CRYOPRESERVATION OF IN VITRO-PRODUCED BOVINE EMBRYOS - EFFECTS OF PROTEIN TYPE AND CONCENTRATION DURING FREEZING OR OF LIPOSOMES DURING CULTURE ON POSTTHAW SURVIVAL

Two experiments were conducted to examine the effect of membrane stabi lization through the modification of in vitro culture medium or freezi ng medium on post-thaw survival of in vitro-produced bovine embryos. I n Experiment 1, Day 7 (Day 0 = day of IVF) late morulae and blastocyst s that developed following culture in SOF/aa/BSA (IVC medium) were fro zen slowly to -35 degrees C in the presence of 1.5 M ethylene glycol p repared in ovum culture medium (OCM) or in OCM supplemented with 10, 2 5 or 50% fetal calf serum (FCS) or 5, 10 or 25 mg/mL BSA. Postthaw sur vival was assessed by re-expansion and/or hatching following 48 h of c ulture in IVC medium + 10% FCS. Overall, survival was significantly (P <0.01) affected by embryo stage, with more hatched blastocysts survivi ng (71%) than blastocysts (59%) or late morulae (51%). Addition of FCS significantly (P<0.01) reduced survival compared with control embryos or those frozen in BSA-supplemented medium (50.48 vs 68.01 vs 63.53%, respectively). There was also a significant interaction between embry o stage and protein type (P<0.05). The survival of late morulae/early blastocysts following freezing was improved in the presence of additio nal BSA but not FCS. In Experiment 2, the IVC medium was supplemented with liposomes containing lecithin, sphingomyelin and cholesterol. Sph ingomyelin and cholesterol at ratios of 1:1, 1:4 and 4:1 were added to 50, 100 or 150 mu g/mL lecithin to yield a final lipid concentration of 200 mu g/mL. A further group contained 200 mu g/mL lecithin only. B lastocysts were frozen in 1.5 M ethylene glycol in OCM, then thawed an d assessed as in Experiment 1. The presence of liposomes during NC did not affect the proportion of cleaved embryos that developed to blasto cysts or survival following freezing. However, the survival of blastoc ysts that developed in the presence of 200 mu g/mL lecithin only was s ignificantly lower than in any other treatment (6%; P<0.03). These stu dies demonstrate that the protein composition of the freezing medium c an significantly affect survival after thawing and that the survival o f late morulae can be improved with additional BSA. The presence of le cithin only in the liposome preparation did not affect embryo developm ent, but significantly reduced survival after freezing, suggesting it can affect post-thaw embryo survival, perhaps by altering embryonic me mbrane composition. (C) 1998 by Elsevier Science inc.