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Title: COMPARISON ON IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED IN KSOM OR SOF AND CRYOPRESERVED BY SLOW FREEZING OR VITRIFICATION

Author
item NEDAMBALE, T - UNIV OF CT
item DINNYES, A - AGR BIOTECH CNTR,HUNGARYR
item Dobrinsky, John
item TIAN, X - UNIV OF CT
item YANG, X - UNIV OF CT

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/20/2003
Publication Date: 8/1/2004
Citation: Nedambale, T.L., Dinnyes, A., Dobrinsky, J.R., Tian, X.C., Yang, X. 2004. Comparison on in vitro fertilized bovine embryos cultured in ksom or sof and cryopreserved by slow freezing or vitrification. Theriogenology. 62(3):437-449.

Interpretive Summary: Unfortunately, developmental competence of cryopreserved in vitro produced (IVP) cattle embryos is not even 40% pregnancy. It was our objective to improve cell-free culture and to compare post-warming development of IVP cattle embryos following cryopreservation by vitrification or conventional slow freezing. Non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture. A KSOM'SOF system improved morulae and blastocyst development when compared to KSOM culture control. Embryos cultured in KSOM + BSA developed more slowly to Day 9. Next, day 7 blastocysts were cryopreserved by vitrification or slow freezing. When cultured in SOF/FCS, higher embryo development and hatching rate was obtained from vitrified/warmed embryos. A 30% pregnancy rate was obtained after single direct transfer of vitrified embryos. Replacing KSOM + BSA with SOF + FCS after 4 d of culture promote high quality blastocyst formation with improved survival potential following cryopreservation. Furthermore, vitrification was a more efficient method to cryopreserve embryos produced by the KSOM-SOF system than was slow freezing. This research shows that advanced embryo culture systems help maintain embryo developmental competence after cryopreservation and transfer.

Technical Abstract: The objectives 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 vs 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 d; (2) SOF + 5% FCS for 4 d and then KSOM + 1% BSA to Day 9; (3) KSOM + 0.1% BSA for 4 d and then KSOM + 1% BSA to Day 9 and (4) KSOM + 0.1% BSA for 4 d and then SOF + 5% FCS to Day 9. Treatment 4 (KSOM'SOF system) improved morulae (47 %), early blastocysts (26 %), Day 7 blastocysts (36 %), cell numbers (127), as well as total hatching rate (79%) compared to KSOM alone (Treatment 3). Embryos cultured in KSOM + BSA developed slowly and most of them hatched late on Day 9, compared to other treatments, In Experiment 2, viability of Day 7 blastocysts, produced by the best culture determined in Experiment 1 (KSOM-SOF or Treatment 4), was tested following either: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rate was obtained by vitrification at 6 h (71%), 24 h (64%) and 48 h (60%) post-warming compared to slow freezing (48, 40 and 31%, respectively). There was 30% pregnancy following single direct transfer of vitrified embryos. Replacing KSOM + BSA with SOF + FCS after 4 d of culture promote high quality blastocyst formation with improved survival potential following cryopreservation. Furthermore, VS3a was a more efficient method to cryopreserve embryos produced by the KSOM-SOF system than was slow freezing.