As oocytes experience prolonged aging, reactive oxygen species (ROS) typically accumulate, the mitochondrial electrochemical potential gradient dissipates, and cumulus cells undergo expansion. Trichostatin A (TSA), used to stimulate aging, delays oocyte maturation by inhibiting germinal vesicle breakdown during meiosis. The objective of this study was to study the effects of aging during in vitro oocyte maturation in pigs. Oocytes (n=881) were matured with or without TSA (100 ng/mL) for 24 or 48 h followed by an additional 16 h of maturation without TSA. At the end of maturation, oocytes (n=446) were evaluated for cumulus cell expansion (CCE). A portion of the oocytes were stained to determine the relative levels of ROS (n=476) or mitochondrial electrochemical potential gradient dissipation (n=405). Fluorescent images of the oocytes were acquired, images were analyzed using ImageJ, and statistical analysis of the data was performed using ANOVA and Tukey’s test. Oocytes matured with or without TSA for 48 h had significantly less (P < 0.05) CCE compared to oocytes matured without TSA for 24 h. Oocytes matured without TSA for 24 h generated significantly different (P < 0.05) levels of ROS compared to oocytes matured with TSA for 48 h. Oocytes matured without TSA for 48 h had significantly higher (P < 0.05) mitochondrial membrane potential compared to the all other treatments. Results indicate that oocytes experiencing prolonged aging have less CCE and a decrease in mitochondrial membrane potential but no consistent or predictable trends in ROS formation. The use of TSA to stimulate aging in pig oocytes remains a valid and a reliable option.
Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n=400) and subsequent embryonic development (n=1340) at 48 and 144h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44h at 38.5°C in an atmosphere of 5% CO2. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2×105 sperm mL−1) and oocytes (30 oocytes/well) lasted for 6 to 8h at 38.5°C in an atmosphere of 5% CO2. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18±10.63%), 0.50 (20.93±9.89%) and 0.75mM (18.07±12.02%) quercetin significantly decreased (P<0.05) polyspermic penetration rates compared with no supplementation (40.00±11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50mM quercetin had a significantly higher percentage (P<0.05) of embryos reaching the 2-cell stage of development by 48h after IVF (75.00±7.89%, 68.75±2.23%, respectively) compared with 0.75mM quercetin supplementation (64.62±3.88%) and no supplementation (62.97±4.11%). Supplementation of 0.25 (44.12±6.23%), 0.50 (43.75±7.02%) and 0.75mM (43.08±2.98%) quercetin to the sperm significantly increased (P<0.05) the percentage of embryos reaching the blastocyst stage of development by 144h after IVF compared with no supplementation (28.27±8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.
The imbalance of reactive oxygen species levels and antioxidants impact oocyte matruation. Elderberries (Sambucus sp.) have been identified as possessing the ability to alleviate oxidative stress. An antioxidant class found in elderberry is anthocyanidin, which includes kuromanin. The objective of this study was to determine the effects of kuromanin supplementation to the media during oocyte maturation. Oocytes (n = 692, r=3) were supplemented with 100 or 200 μM kuromanin during 40-44 h of maturation and then evaluated at the end of maturation for the formation of reactive oxygen species, fertilization characteristics, and rates of embryonic cleavage and blastocyst development at 48 h and 144 h after IVF, respectively. There were no significant differences between no kuromanin supplementation and 100 μM kuromanin supplementation when comparing reactive oxygen species generation at the end of oocyte maturation. Supplementation of 200 μM kuromanin significantly increased (P < 0.05) reactive oxygen species generation in oocytes compared to the other groups. There were no significant differences between no kuromanin supplementation and 100 μM kuromanin supplementation when comparing penetration and polyspermic penetration rates and male pronuclear formation. Supplementation of 200 μM kuromanin significantly decreased (P < 0.05) penetration and polyspermic penetration rates and male pronuclear formation compared to the other groups. There were no significant differences between no kuromanin supplementation and 100 μM kuromanin supplementation when comparing the percentage of cleaved embryos by 48 h after IVF and the percentage of those reaching the blastocyst stage by 144 h after IVF. Supplementation of 200 μM kuromanin significantly decreased (P < 0.05) the cleavage rates by 48 h after IVF and the blastocyst formation rates by 144 h after IVF compared to all other treatment groups. These results indicate that supplementing 200 μM kuromanin is detrimental to oocyte maturation and lower levels (100 μM) do not have a significant effect compared to not supplementing the oocytes when evaluating in vitro fertilization and early embryonic development characteristics.
An aged oocyte is one that was not fertilized during the optimal time window after ovulation and potentially has diminished fertilization and embryonic development success. Oocytes (n=2562) were matured with or without trichostatin A (TSA; 100ng mg−1), a known meiotic inhibitor, for 24 or 48h (OMI), then for an additional 16h (OMII) without TSA or hormones. Oocytes were measured for meiosis before maturation (n=95), after OMI (n=365), and after OMII (n=230) as well as cumulus cell expansion (CCE). Oocytes (n=800) were fertilized with frozen–thawed boar sperm and potential embryos were evaluated for IVF characteristics and cleavage and blastocyst formation 48 and 144h after IVF, respectively. Apoptosis was determined in the maturing oocytes by levels of reactive oxygen species (ROS; n=476), mitochondrial electrochemical potential gradient dissipation (n=405), caspase 3 (n=405), phosphatidylserine (n=453), and DNA breakdown (n=192). Data were analysed by ANOVA and Tukey’s test. Oocytes matured without TSA for 48h had a significantly higher (P<0.05) percentage of oocytes at the MI stage of meiosis compared to all other treatment groups (16.00±6.80) and oocytes matured without TSA for 24h had a significantly higher (P<0.05) percentage of oocytes at the MII stage of meiosis compared to all other treatment groups (14.7±11.30). Oocytes matured with or without TSA for 48h had significantly less (P<0.05) CCE compared to oocytes matured without TSA for 24h. Supplementation of TSA to OMI significantly decreased (P<0.05) fertilization penetration rates compared with not supplementing TSA for 24h. Percent of embryos cleaved by 48h and those reaching blastocyst by 144h after IVF were significantly higher (P<0.05) in oocytes matured for 24h compared with those matured for 48h. Oocytes matured without TSA for 24h generated significantly different (P<0.05) levels of ROS compared with oocytes matured with TSA for 48h. Oocytes matured without TSA for 48h had significantly higher (P<0.05) mitochondrial membrane potential compared with all other treatments. Levels of caspase 3 activation and phosphatidylserine (which translocates in response to apoptosis) differed (P<0.05) between treatments. Results from the TUNEL assay indicate oocytes matured through OMII with a short OMI and TSA supplemented (23.8±2.99%) and through OMI with a long OMI and TSA supplemented (24.3±1.11%) had significantly greater (P<0.05) percent of oocytes with fragmented DNA than the other treatments, except for oocytes matured through OMII with a long OMI and TSA supplemented (35.0±3.55%), which was significantly greater (P<0.05) than all other treatments. Results indicate that use of TSA to stimulate aging in pig oocytes is a valid and a reliable option.
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