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Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.

This study elucidated the molecular markers that decrease oocyte quality during in vitro culture, restricting optimal developmental potential. Here, we evaluated the transcriptomic differences betw...

Lupeol is a triterpene with various pharmacological properties. This study investigated the effect of lupeol on the in vitro development of bovine embryos. Oocytes (270 per group, 1620 in total) obtained from slaughterhouse-derived ovaries were matured and fertilized in vitro and then cultured for 8 days in a humidified atmosphere of 5% CO2 in air at 38.5 °C. The in vitro maturation medium was supplemented with 0.5, 1.0, 2.0, 3.0, and 4.0 μM lupeol. Treatment with 2.0 μM lupeol significantly (P

2-Methoxystypandrone (2-MS), a naphthoquinone from Polygonum cuspidatum, has been reported to exhibit antioxidant activities. However, its pharmacological properties have not been fully studied in vitro culture system of oocytes and embryos development. In this study, we examined the effects of 2-MS on bovine blastocyst developmental competency during preimplantation using TUNEL assay, qRT-PCR and immunocytochemistry. Oocytes were treated with 1 μM 2-MS during maturation, followed by in vitro fertilized and the zygotes were cultured. We observed that blastocyst development was significantly higher in the 2-MS treated oocytes (45.29%) compared to control groups (32.21%). We also found that 2-MS treatment significantly increased total cell number and decreased the apoptotic cells in Day-8 blastocyst. Finally, we examined the signal pathway affecting the embryo development after the 2-MS treatment. We detected a significant decrease in the mRNA levels of NFKB1, IKBKB, COX2, NOS2, BAX, CASP3, and JAK2 after 2-MS treatment, however, the mRNA level of the anti-apoptotic gene BCL2 was significantly higher than that in the control. Moreover, protein expression levels of NFKB1, IKBKB, and COX2, and 8-oxoG DNA lesion decreased significantly in the 2-MS treatment group, compared to the control. In summary, our findings suggested that treatment of oocytes with a potential anti-oxidant, 2-MS during IVM improved in vitro developmental competence of bovine embryos.

Sex preselection has always generated great interest among livestock producers. Among the prevalent sperm sorting methods, there is much evidence that sex sorting has a negative effect on sperm quality with an altered pattern of sperm motility, ultimately reducing the period of cell viability. In this study, we have established a new approach for the preselected embryo production by using WholeMom®; a monoclonal antibody developed against bull sperm epitopes for simple and easy separation of X- and Y-sperm. There were no significant differences (P 0.05) in the percentage of presumptive zygotes between the control and the X-sperm sorted group, but there was a difference in early cleaving embryos with there being 81.2 ± 1.4%, 78.3 ± 1.0%, and 66.7 ± 1.1% for the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Similarly, the percentage of embryos that developed to the blastocyst stage (Day 7) were also greater (P 0.05) in the control and X-sperm sorted group compared with the Y-sperm sorted group being 34.8 ± 1.0%, 32.1 ± 0.8%, and 23.7 ± 1.0% in the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Furthermore, B-SRY F2 and B-SRY R2 gene expression data indicated there was a detection accuracy of 81.0% for the female embryos and 72.5% for the male embryos produced in vitro. In conclusion, in cattle in vitro derived embryo production using pre-selected sexed semen and subsequent embryo transfer can facilitate the mass production of individuals that are genetically superior.

This study investigated the use of bovine serum albumin (BSA) plus insulin–transferrin–sodium selenite (ITS) and/or epidermal growth factor (EGF) as alternatives to fetal bovine serum (FBS) in embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, gene expression and cryotolerance, as well as the invasion ability of trophoblasts. The percentage of embryos that underwent cleavage and formed a blastocyst was higher (P

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 μM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p

Heat stress has large effects on reproduction including conception rate in cattle. In this study, we examined the effects of coagulansin-A (coa-A), a steroidal lactone, on acquired thermo tolerance duringin vitroproduction of bovine embryos. Oocytes were incubated inin vitromaturation (IVM) media with or without coa-A at two different temperatures, 40.5˚C and 42˚C, for 20 h. The treatment of coa-A significantly improved blastocyst development only at 40.5˚C (P HSP70andPI3Kwas elevated, whereas expression ofNF-κB,COX2and inducible nitric oxide synthase (iNOS) was significantly (P in vitrobut also increased their resistance to heat stress condition through induction ofHSP70/PI3K.

