Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones.
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.
George
F
F
Catholic University of Louvain, Institut des Sciences de la Vie, Unité des Sciences Vétérinaires, Place Croix du Sud 5, Box 10, B-1348 Louvain-la-Neuve, Belgium.
Vrancken
M
M
Verhaeghe
B
B
Verhoeye
F
F
Schneider
Y-J
YJ
Massip
A
A
Donnay
I
I
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D002417:Cattle / Q000196:embryology*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D003470:Culture Media / Q000737:chemistry*; D016895:Culture Media, Serum-Free; D046149:Embryo Culture Techniques / Q000379:methods / Q000662:veterinary*; D004622:Embryo, Mammalian / Q000187:drug effects / Q000502:physiology; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary; D010461:Peptones / Q000494:pharmacology*; D013997:Time Factors
D003470:Culture Media; D016895:Culture Media, Serum-Free; D010461:Peptones