166 MOUSE EMBRYONIC DEVELOPMENT FROM ONE-CELL STAGE TO BLASTOCYST, UTILIZING KSOM SUPPLEMENTED WITH AMINO ACIDS IN A MICROCHANNEL DEVICE

Abstract

Great efforts have been made to improve in vitro culture for enhancement of embryonic development. However, in vitro development of mammalian embryos still remains a challenge for the scientific community. Recently, the use of microfluidic culture devices, as an alternative technique compared to the standard drop, has allowed mammalian embryos to develop to the hatched blastocyst stage. With the use of a different medium, mouse strain, and microchannel device than previously reported (Raty S et al., 2001 Theriogenology 55, 241 abst), this study was undertaken to determine if a microchannel device fabricated from borosilicate and poly-dimethylsiloxane would support development of mouse embryos from one-cell to blastocyst, as an alternative to standard microdrop culture. Mice (F1 inbred C57BL/6CRL X SJL) from 3 to 8 weeks old were superovulated with 5 IU of PMSG and 5 IU of hCG. The female SJL strain of the mice has demonstrated low reproductive performance. One-cell embryos were collected in M2 medium (Sigma, St. Louis, MO, USA.). For each treatment, 240 embryos in 24 replicates were cultured. Groups of 10 embryos were cultured in the microchannel device using 500 μL of KSOM with amino acids (MR-106-D, Speciality Media, Phillipsburg, NJ, USA.); no additional supplements were added. Groups of 10 embryos were cultured in standard microdrops (control) using 30 μL of the same medium covered with mineral oil. Embryos were cultured in a 100% humidified, 5% CO₂ in air atmosphere at 37°C for 96 h. Embryos were allocated to the control treatment or the microchannel device treatment using a randomized block design. The percentage of embryos at each stage of development was evaluated at 24-h intervals. The stage of embryo development at each observation was analyzed by ANOVA using the general linear model in SAS (PROC GLM, type I sum of squares). Blastocyst development in the microchannel device was not different when compared to results obtained in the standard drop. The percentage of blastocysts developing, when analyzed from one-cell stage, was 29 ± 5% for the control and 26 ± 6% for the microchannel. The percentage of blastocysts, when analyzed from cleavage, was 35 ± 5% for the standard drop and 31 ± 7% for the microchannel device. The results obtained are encouraging, when considering the non-optimized medium and mouse strain utilized in this experiment. In conclusion, the results show the microchannel device may be considered an alternative technique for use in embryo culture as it supports development of mouse embryos from one-cell stage to blastocyst.