Allicin, a chemical component of garlic, has strong antioxidant activity and is thought to exert antiaging effects in vitro. We investigated whether allicin treatment would protect porcine oocytes and embryos from postovulatory aging mediated by apoptosis and autophagy. The rates of oocyte survival and polar body extrusion in samples treated with 1 µM allicin (1 AL) were significantly higher than in untreated samples (0 AL). In addition, 1 AL prevented defects in spindle formation and chromosome alignment, as well as decreases in the expression of maturation markers, during in vitro aging. In this study, we considered allicin to be a regulator of autophagy rather than an antioxidant or antiapoptotic agent. At the embryo level, although the cleavage rate after parthenogenetic activation was similar in all groups, the blastocyst formation rate was higher in the 1 AL group than in the 0 AL group. Our findings demonstrate that allicin effectively prevents the deterioration of porcine oocytes during aging in vitro, and could therefore be used to improve the quality of aged oocytes used in in vitro experiments.
Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (<i>in vivo</i> aging) or in culture (<i>in vitro</i> aging) will deteriorate over time. Icariin (ICA), found in all species of <i>Epimedium</i> herbs, has strong antioxidant activity, and is thought to exert anti-aging effects <i>in vitro</i>. We asked whether ICA protects oocytes against age-related changes <i>in vitro</i>.Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA.Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [<i>SOD1</i>], <i>SOD2</i>, peroxiredoxin 5, and nuclear factor erythroid 2‐like 2), during aging <i>in vitro</i>. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto‐oncogene, serine/threonine kinase, and growth differentiation factor‐9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group.Conclusion: ICA protects oocytes against damage during aging <i>in vitro</i>; therefore, it can be used to improve assisted reproductive technologies.
To optimize the reproduction of Jeju black cattle (JBC), freezing conditions for sperm were established and sperm motility, vitality, morphology, and fertility were evaluated to select the optimal bull for breeding. Semen samples from five JBC bulls were individually mixed with freezing medium at a final concentration of 1 × 108 sperm/mL and frozen in liquid nitrogen vapor at a height of 3 or 7 cm (referred to as 3 cm sperm and 7 cm sperm, respectively). When the freezing conditions were compared, the motility of 7 cm sperm was significantly higher than that of 3 cm sperm for the JBC-A bull. The motility, curvilinear velocity, straight-line velocity, and average path velocity of fresh and frozen–thawed sperm were the highest for the JBC-A bull. The vitalities of fresh and frozen–thawed sperm were the highest for the JBC-A/E and JBC-A bulls, respectively. The percentage of normal cells in fresh sperm was the highest for the JBC-D bull. The rates of the normal formation of two pronuclei and total sperm penetration were the highest in zygotes fertilized with sperm from the JBC-A bull. The sperm from the JBC-A bull had superior qualities and are thus the most appropriate choice for the preservation and reproduction of these endangered cattle.
Summary Our previous studies have already revealed that β-cryptoxanthin (BCX), hesperetin (HES), and icariin (ICA) antioxidants are effective for in vitro maturation (IVM) of porcine oocytes. In this study, we investigated which of BCX, HES, or ICA was more effective for IVM of porcine oocytes. The antioxidant properties were assessed with aged porcine oocytes and embryos by comparing 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH), reducing power, and H2O2 scavenging activity assays. The chemical assay results demonstrated that BCX had a greater DPPH scavenging activity and reducing power than HES and ICA, compared with controls. However, the H2O2 scavenging activity of the antioxidants was similar when tested at the optimal concentrations of 1 μM BCX (BCX-1), 100 μM HES (HES-100), and 5 μM ICA (ICA-5). The biological assay results showed that BCX-1 treatment was more effective in inducing a significant reduction in reactive oxygen species (ROS), improving glutathione levels, and increasing the expression of antioxidant genes. In addition, BCX-1 inhibited apoptosis by increasing the expression of anti-apoptotic genes and decreasing pro-apoptotic genes in porcine parthenogenetic blastocysts. BCX-1 also significantly increased the blastocyst formation rate compared with the ageing control group, HES-100 and ICA-5. This study demonstrates that damage from ROS produced during oocyte ageing can be prevented by supplementing antioxidants into the IVM medium, and BCX may be a potential candidate to improve assisted reproductive technologies.
This study investigated the effect of the antioxidant dieckol, a component of Ecklonia cava, on maturation and developmental competence of porcine oocytes exposed to oxidative stress in vitro. Oocytes were matured in in vitro maturation (IVM) medium containing various concentrations of dieckol. The blastocyst formation rate was highest in the 0.5 μM dieckol‐treated (0.5 DEK) group. The reactive oxygen species level was decreased, and the level of glutathione and expression of antioxidant genes (NFE2L, SOD1, and SOD2) at metaphase II were increased in the 0.5 DEK group. Abnormal spindle organization and chromosome misalignment were prevented in the 0.5 DEK group. Expression of maternal markers (CCNB1 and MOS) and activity of p44/42 mitogen‐activated protein kinase were increased in the 0.5 DEK group. After parthenogenetic activation, the total number of cells per blastocyst was increased and the percentage of apoptotic cells was decreased in the 0.5 DEK group. Expression of development‐related genes (CX45, CDX2, POU5F1, and NANOG), antiapoptotic genes (BCL2L1 and BIRC5), and a proapoptotic gene (CASP3) were altered in the 0.5 DEK group. These results indicate that the antioxidant dieckol improves IVM and subsequent development of porcine oocytes and can be used to improve the quality of oocytes under peroxidation experimental conditions.
Optimization of culture conditions is important to improve oocyte maturation and subsequent embryo development. In particular, this study analyzed the effects of increasing concentrations of PIO in the maturation medium on spindle formation and chromosome alignment, glutathione, and intracellular ROS levels and expression of selected genes related to maternal markers, apoptosis, and lipid metabolism. The percentage of oocytes displaying normal spindle formation and chromosome alignment was higher in the 1 µM PIO (1 PIO)‐treated group than in the control group. The glutathione level was significantly higher in the 1 PIO‐treated group than in the control group, while the reactive oxygen species level did not differ. Expression of maternal marker (MOS and GDF9), antiapoptotic (BIRC5), and lipid metabolism‐related (ACADS, CPT2, SREBF1, and PPARG) genes was higher in the 1 PIO‐treated group than in the control group, while expression of a proapoptotic gene (CASP3) was lower. The blastocyst formation rate and the percentage of blastocysts that reached at least the hatching stage on Days 6 and 7, and the percentage of blastocysts containing more than 128 cells were significantly higher in the 1 PIO‐treated group than in the control group. These results indicate that PIO treatment during in vitro maturation improves porcine oocyte maturation and subsequent parthenogenetic embryo development mainly by enhancing lipid metabolism and antioxidant defense in oocytes.
SummaryThis study investigated the effect of the flavonoid-based compound isorhamnetin (ISO) on maturation and developmental competence in oxidative stress-exposed porcine oocytes in vitro. Treatment with 2 μM ISO (2 ISO) increases the developmental rate of oxidative stress-exposed porcine oocytes during in vitro maturation (IVM). The glutathione level and mRNA expression of antioxidant-related genes (NFE2L2 and SOD2) were increased in the 2 ISO-treated group, whereas the reactive oxygen species level was decreased. Treatment with 2 ISO increased mRNA expression of a cumulus cell expansion-related gene (SHAS2) and improved chromosomal alignment. mRNA expression of maternal genes (CCNB1, MOS, BMP15 and GDF9) and mitogen activated protein kinase (MAPK) activity were increased in the 2 ISO-treated group. The total cell number per blastocyst and percentage of apoptotic cells were increased and decreased in the 2 ISO-treated group, respectively. Treatment with 2 ISO increased mRNA expression of development-related genes (SOX2, NANOG, and POU5F1) and anti-apoptotic genes (BCL2L1 and BIRC5) and decreased that of pro-apoptotic genes (CASP3 and FAS). These results demonstrate that 2 ISO improves the quality of porcine oocytes by protecting them against oxidative stress during IVM and enhances subsequent embryo development in vitro. Therefore, we propose that ISO is a useful supplement for IVM of porcine oocytes.
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