Defined media for vitrification, warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos.
The purpose of this study was to assess the viability (rates of re-expanding and hatching in vitro), of in vitro derived ovine blastocysts using vitrification and warming/rehydration media containing fetal calf serum (20% FCS) or polyvinyl alcohol (0.1% PVA), and the incorporation of labelled methionine in protein synthesised during the first 4h after cryopreservation. In experiment 1, after 60 h culture in TCM-199 supplemented with 10% FCS, the hatching rates of blastocysts that had been vitrified, warmed, and rehydrated in media containing only PVA (p/p) were significantly (P<0.05) lower than those vitrified in medium containing PVA with warming and rehydration in medium containing FCS (p/s). Blastocysts that were vitrified in medium containing FCS and warmed and rehydrated in medium with PVA (s/p) had hatching rates that were significantly lower (P<0.01) than those vitrified, warmed, and rehydrated in media with only FCS (s/s). After warming, the number of dead cells in the p/p group was significantly (P<0.05) lower than in all other groups. In experiment 2, the [35S]methionine uptake by embryonic cells of the s/p group was significantly (P<0.01) higher than in other groups. The incorporation of labelled methionine into newly synthesised proteins was significantly lower in the p/p group (P<0.01) than in all other groups. No differences in the newly synthesised proteins were observed between groups. In conclusion, these results suggest that it is possible to replace serum with defined macromolecules in vitrification and warming/rehydration media for in vitro derived ovine blastocysts but this leads to a decrease in viability and a reduction in protein synthesis after warming.
Leoni
Giovanni
G
Department of Animal Biology, University of Sassari, 07100 Sassari, Italy. vetfis@ssmain.uniss.it
Bogliolo
Luisa
L
Berlinguer
Fiammetta
F
Rosati
Irma
I
Pintus
Pier Paolo
PP
Ledda
Sergio
S
Naitana
Salvatore
S
D000818:Animals; D001757:Blastomeres / Q000166:cytology*; D001798:Blood Proteins / Q000494:pharmacology; D002470:Cell Survival; D015925:Cryopreservation; D003470:Culture Media / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro; D005326:Fetal Proteins / Q000494:pharmacology; D005440:Fluid Therapy; D006358:Hot Temperature; D009865:Oocytes / Q000166:cytology; D011142:Polyvinyl Alcohol / Q000494:pharmacology*; D012756:Sheep
D001798:Blood Proteins; D003470:Culture Media; D005326:Fetal Proteins; D011142:Polyvinyl Alcohol