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.

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages