Metabolism, protein content, and in vitro embryonic development of goat cumulus–oocyte complexes matured with physiological concentrations of glucose and L ‐lactate
Jason R. HerrickMichelle LaneDavid K. GardnerE. BehboodiErdoğan MemiliS. BlashYann EchelardRebecca L. Krisher
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Abstract:
No information is available concerning how the maturation environment controls the metabolism of goat oocytes. The objectives of this experiment were to: (1) Determine the concentrations of glucose, lactate, and pyruvate in caprine follicular fluid; and (2) Investigate the effects of physiological concentrations of glucose and lactate in the in vitro maturation (IVM) medium on the metabolism (glycolysis and pyruvate oxidation), protein content, and developmental competence of caprine oocytes and cumulus-oocyte complexes (COCs). Abattoir-derived COCs were matured for 18-20 hr in a defined, SOF-based medium containing 0.75, 1.5 (follicular fluid = 1.4 mM), or 3.0 mM glucose, and 3.0, 6.0 (follicular fluid = 7.1 mM), or 12.0 mM L-lactate. The protein content of oocytes and COCs was not affected (P > 0.05) by the concentration of glucose and lactate in the maturation medium. Increasing glucose and lactate decreased (P < or = 0.05) glycolytic activity of oocytes, without affecting (P > 0.05) pyruvate oxidation. In COCs, increasing glucose concentrations tended (P = 0.07) to decrease glycolysis. When metabolic activity was corrected for protein content (pmol/microg protein/3 hr), increasing glucose or lactate concentrations in the medium decreased (P < or = 0.05) pyruvate oxidation in oocytes, but increased (P < or = 0.05) pyruvate oxidation in COCs. Embryonic development (cleavage and blastocyst development, hatching, and cell number) was not affected (P > 0.05) by the glucose and lactate concentrations tested. These results indicate that concentrations of glucose and lactate in the medium have cell type-specific effects on metabolism of oocytes and COCs, but do not affect developmental competence within the range of concentrations tested.Keywords:
Follicular fluid
Carbohydrate Metabolism
In vitro maturation
To optimize the most efficient method for porcine in vitro maturation (IVM), we compared the effects of supplementing extracellular vesicles (EVs) derived from porcine follicular fluid (pFF). The cumulus oocyte complexes were grouped into 4 groups with different supplementations as following: pFF (G1), pFF-depleted EVs (G2), EVs (G3) and control (G4) groups. After IVM with different supplementations, maturation rates and the developmental competences of porcine oocytes and blastocyst development were investigated. Additionally, glutathione (GSH) and reactive oxygen species (ROS) levels were measured in mature oocytes. The EVs were isolated and characterized with cryo-TEM and nanoparticle tracking analysis. The pFF significantly affected the maturation rate, whereas the presence of EVs did not show notable difference in the maturation rates. Although there were numerical increases in the measured parameters in EV and pFF-depleted EVs groups, no significant differences were observed between them. The EV group showed similar oocyte maturation rate for both positive and negative control groups. The GSH was not different among the groups, but ROS levels were significantly lower in pFF-supplemented group when compared with other groups with the highest level in the control group. G2 group wasn’t significantly different G1 and G3 group. G3 group wasn’t significantly different from G2 and G4 group. This suggests that EVs in IVM medium which probably effected partially to protect against oxidative stress and potentially enhance the quality of oocytes. This study indicates that the EVs in pFF play a significant role in improving the efficiency of oocyte maturation in porcine.
Follicular fluid
In vitro maturation
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The present study was carried out to evaluate if the addition of cysteamine to the culture medium during in vitro maturation of bovine oocytes increased the glutathione (GSH) levels in the mature oocytes, and if these changes may promote an improvement on in vitro development to the blastocyst stage. Follicular oocytes from slaughterhouse ovaries were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and O (control), 25, 50, or 100 muM of cysteamine for 24 hr. After in vitro maturation the oocytes were fertilized and cultured for 8 days. The percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.01) for oocytes matured in medium containing 100 muM of cysteamine than for those matured in control medium. Moreover, the intracellular GSH levels were increased (P < 0.05) in oocytes matured with 100 muM of cysteamine with respect to control. No differences were observed in maturation and cleavage rates, and in the mean cell numbers per blastocyst among treatments (P > 0.05). These results indicate that the addition of thiol compounds such as cysteamine to maturation medium increases the efficiency of in vitro blastocyst production from immature bovine oocytes. The higher levels of GSH in oocytes matured in the presence of cysteamine suggest that the beneficial effects of cysteamine on in vitro maturation and subsequent development after in vitro fertilization are mediated by GSH.
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To evaluate the effects of supplementation of Insulin and leukemia inhibit factor(LIF) in culture media on in vitro nuclear maturation of porcine oocytes and in vitro development of porcine parthenotes.1) Insulin or LIF was added into the medium of porcine oocytes in vitro maturation;and the matured oocytes with the first poly body were recorded and parthenogentically activated followed by being cultured in vitro in normal medium;2) Insulin or LIF was supplemented into parthenotes in vitro culture medium;and the rates of cleavage and blastocyst formation were recorded.For in vitro maturation,supplementation of 5μg/mL insulin significantly increased the rate of in vitro nuclear maturation of pig oocytes although subsequent in vitro development of these oocytes was similar to the control after parthenogenetic activation.When LIF was added,no obvious increase of nuclear maturation was found whereas the blastocyst rate decreased drastically of such oocytes after parthenogenetic activation.When insulin was only added to culture medium of parthenotes,no beneficial effect was observed regarding to cleavage and blastocyst formation.When LIF was singly supplemented to embryo culture medium,very slight,and not significant increase of the cleavage and blastocyst rates was found.Insulin was confirmed conducive to the maturation of porcine oocytes,but further studies are required to investigate if other concentrations or treatments of insulin are helpful to porcine parthnotes development in vitro.Regarding to LIF,it is harmful to blastocyst development in vitro.However,more concentrations and treatments of LIF are deserved to be examined whether it could promote maturation and in vitro development of porcine embryos or not.
In vitro maturation
Parthenogenesis
Cleavage (geology)
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The objective of the present study was to investigate the in vitro developmental competence of porcine in vitro matured (IVM) oocytes vitrified after removal of cytoplasmic lipid droplets (delipation). After vitrification and warming, the delipated porcine IVM oocytes were inseminated and subsequently cultured in vitro. The rate of development to the blastocyst stage of delipated, vitrified oocytes (5.9%) was significantly lower than that of control oocytes (untreated oocytes) (26.2%). We also examined the influence of delipation of porcine IVM oocytes on development to the blastocyst stage following in vitro fertilization (IVF). Delipated porcine IVM oocytes (not vitrified) were inseminated and subsequently cultured in vitro. The rates of development to the blastocyst stage were similar for delipated and undelipated oocytes (21.1% and 26.2%, respectively). The results of the present study showed that delipated, vitrified porcine IVM oocytes can develop to the blastocyst stage following IVF, though blastocyst formation rate was low, and that delipation of porcine IVM oocytes did not negatively affect their development to blastocyst stage.
