Post-thaw viability, developmental and molecular deviations in in vitro produced bovine embryos cultured with l-carnitine at different levels of fetal calf serum.

l-carnitine is a well-known an antioxidant that enhanced lipid metabolism. Therefore, this study investigated the influence of supplementing l-carnitine (LC) to in vitro culture medium on preimplantation development, quality, cryotolerance and transcription profile of candidate genes. Following in vitro fertilization, embryos at zygote stage were cultured with medium supplemented with LC at 1.5 mM and fetal calf serum (FCS) at 0, 2.5, 5, 7.5 and 10% of the CR1-aa culture media. Intracellular quality of produced embryos was measured using different fluorescent stains that measured reactive oxygen species (ROS), lipid and mitochondria intensities. In addition, total cell number and total apoptotic cells were counted per embryo. Quantitative expression of candidate genes was conducted to find out molecular response of embryos after treatment. Moreover, vitrification was done at day 8 of preimplantation development to evaluate post-thaw embryo viability. The results indicated improved blastocyst formation rate at day 8 of preimplantation development (day zero = day of IVF) when embryos cultured with LC supplementation at low FCS at levels of 2.5% (35.3%) and 5% (34.7%) compared to control (25.9%), LC + FCS 7.5% (26.5%) and LC + FCS 10% (28.1%) groups. The total number of blastocyst cells that were cultured with LC + FCS 2.5% and LC + FCS 5% was increased and the number of dead cells (apoptotic) was decreased compared to control counterparts. Intracellular mitochondria activity was enhanced and resulted in reduction of cytoplasmic lipid in embryos treated with LC + FCS 2.5% and LC + FCS 5% compared with other experimental embryo groups. In addition, intracellular reactive oxygen species level was reduced in LC + FCS 2.5%, LC + FCS 5% and LC + FCS 7.5% compared to control and LC + FCS 10% groups. The expression profile of genes regulating embryo quality (BCL2), metabolic activity (GLUT1, CPT2 and TFAM), lipolysis (LIPE, AMPKa1 and ACCα), resistance to stress (SOD2) and ability to induce pregnancy (IFNt) was up-regulated under low FCS (2.5% and 5%) combined with LC supplementation. On the other hand, genes regulating lipogenesis were down-regulated (ACSL3 and S1PR). It can be concluded that LC is an efficient culture media supplement when added with FCS at 2.5 and 5% which improved blastocyst development rate and quality. These improvements are due to enhanced utilization of intracellular embryo lipid that subsequently increased cryotolerance through orchestrating genes involved in various activities of bovine embryos.

Coagulansin-A has beneficial effects on the development of bovine embryos in vitro via HSP70 induction.

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans which belong to Solanaceae family. The present study investigated the effects of coagulansin-A on bovine oocyte maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To determine whether coagulansin-A has beneficial effects on bovine oocyte maturation in vitro, 355 oocytes per group (control and treated) in seven replicates were subjected with different concentrations (1, 2.5, 5, 7.5 and 10 µM) of coagulansin-A. The coagulansin-A was added in the in vitro maturation (IVM) media followed by in vitro fertilization (IVF) and then in vitro culture (IVC). Only treatment with 5 µM coagulansin-A remarkably (P<0.05) improved embryos development (Day 8 blastocyst) having 27.30 and 40.01% for control and coagulansin-A treated groups respectively. Treatment with 5 µM coagulansin-A significantly induced activation of heat shock protein 70 (HSP70) (P<0.05). Immunofluorescence analysis revealed that 5 µM coagulansin-A treatment also significantly inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing 8-oxoguanosine (8-OxoG) (P<0.05) and nuclear factor-κB (NF-κB) (P<0.05). The expressions of HSP70 and NF-κB were also conformed through real-time PCR (RT-PCR). Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay confirmed that coagulansin-A treatment significantly improved the embryo quality and reduced bovine embryo DNA damage (P<0.05). The present study provides new information regarding the mechanisms by which coagulansin-A promotes bovine embryo development in vitro.

Effect of peroxiredoxin II on the quality and mitochondrial activity of pre-implantation bovine embryos.

