Effects of Delipidation and Oxygen Concentration on In Vitro Development of Porcine Embryos
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The effects of delipidation and the oxygen (O(2)) concentration in the atmosphere during culture on in vitro development and H(2)O(2) content were investigated in porcine in vivo fertilized embryos and embryos after in vitro maturation and in vitro fertilization (IVM/IVF embryos). There was no significant difference in the developmental rates to the blastocyst stage between the intact and delipidated IVM/IVF embryos. However, the mean number of cells in blastocysts derived from delipidated IVM/IVF embryos (19.8 +/- 0.8 cells) was significantly smaller than that from intact embryos (24.2 +/- 1.2 cells). Although there were no significant differences in the developmental rates to the blastocyst stage of intact and delipidated IVM/IVF embryos between the cultures under 5% O(2) and 20% O(2), the developmental rate of intact IVM/IVF embryos cultured under 5% O(2) (27.1%) was significantly higher than that of the delipidated embryos cultured under 20% O(2) (19.3%). On the other hand, there was no difference in the developmental rate to the blastocyst stage between in vivo fertilized embryos cultured under 5% O(2) and 20% O(2). Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), is thought to cause damage to embryos. The H(2)O(2) content per embryo derived from oocytes cultured under 5% O(2) (in vivo fertilized, 58.0 +/- 2.5 pixels; IVM/IVF, 79.6 +/- 3.2 pixels) was significantly lower than that (in vivo fertilized, 100.2 +/- 3.8 pixels; IVM/IVF, 103.9 +/- 3.2 pixels) under 20% O(2). Furthermore, the level of H(2)O(2) in delipidated IVM/IVF embryos (94.7 +/- 3.9 pixels) was significantly lower than that in intact embryos (103.9 +/- 3.2 pixels) cultured under 20% O(2). The present results indicate that the delipidation of porcine IVM/IVF embryos and reduction of the O(2) concentration decreased the H(2)O(2) level rather than the in vitro developmental rate to the blastocyst stage.Keywords:
In vitro maturation
During in vitro maturation of bovine oocytes, effects of gonadotropins (FSH, LH) and growth factors such as epidermal growth factor (EGF) vary among studies. Now that we can use defined or semi-defined medium, it becomes possible to evaluate recombinant products to assess their roles. Therefore, this study was designed to evaluate the effect of purified porcine (pFSH) or recombinant human (r-hFSH; 5, 50, or 500 ng/ml) follicle stimulating hormone, luteinising hormone (LH; 50, 500 or 5000 ng/ml) and epidermal growth factor (EGF; 5, 10, 30 or 50 ng/ml) on subsequent embryonic development of in vitro matured bovine oocytes. In addition, the presence of bovine serum albumin (BSA; 8 mg/ml) as a protein supplement during in vitro maturation (IVM) was studied. For all treatments, cumulus-oocyte complexes were matured in defined maturation medium consisting of synthetic oviduct fluid. Addition of LH to the maturation medium at all concentrations studied did not increase the proportion of oocytes developing to the morula and blastocyst stages. However, morula and blastocyst yield were improved ( p < 0.05) after addition of EGF (30 ng/ml) as compared with maturation medium alone (29.3% vs 18.0%, respectively). Addition of r-hFSH to the maturation medium in the presence of 17β-estradiol (E 2 ) significantly ( p < 0.0001) increased the morula and blastocyst rate compared with maturation medium alone (40.3% vs 19.3%, respectively). The presence of BSA alone during IVM significantly reduced the developmental competence of oocytes as reflected by the morula and blastocyst yield. These results demonstrate the essential role of FSH, EGF and E 2 on the kinetics of nuclear and cytoplasmic maturation that are essential for the formation of an egg capable of fertilisation and development. Also, supplementation of r-hFSH and E 2 during IVM under our conditions increases morula and blastocyst yield following in vitro fertilisation and in vitro culture in defined medium. Finally, the presence of BSA as the only protein supplement during IVM may be detrimental to oocyte maturation.
