142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

Abstract

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO₂. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO₂, 5% O₂ and 90% N₂. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.