title: Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification.
abstract: The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.
authors.affiliation: Connecticut Center for Regenerative Biology, University of Connecticut, 1392 Storrs Rd., U-4233, Storrs, CT 06269-4223, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Nedambale
authors.forename: A
authors.initials: A
authors.lastname: Dinnyés
authors.forename: W
authors.initials: W
authors.lastname: Groen
authors.forename: J R
authors.initials: JR
authors.lastname: Dobrinsky
authors.forename: X C
authors.initials: XC
authors.lastname: Tian
authors.forename: X
authors.initials: X
authors.lastname: Yang
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology; D001826:Body Fluids; D002417:Cattle / Q000196:embryology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003470:Culture Media; D046508:Culture Techniques; D004624:Embryo Transfer / Q000662:veterinary; D005187:Fallopian Tubes; D005260:Female; D005307:Fertilization in Vitro; D009028:Morula / Q000502:physiology; D011188:Potassium; D011247:Pregnancy; D015053:Zygote / Q000254:growth & development / Q000502:physiology*
chemical_list: D003470:Culture Media; D011188:Potassium