In vitro maturation
Oviduct
Chemically defined medium
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Contents Nowadays, the use of foetal calf serum ( FCS ) during in vitro embryo culture is very controversial. Whilst some authors have encouraged its use, others reject it because of its harmful effects. Although in vitro embryo production in red deer is a promising assisted reproductive technique, it is still in its infancy and a great effort is needed to update the protocols used. The aim of this study was to assess whether FCS supplementation in red deer embryo culture medium is necessary to produce blastocyst and, if so, when is the best time to add it in terms of blastocyst production and quality. In vitro blastocysts were cultured with FCS added at 24, 48 or 96 hours post‐insemination (hpi). In addition, a treatment without FCS was used as control. Six hundred and ninety‐four cumulus–oocyte complexes were collected for in vitro fertilization. Cleavage rate was examined at 48 hpi, and blastocyst yield was recorded on days 6, 7 and 8. FCS had no influence on cleavage and blastocyst rate for any of the treatments studied. However, the number of cells was higher ( p = .025) in those blastocysts cultured with FCS from 48 hpi compared with FCS ‐free culture media (93.88 ± 7.76 vs. 54.11 ± 8.36). In conclusion, the addition of FCS to the embryo culture medium at 48 hpi improves the quality of red deer blastocyst, although it does not affect the percentage of embryos obtained.
Embryo quality
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Objective To inhibit NO production in embryonic cells using NO synthase(NOS) inhibitor L-NAME and to explore the regulatory function of NO as an important cellular messenger molecule in early embryonic development in vitro.Methods The embryos were produced by in vitro fertilization and then cultured in the embryonic culture medium including different concentrations of NOS inhibitor L-NAME.The cleavage rate of embryos on day 2 of culture,and the blastocyst rate and blastocyst total cell number on day 4 of culture were evaluated.Results The effect of 0.1 mmol/L L-NAME on the embryonic cleavage rate and blastocyst rate was not significant,but the total cell number of blastocysts was significantly decreased with increasing concentrations of L-NAME.One and 10 mmol/L L-NAME significantly reduced the embryonic cleavage rate,blastocyst rate and blastocyst total cell number.Conclusion NO plays an important regulatory role in the early embryonic development.The inhibition of NO production in embryonic cells has much effect on the early embryo development and their quality.
Cleavage (geology)
In vitro maturation
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Based on maturation in vitro of bovine oocytes, our research mainly focused on the effect of oocyte maturation in vitro on parthenogenetic embryos development competence in bovine. The results showed that The development rate to the blastocyst stage of oocytes activated after maturation for 22~24 h in medium prepared with Milli-Q water (29.7%) was significantly higher than with three-distilled water (14.1%, P0.05). The addition of 10% or 20% BFF (bovine follicle fluid) in maturation medium obtained higher blastocyst rate (32.3% or 30.9%) than that in 5% or control treatment (21.8% or {22.7%}, P0.05). In addition, the addition of 20% BFF in maturation medium inhibited the hatching of blastocyst. It suggested that oocytes matured for 28 h or 30 h before pathenogenetic activation resulted in higher blastocyte rate (30.5% or 29.4%) than those matured for 24 h or 26 h (20.5% or 22.1%, P{0.05}).;
In vitro maturation
Parthenogenesis
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Co-culture with numerous cell lines has been shown to improve in-vitro embryo development. It is usually performed in open culture without an oil overlay, or in relatively large volumes of medium (e.g. 0.5 ml) under oil. We compared the efficacy of open and microdrop co-culture systems using human endometrial and tuba] cell lines and mouse zygotes; Although the mean pH values of the media from the tubal cell cultures (both open and oil-covered) decreased significantly over 5 days of culture, this did not appear to impair embryo development Both co-culture and microdrop culture significantly improved blas-tocyst and hatching blastocyst formation rates. The combination of the two techniques (microdrop and co-culture) demonstrated the highest blastocyst formation and hatching blastocyst formation rates, as well as the highest mean cell numbers in hatching blastocysts. Co-culture in a microdrop is a superior system for mouse embryo culture.
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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.
Cysteamine
In vitro maturation
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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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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.
Cysteamine
In vitro maturation
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