In vitro maturation
Vitrification
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In vitro maturation
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The present study evaluated the role offollicular fluid exosomes on the developmental competence of bovine oocytes. Ovaries from slaughtered crossbred cattle were collected, all visible surface follicles aspirated, and culture-grade oocytes were subjected to further study. Exosomes were isolated from bovine follicular fluid by differential ultracentrifugation. A total of 358 oocytes selected for the study were randomly divided into two groups. Group I constituted 111 oocytes, in which normal maturation was carried. Group II constituted 247 oocytes, in which in vitro maturation (IVM) medium was supplemented with follicular fluid exosomes at 1 μL/100 μL IVM medium. Maturation was assessed after 24h of culture in a CO2 incubator maintained at 38.5°C in 95 per cent humidified atmosphere of 5 per cent CO2. Following IVM of oocytes for 24 h, in vitro fertilisation (IVF)was carried out by co-incubating with capacitated spermatozoa for 18 h and embryo culture was carried out subsequently. In group II oocytes supplemented with exosomes, a significantly higher maturation rate (p ≤0.01) (95.80 ±1.67 vs76.10 ± 0.95), fertilisation rate (53.68 ± 3.02vs37.85 ± 7.01) and cleavage rate (p ≤0.01) (43.66 ± 2.13vs(32.47 ± 5.23) was noticed compared to oocytes in group I without any supplementation.The present study established that supplementation of follicular fluid exosomescould improve the developmental competence of bovine oocytes.
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In vitro maturation
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The aim of this study was to investigate the effect of time on in vitro maturation of bovine oocytes and of the addition of follicular fluid on meiotic progression. The cumulus-oocyte complexes (COCs) collected from 3 to 6 mm follicles were obtained from ovaries of slaughtered female animals. The medium of maturation was supplemented or not with 20 μL follicular fluid (FF); 661 oocytes were matured in vitro (extrusion of the first polar corpuscle) for 22 hours with added follicular fluid (AFF) (72.01%) or without follicular fluid (WFF) (67.53%) and 679 oocytes were matured in vitro for 26 hours (extrusion of the first polar corpuscle) with AFF (92.1%) and WFF (77.15%). The results of extrusion of the second polar corpuscle as an event related to the fertilization percentages showed that the increase in the fertilization rate is maintained at 26 hours with AFF (79.45%), but the percentage decreases WFF (65.08%). After 22 hours, the fertilization rate was 62.38% AFF and 53.40% WFF. The developmental competence of bovine oocytes is affected by the duration of maturation in vitro and the inclusion in the FF culture medium. The use of follicular fluid in the in vitro maturation medium may be a biological strategy to increase the cumulus expansion, the nuclear maturation and the in vitro fertilization.
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In vitro maturation
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The aim of the present study was to investigate the effects of exposure time to gonadotrophin (FSH and LH) and porcine follicular fluid (pFF) on maturation in vitro of porcine oocytes.The results indicate that exposure of porcine cumulus-oocyte-complexes to gonadotrophin supplements for the first 2324 hours improves cumulus expansion but has no significant effect on oocyte nuclear maturation compared with that for 4648 hours (P0.05),and supplementation of a certain amount(10%) of porcine follicular fluid in maturation medium can enhance in vitro maturation of porcine oocytes, but excessive supplementation will relatively inhibit oocyte nuclear maturation process.
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In vitro maturation
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In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.
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In vitro maturation
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Current approaches to in vitro maturation (IVM) may result in low efficiency and inadequate quality of the oocytes due to insufficient cytoplasmic maturation. Although positive effects of the cysteamine supplementation in IVM medium for oocyte nuclear maturation or male pronuclear formation have been confirmed, it is still controversial whether the cysteamine addition affects embryo development after IVM. We aimed here to confirm the effect of cysteamine addition into IVM medium for subsequent embryo development in vitro.We administered the cysteamine to the IVM culture of rabbit immature oocytes at various concentrations and observed the developmental rate, speed to reach blastocyst stage and cell numbers at the blastocyst stage.Cysteamine supplementation improved developmental rate to blastocyst stage of the IVM oocytes. On the other hand, addition of glutathione (GSH) inhibitor buthionine sulfoximine inhibited GSH accumulation in the oocytes and subsequent embryo development to the blastocyst stage.Controlling the GSH quantity of IVM oocytes may be an important factor for success of embryo development, and it is quite probable that a cysteamine supplementation can contribute to an increase of GSH content in oocyte.
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In vitro maturation
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