IN VITRO CULTURE OF BOVINE EMBRYOS WITH A SERUM REPLACER IMPROVES SURVIVAL FOLLOWING VITRIFICATION AND WARMING

Abstract

Use of fetal bovine serum during in vitro embryo culture is known to be detrimental to bovine embryo cryosurvival. Previously, a serum replacer (KNOCKOUT SR, Gibco) was shown to be beneficial for the in vitro culture of bovine embryos, so we tested whether this culture supplement could also improve survival of embryos following vitrification and warming. Cumulus oocyte complexes were retrieved by slashing 2–8 mm follicles on ovaries obtained from the abattoir. They were matured, fertilized (day 0), and then cultured for 3 days in KSOM. On day 3 postfertilization, embryos were divided into four treatments: 1) KSOM static culture (KNM; n = 533); 2) embryos moved to fresh KSOM (KD3; n = 537); 3) embryos moved to KSOM+SR (10%; n = 534); or 4) embryos moved to KSOM+FBS (10%; n = 534), and were cultured for the remainder of the 7 day culture period, in a humidified low oxygen environment (5/5/90). Embryos were then graded for stage and quality. Grade 1 and 2 blastocysts were vitrified in groups of 3–5 within treatments in a two step process, loaded into gel loading tips in 16.5% EG/16.5%DMSO/0.5M sucrose and quickly plunged into liquid nitrogen for storage. Upon warming and rehydration, embryos were cultured for an additional 48h under the same culture conditions by treatment listed above. Data were analyzed by ANOVA using SAS, and differences were determined using LSMeans, with significance deemed P < 0.05. Development to blastocyst on day 7 was highest for KSOM+SR (37%), but was not different from other treatments. The percentage of freezable quality blastocysts (grades 1 and 2) by treatment were 70, 60, 58 and 50% for KNM, KD3, KSOM+SR and KSOM+FBS, and differed between KNM and KSOM+FBS, and KNM and KSOM+SR (P < 0.05). Survival was defined as re-expansion and continued development with normal morphology in culture following vitrification and warming, and was significantly greater for KSOM+SR than all other treatments at 24h (64.7%, P < 0.05). At 48h post warming, KSOM+SR still had the highest survival, and was significantly greater than KSOM+FBS (46% vs. 19%, P < 0.05). KSOM+SR gave the highest percentage hatching/hatched blastocysts in culture post-warming, but was not different from the other treatments (P > 0.10). All treatments had significantly lower hatching in culture than non-vitrified controls cultured in fresh drops of KSOM+SR from day 7 to day 9 post-fertilization (90%, P < 0.05). These data suggest that culture with SR can improve the cryosurvival of in vitro produced and vitrified bovine embryos, but is still not comparable to non-vitrified controls. Future work will determine if SR can also replace FBS in vitrification solutions to get further improvements in bovine embryo cryosurvival. This project was supported by the Florida Agricultural Experiment Station. (poster)