ABSTRACT
Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrations<18 mg/mL on in vitro bovine blastocysts. Increasing concentrations of peptones were then added in the freezing medium. The surviving and hatching rates were not affected by comparison with those observed with BSA. No significant difference was observed between groups either for the total number of cells or for the ratio ICM/Total cell, nor for the rate of apoptosis in surviving embryos. When embryos were cryopreserved in 1.8 mg/mL peptone, the hatching rate and embryo quality as assessed at 48 h post-thawing were not significantly different from those of unfrozen embryos. In a second experiment two additives were added in this animal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.
Subject(s)
Blastocyst/physiology , Cattle/embryology , Culture Media/chemistry , Embryo Culture Techniques/veterinary , Embryo, Mammalian , Peptones/pharmacology , Animals , Blastocyst/cytology , Cell Survival , Cryopreservation/methods , Cryopreservation/veterinary , Culture Media, Serum-Free , Embryo Culture Techniques/methods , Embryo, Mammalian/drug effects , Embryo, Mammalian/physiology , Fertilization in Vitro/methods , Fertilization in Vitro/veterinary , Time FactorsABSTRACT
Familial hypercholesterolemia (FH) results from an inherited functional defect of the low density lipoprotein (LDL) receptor and is complicated by premature atherosclerosis. FH diagnosis is obtained by sophisticated techniques or is suggested by clinical criteria. We have developed a technique based on flow cytometry for the measurement of DiI-labeled LDL uptake in human peripheral blood T lymphocytes left for 2 days in a lipoprotein-deficient culture medium. Flow cytometry allowed us to discriminate the uptake of DiI-LDL by T lymphocytes subpopulation from the uptake by the whole mononuclear population using a T cell-specific anti-CD3 antibody. The method appeared to be highly specific for the receptor-mediated pathway of LDL uptake as DiI-LDL uptake was inhibited in the presence of a 10-fold excess of unlabeled LDL and by EDTA. A good relationship was found between the uptake of DiI-LDL and 125I-labeled LDL degradation. The test was applied in three groups of patients: patients with normal cholesterol levels, patients with heterozygous FH, and patients with high cholesterol levels but without clinical criteria of FH. The mean fluorescence intensities were 23.1 +/- 8.9, 6.3 +/- 1.7, and 17.1 +/- 3.5 (mean +/- standard deviation), respectively. The ability to measure the fluorescence in T lymphocytes improved the discrimination between FH and non-FH subjects when compared with values obtained from the whole mononuclear cell population. These results suggest that our method could be useful for LDL receptor defects screening.