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

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

The aim of the present study was to investigate the beneficial effect of polydatin (PD), the glycoside form of resveratrol, on embryo development in vitro. Oocytes were aspirated from ovaries of Korean Hanwoo cows and cultured until Day 8 in a humidified atmosphere of 5% CO2 in air at 38.5°C. Protein and gene expression levels were determined through confocal microscopy and reverse transcription–polymerase chain reaction respectively, whereas the number of total and apoptotic cells in Day 8 blastocysts was determined using Hoechst 33342 staining and terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling. Of the different concentrations of PD (0.5, 1.0 and 2.0 µM) added to the IVM medium, only 1.0 µM PD significantly improved blastocyst development. Immunofluorescence analysis confirmed that protein levels of sirtuin 1 (Sirt1) increased significantly (P

The domestic cat (Felis catus) is a useful animal model for biomedical research because it shares many devastating diseases with humans. For example, feline immunodeficiency virus-1 is structurally and functionally similar to human immunodeficiency virus-1 (HIV-1). In vitro, the replication of HIV-1 in cat cells is restricted by the feline APOBEC3H (fA3H) and APOBEC3CH (fA3CH). Accordingly, we hypothesised that cats could be used to study HIV-1 infection in humans if fA3H and fA3CH were knocked out. However, due to limited availability of genomic editing tools in the cat, genetic modification has not been widely reported in this species. Here, we show that the fA3H/fA3CH locus could be knocked out in cat using the CRISPR/Cas9 system. Taking advantage of the fact that the fA3H and fA3CH genes share their last four exons (exons 2–5 for fA3H and exons 5–8 for fA3CH), we PCR-amplified and analysed the partial sequence of fA3H and fA3CH and designed a single guide (sg) RNA targeting exon 3 of fA3H (exon 6 of fA3CH) to achieve double knockout of these 2 genes. After transfecting cells with a sgRNA/Cas9 expression DNA vector or co-transfecting the sgRNA along with Cas9 mRNA, which were prepared by in vitro transcription into cat embryonic fibroblast cells, in vitro gene-targeting analysis revealed that the CRISPR/Cas9 system could introduce indels with high efficiency: 23 and 41% of cat fibroblast cells were targeted in the fA3H/fA3CH locus when Cas9 was introduced in the DNA and mRNA forms, respectively. We chose to perform cytoplasmic microinjection injections 6 h post-IVF with 10 pL of injection solution containing 50 ng µL−1 for sgRNA and 100 ng µL−1 for Cas9 mRNA at a volume ratio of 1:1. We achieved a 95.5% embryo survival rate and a 22.9% blastocyst formation rate. High gene-targeting efficiency (62.5%, 15/24) from single blastocyst was achieved in targeting the fA3H/fA3CH locus by PCR-RFLP assay. We transferred 45 injected embryos to the uterine tubes of 4 pseudo-pregnant queens within 2 h of injection. Two of the recipients were determined to be pregnant by ultrasound on Day 30 after embryo transfer, and gave birth to 6 live kittens on Day 65. Genotyping analysis revealed that 2 kittens were successfully targeted in the fA3H/fA3CH locus: kitten 1 was biallelically targeted, whereas kitten 5 was monoallelically targeted. Sanger sequencing of the PCR products subcloned into TA cloning vectors showed that kitten 1 carried the same 1-nucleotide deletion on both alleles, and that kitten 5 also carried the same 1-nucleotide deletion monoallelically. Therefore, we conclude that the fA3H/fA3CH locus in the 2 kittens is specifically targeted by the CRISPR/Cas9 system. To our knowledge, this is the first report of successful site-specific genetic modification in the domestic cat achieved using the CRISPR/Cas9 system and the production of live fA3H/fA3CH double-knockout kittens to study the HIV-1 infected diseases. Supported by the BioGreen 21 (PJ01107703, PJ01107704), Felix Pets, and BK21+ program, Republic of Korea.