Biomedical subjects

C Guyader-Joly

Publications and source records attributed to C Guyader-Joly.

7 recordsLinked to original sources

PubMed2001

Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability.

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.

Animals↗

PubMed1999

Effect of lecithin on in vitro and in vivo survival of in vitro produced bovine blastocysts after cryopreservation.

The use of soybean lecithin in an glycerol-based solution for slow freezing of in vitro matured, fertilized and cultured (IVMFC) bovine embryos was examined. Embryos were developed in vitro in INRA Menezo's B2 medium supplemented with 10% fetal calf serum (FCS) on Vero cells monolayers. Day 7 blastocysts were frozen in a two-step protocol consisting of exposure to 5% glycerol and 9% glycerol containing 0.2 M sucrose in F1 medium + 20% FCS. Soybean lecithin was either added or not to the freezing solutions at a final concentration of 0.1% (w/v). In Experiment 1, blastocysts were equilibrated in cryoprotectant solutions without cooling. Cryoprotectant was diluted from embryos with 0.5 M and 0.2 M sucrose. The percentages of fully expanded and hatched blastocysts treated with or without lecithin after 24 and 48 h in culture were not significantly different (100 versus 100% and 93.3 versus 100%, respectively). In Experiment 2, the in vitro survival of frozen-thawed IVMFC blastocysts was compared when cryoprotectant solutions were either supplemented or not with lecithin. No significant effect of lecithin was found on the ability of frozen-thawed blastocysts to re-expand after 48 h in culture (65.6 and 54.2%, respectively). However, the post-thaw hatching rate of embryos cryopreserved in the presence of 0.1% lecithin was significantly higher after 72 h in culture (52 and 31.8%, respectively). In Experiment 3, the ability of frozen-thawed IVMFC blastocysts to establish pregnancy following single embryo transfer was determined. Transfers of 58 and 66 frozen-thawed embryos cryopreserved with or without lecithin resulted in 6 and 10 (10.3 and 15.1%, respectively) confirmed pregnancies at Day 60. Addition of lecithin to cryoprotectants did not improve the in vivo development rate of cryopreserved IVMFC bovine blastocysts.

Animals↗

PubMed1999

[Sequential media: why and how?].

Blastocyst transfer is highly recommended for the following indications to avoid repeated failure of implantation: related to maternal, paternal and cytogenetic aspects on the embryonic side and/or uterus motility. Sequential media have now replaced coculture in order to produce blastocysts. The basic idea of sequential media is to follow embryo's need. There are 2 phases during preimplantation period: the first one corresponds to a development through maternal stores, gathered during maturation, before genomic activation. This requires a complete protection against free radicals (through EDTA), and a decrease in the concentration of glucose and phosphate: mitocondrial function is impaired. Regulation of the endogenous pool is not perfect. Then at the time of genomic activation, there is an increased need for numerous metabolites. Insulin can be added as the I-Receptor appears at the 8-cell stage. The medium used during this phase has to be rich.

Coculture Techniques↗

PubMed1998

Precursors of taurine in female genital tract: effects on developmental capacity of bovine embryo produced in vitro.

Two precursors of taurine have been studied: cysteamine and hypotaurine. Cysteamine has been quantified in genital secretions and found in follicular fluids of all species tested. On the contrary cysteamine was not detected (or traces) in tubal fluids of the same species. Addition of 50, 100 or 250 microM of cysteamine to the maturation medium used in the culturing of bovine oocytes did not improve the cleavage rate nor the embryo's developmental potential in vitro. Furthermore, at 250 microM, cysteamine seems to be toxic to the embryo. Addition of 0.5-1 mM hypotaurine to the bovine embryo culture medium improved significantly blastocyst production and quality. The respective roles of these 2 taurine precursors on maturation and embryo development are discussed.

Animals↗

PubMed1997

Glycine and methionine transport by bovine embryos.

As glycine is one of the most concentrated amino acids in the female genital tract, we investigated its uptake by bovine in vitro matured/in vitro fertilised blastocysts in the presence of increasing concentrations of radiolabelled glycine. We also determined methionine uptake by in vitro and in vivo produced embryos. In our study, the hypothesis of more than one site of enzyme activity for glycine substrate was not validated. We determined a Vmax of 23.4 fmol/min per embryo and a K(m) value of 13.3 microM. No significant difference was observed either between in vivo and in vitro derived embryos or between grade 1 and grade 2 embryos for methionine uptake. The methionine and glycine uptake of a day 7 bovine was similar to that of a day 4 mouse blastocyst. This is rather low if we consider the relative cell numbers.

Animals↗

PubMed1997

Beneficial effects of Vero cells for developing IVF bovine eggs in two different coculture systems.

A Vero cell line was used for coculture of bovine in vitro fertilized eggs up to blastocyst stage in comparison with bovine oviductal epithelial cells (BOEC) in two culture systems: monolayers or microdrops. Inseminated oocytes cocultured for 7 days with Vero cells in microdrops resulted in a significantly higher blastocyst rate compared to BOEC (29.5% vs 21.1%, respectively; P < 0.01). This difference was not significant in the monolayer coculture system although the blastocyst rate tended to be higher with Vero than with BOEC monolayers (27% vs 22.3%, respectively). Interestingly, the coefficient of variation between replicates was lower in both Vero cell groups than in BOEC groups indicating that Vero cells may help reduce variability. Medium conditioned by Vero cells partly supported embryo development compared to coculture itself (14.6% vs 26.5%, respectively; P < 0.01). Blastocysts developed on Vero cells contained significantly more cells (142 +/- 39) than those developed on BOEC (88.8 +/- 32.8, P < 0.001). Viability of blastocysts developed on Vero cells was evaluated by single transfer to 26 recipient heifers. Confirmed pregnancy rate after 3 months was 58%, demonstrating their high viability.

Animals↗

PubMed1996

Comparative glucose and fructose incorporation and conversion by in vitro produced bovine embryos.

We have investigated the quality of bovine IVM/IVF embryos co-cultured on Vero cells. Blastocyst cell numbers are very similar to those obtained in vivo, and higher than those obtained by co-culture with oviduct cells. The metabolism and conversion of fructose and glucose are not equivalent even though carbon dioxide production is similar and increasing from morula to blastocyst. Formation of free amino acids and incorporation into proteins are higher and faster for glucose than for fructose, but this conversion is rather stable with embryonic growth. Moreover, the by-products formed are not the same. Glucose at physiological concentrations (i.e. 2 mM) seems to be a more appropriate fuel for the burst of embryonic development at the blastocyst stage in preparation for hatching.

Amino Acids↗