ONE-STEP VITRIFICATION METHOD FOR CRYOPRESERVATION OF BOVINE EMBRYOS TRANSFERRED DIRECTLY INTO RECIPIENTS

A one-step vitrification procedure for cryopreservation of embryos tra nsferred to recipients directly (without postthawing morphological con trol) was developed. Intact day 7 embryos - morulae and blastocysts - recovered non-surgically from superovulated donors were equilibrated i n medium composed of PBS + 15% lyophilized growth protein of calf seru m (commercial preparation SEVAC Prague, C.S.F.R.) with 10% (v/v) glyce rol for 10 min. Then they were transferred into a drop of vitrificatio n medium (30% (v/v) glycerol, 20% (v/v) FCS, 50% (v/v) 2 M sucrose in tridistilled water) for 1-2 min. Transfer into 2 drops of vitrificatio n medium was followed immediately by filling embryos into 0.25 ml stra ws (IMV, France). Straws were closed with warm forceps and dipped into LN2 bath (plug upwards). Thawing was made by rubbing straws in hands; vitrification medium and wash medium crystals having been thawed, the straw content was mixed by shaking. Washing was done by turning the s traw up several times. Embryos were transferred non-surgically into re cipients on day 5 or 7 after synchronized or spontaneous estrus. The s urvival rate after in vitro cultivation for 48 h was 50% and 60% respe ctively (washing in 1 M resp. 0.5 M sucrose in medium) in experimental group; the corresponding values in control embryos frozen by dropping into LN2 were 61.5% and 65.2% respectively (P > 0.05). Conception rat e of vitrified embryos frozen by one-step method and washed in 1 M suc rose in culture medium was significantly lower (27.9%, n = 12/43, P < 0.05) than in embryos washed in 0.8 M sucrose (56.7%, n = 17/30) and e mbryos washed in 0.5 M sucrose (50%, n = 7/14). Conception rate in the control recipients (embryos frozen by routine one-step method in PLAN ER R 204 freezer) were 60% (n = 12/20). ET performed on day 5 followin g postovulatory bleeding resulted in significantly lower conception ra tes (36.9%, n = 24165, P < 0.05) than ET performed on day 7 after sync hronization (45.5%, n = 5/11) or spontaneous estrus (63.6%, n = 7/11). One-step vitrification method of bovine embryo cryopreservation prese nts a simple, inexpensive, undemanding procedure for practical use.