Endogenous peroxiredoxin II (PRDX II) protein plays a vital role in early embryonic development. This study assessed the beneficial effects of exogenous PRDX II on bovine embryo development at the cellular and molecular levels. To this end, in vitro maturation (IVM) medium was supplemented with various concentrations of PRDX II (0, 6.25, 12.5, 25, 50, and 100µg/mL). Of these, 12.5µg/mL PRDX II was the most effective and significantly promoted embryonic development. Therefore, this concentration of PRDX II was used in subsequent experiments. The percentage of embryos that developed into Day 8 blastocysts and the total number of cells per blastocyst (38.2% and 150.6±5.1) was higher in the PRDX II-treated group than in the control (26.4% and 128.9±3.9, respectively). Moreover, the percent of TUNEL positive cells was higher (P<0.05) in the control than in the PRDX II-treated. Furthermore, PRDX II added to the IVM media increased mitochondria content in blastocysts and decreased the intracellular ROS levels in oocytes and blastocysts compared with the control (P<0.05). The expression of genes associated with blastocyst quality (CDX2 and IFNτ), antioxidant activity (SOD2), and mitochondrial activity (TFAM) was higher, whereas the expression of a gene involved in the apoptotic pathway (c-FOS) was lower, in the PRDX II-treated than in the control group. In conclusion, supplementation of IVM medium with PRDX II promotes development to the blastocyst stage and improves blastocyst quality through reducing ROS, enhancing embryonic mitochondrial activity, and modulating development- related target genes expression.

Differential expression of selected candidate genes in bovine embryos produced in vitro and cultured with chemicals modulating lipid metabolism.

Lipid accumulated in embryos produced in vitro has been linked to reductions in both quality and postcryopreservation viability. Therefore, the objective of the present study was to investigate the influence of lipid-reducing chemicals on embryo development, quality, and postcryopreservation viability, in addition to expression profiles of selected lipid metabolism-regulating genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro; eight-cell stage embryos were cultured in IVC medium supplemented with phenazine ethosulfate (PES), L-carnitine (LC), PES + LC, or no supplementation (control). Culturing embryos in medium with LC increased (P < 0.05) blastocyst rate (38.8%) compared with the other groups (control = 28.1%, PES = 27.1%, PES + LC = 26.3%). Embryos cultured with supplements had greater total cell number and fewer apoptotic cells than the control. Cytoplasmic lipid content was reduced, whereas mitochondria density was increased in embryos treated with culture supplements; this was linked to altered expression profiles of selected genes regulating lipid metabolism. For example, transcript abundance of transmembrane lipid gene (SGPP1) was greater in LC- and PES-treated embryos, and they had increased postcryopreservation hatching ability (indicative of embryo cryotolerance). In conclusion, the two lipid metabolism regulators added to the culture media had improved embryo quality and cryotolerance, but embryo development rate and downstream lipid metabolism-regulating genes were more influenced with LC supplementation.

Mitochondrial content and gene expression profiles in oocyte-derived embryos of cattle selected on the basis of brilliant cresyl blue staining.

The aim of this study was to investigate the developmental rate, lipid and mitochondrial distribution and gene expression in oocyte-derived embryos selected on the basis of brilliant cresyl blue (BCB) staining. Lipid content and mitochondrial distribution in Day 8 blastocysts were evaluated by fluorescence intensity, while gene expression was analyzed by real-time PCR. The proportion of blastocysts (30.9%) was greater (P<0.05) in BCB+ than in BCB- oocytes (13%) but not different (P>0.05) from the control group (28.2%). Total cell number was also greater in BCB+ (155.1 ± 36.2) than in BCB- (116.6 ± 40.5) and control (127.5 ± 35.7) blastocysts. Furthermore, the apoptotic cell number was less in BCB+ (3.7 ± 4.4) than in BCB- blastocysts (8.7 ± 8.7) but not different from the control group (5.9 ± 3.9). BCB+ embryos contained more mitochondria compared to BCB- embryos (P<0.05). There was no significant difference in intercellular lipid accumulation in embryos from all groups. Interferon-τ (IFNτ), transforming growth factor ß1 (TGFB1) and secreted seminal-vesicle Ly-6 protein 1 (SSLP1) gene expression was greater in BCB+ than in BCB- blastocysts. By contrast, Bcl2-associated X protein (BAX) and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) gene expression was greater in BCB- than in BCB+ and control embryos. In conclusion, oocyte-derived embryos selected on the basis of BCB staining showed differences in developmental rate, quality, mitochondrial content and target gene expression compared to control embryos.