Embryo culture media typically contain undefined biologicals such as BSA. Our goal is to develop chemically defined culture media that are based on the biology and physiology of the embryo. To that end we evaluated the presence of message in embryos at various stages of development and determined that the message for the low density lipoprotein receptor (LDLR) increased from the germinal vesicle and 4-cell stage to the blastocyst stage of porcine embryogenesis. Thus, this study was conducted to determine if the addition of low density lipoprotein (LDL) would enhance the development and quality of in vitro produced porcine embryos in an already chemically defined culture medium. Slaughterhouse ovaries were aspirated, cumulous–oocyte complexes (COC) identified, and the COC were matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were then selected. Fertilization was then preformed in modified Tris buffered medium and cocultured with 0.25 � 106/mL frozen thawed porcine semen for 5 h. The presumptive zygotes were then transferred to either porcine zygote medium with 0.3% BSA or 0.1% PVA (PZM3, PZM4). After 28 h, cleaved embryos were then sorted into six treatment groups (1. PZM3, 2. PZM3 + 20 µg mL–1 LDL, 3. PZM4, 4. PZM4 + 10 µg mL–1 LDL, 5. PZM4 + 20 µg mL–1 LDL, 6. PZM4 + 50 µg mL–1 LDL). The embryos were cultured in 5%O2 5%CO2 90%N until day 7. The percentage of development to the blastocyst stage was determined and analyzed with the SAS Proc GENMOD Procedure (a–cP ≤ 0.05). The percentage blastocyst was 51.3 � 0.09a, 51.6 � 0.09a, 33.1 � 0.99c, 35.8 � 0.09c, 36.9 � 0.09c, and 41.3 � 0.06b, for treatments 1–6, respectively. Culture in PZM4 (without BSA) significantly reduced development. However, addition of 50 µg mL–1 of LDL to PZM4 improved development above PZM4 alone. We interpret these data to indicate that a high concentration of LDL in the PZM4 media did improve embryo development and that LDL could partially substitute for BSA. Differential staining was performed on the blastocysts, and preliminary results suggest that the ICM to trophectoderm ratio in the high LDL treatment group is closer to the ratio found in in vivo produced embryos.
This project was supported by USDA CSREES NRI (2006-35203-17282) and Food for the 21st Century.
Growth of Paracoccidioides brasiliensis yeast phase in a chemically defined culture medium.
