA levels of lipid metabolism-related genes, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, and the cholesterol metabolism related gene hydroxymethylglutaryl-CoA reductase were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of survival gene sirtuin 1, antioxidant gene superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen–thawed blastocysts were significantly (P < 0.05) higher in the CDS FBS group than in the HI FBS group; however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. In conclusion, these data suggest that supplementation of in vitro culture medium with CDS FBS improves in vitro bovine embryo developmental competence and the quality of blastocysts in terms of their crytolerance and gene expression. This research was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets), BK21plus, and KGSP.
Melatonin, the antioxidant pineal hormone, is a strong regulator for various cellular processes essential for reproduction. Although the protective role of 0.1µM melatonin against the toxicity of different anti-developmental compounds has been elucidated in numerous studies, its effect on the autophagy level in invitro-produced blastocysts has not been entirely clarified. In this study, oocytes were incubated for 24h in the presence and absence of melatonin, administered during IVM, to investigate the effect of 0.1µM melatonin on the developmental competence of bovine oocytes and pre-implantation embryos, autophagy, and quality of embryos. The developmental potential of embryos were basically the stages from oocytes fertilization to blastocyst production. Gene expression levels were evaluated in matured oocytes, whereas blastocysts were used for immunofluorescence experiments. The differences between treated and control groups were analysed using Student’s t-test (GraphPad Prism version 6; GraphPad Inc.), where P-values <0.05 were considered significant. Results showed that oocyte maturation, Day-4 total cleavage, and Day-8 blastocyst development rates were not significantly improved (melatonin: 72±2 vs. control: 69±2 for cleavage rate, and melatonin: 33±1 vs. control: 31±2 for control for Day-8 blastocyst; P>0.05), whereas the level of reactive oxygen species (ROS) was reduced (P<0.05) with addition of melatonin. Using RT-qPCR, cumulus cells-related (HAS2) and apoptosis-related (Bcl2 and SOD2) genes were upregulated, whereas BAX was downregulated in melatonin-treated oocytes. Using immunofluorescence, apoptosis (caspase-3) and autophagy (Beclin-1 and LC3) markers were underexpressed, whereas the PI3K survival protein (P<0.05) and matrix metalloproteinases (MMP-2 and MMP-9; P>0.05) were overexpressed, in Day-8 embryos of melatonin-treatment. Additionally, the total number of cells per blastocysts, inspected via nuclei-based 4′,6-diamidino-2-phenylindole (DAPI) staining was higher in the melatonin-treated group (P<0.05). Taken together, our study demonstrates that 0.1µM melatonin treatment during IVM does not interfere with developmental competence, but improves the quality of IVF-produced embryos by lowering the incidence of autophagy.
The 2-methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum. The objective of this study was to investigate the effects of 2-MS on oocyte maturation, blastocyst development, and embryo quality in terms of cell number and gene expression in vitro. A total of 2364 oocytes were cultured in TCM-199 supplemented with 10% fetal bovine serum, 1 μg mL−1 oestradiol-17β, 10 μg mL−1 FSH, 10 ng mL−1 epidermal growth factor, 0.6 mM cysteine, and 0.2 mM sodium pyruvate and supplemented with different concentrations of 2-MS as following: 1.5 μM (n = 458), 1.0 (n = 493), 0.5 (n = 468), 0.1 (n = 470), and 0 μM (control, n = 475) followed by IVF and then culture in CR1-aa medium supplemented with 44 μg mL−1 sodium pyruvate, 14.6 μg mL−1 glutamine, 10 μL mL−1 penicillin-streptomycin, 3 mg mL−1 BSA, and 310 μg mL−1 glutathione for the first 3 days, and then the BSA was replaced with 10% FBS until Day 8. The differences in embryo development between experimental groups were analysed by one-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The results showed that the addition of 2-MS at 1.0 mM significantly improved (P < 0.05) the percentage of MII oocytes, which were identified by aceto-orcein staining, compared with that in the control (76.5 v. 65.4%, respectively), and remarkably (P < 0.05), improved blastocyst development rates (45.29%) compared with control (32.21%). Additionally, TUNEL assay demonstrated that treatment with 1.0 μM of 2-MS significantly improved the embryo quality by increasing total number of cells and reducing DNA damage. Immunofluorescent analysis showed that the protein levels of nuclear factor-kappa B (NFkB), inhibitor of kappa B kinase β (IkKβ), 8-oxoguanine, and cyclooxygenase-2 (COX2) declined significantly (P < 0.05) after 2-MS treatment compared with the control. These results were confirmed by qRT-PCR, which showed a significant decrease in the mRNA levels of NFkB, IkKβ, COX2, inducible nitric oxide synthase (iNOS), BCL2-associated X protein (BAX), caspase-3, and Janus kinase2 (JAK2) after 2-MS treatment; however, the mRNA level of the anti-apoptotic gene B-cell lymphoma2 (BCL2) was significantly higher than that in the control. In conclusion, the addition of 2-MS at the indicated concentration dramatically improves the developmental competence of bovine in vitro-produced embryos. This work was supported by a grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017–5), BK21plus, and KGSP.
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