136 N-METHYL-D-ASPARTIC ACID AND HOMOCYSTEINE CAN BE USED TOGETHER AS A REPLACEMENT FOR BOVINE SERUM ALBUMIN IN EARLY PORCINE EMBRYO CULTURE

Abstract

Most mammalian embryo culture media contain some form of unidentifiable biological contaminant, usually associated with fetal bovine serum (FBS) or bovine serum albumin (BSA). Such factor(s) confound experiments attempting to evaluate culture media composition and decrease the repeatability of experiments when different lots or batches of FBS or BSA are used. The goal of this study was to formulate a completely chemically defined culture media for development of early porcine embryos based on adding ligands for which there is the presence of mRNA for the corresponding receptors in the blastocyst. Cumulus–cell oocyte complexes were matured for 42 h in M199 supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris-buffered media for 4 h. Presumptive zygotes were then placed into porcine zygote media with 0.3% BSA (PZM3) or 0.1% polyvinyl alcohol (PZM4). At 28 h post-fertilization, 2- to 4-cell stage embryos were selected and placed into treatment groups for 5 days. Fifteen embryos were put into 25 μL of media and cultured in 5% CO₂, 5% O₂, and 90% N at 38.5°C. The treatment groups were as follows: 1. PZM3, 2. PZM4, 3. PZM4 + 0.5 mM N-methyl D-aspartic acid (NMDA), 4. PZM4 + 0.5 mM NMDA + 10 μM homocysteine (HC), and 5. PZM4 + 10 μM HC. There were 120, 135, 120, 135, and 120 embryos for each treatment, respectively. The percentages of embryos that developed to the blastocyst stage were 63.3%^a, 29.7%^b, 46.1%^c, 55.6%^ac, and 49.2%^ac, respectively [SAS; SAS Institute, Cary, NC, USA) Proc GLM (^a,b,cP < 0.05)]. Total cell number was determined using bisbenzimide to stain the nuclei, and the data were analysed by SAS Proc GLM. There was no difference in cell number among treatments with a mean cell number of 31.4. To further investigate the equality of the chemically defined media, the surface area of the blastocysts was measured by using Nis Elements BR3.0 software under 20× magnification. There was less surface area in treatment 5 compared with 4 [296 180^ab, 295 114^ab, 303 451^ab, 271 913^b, and 316 773^a arbitrary units (^a,bP < 0.05), with n = 33, 26, 37, 31, and 32 embryos in each treatment, respectively]. Because HC has been shown to affect global DNA methylation of bovine embryos, we stained our embryos for 5-methylcytidine (Eurogentec anti 5-MECY-0100) and embryos were visualised by UV light with a Texas red filter, and intensity was measured by Nis Elements BR 3.0 software under 20X magnification. The mean intensities were lower for the NMDA treatment [26.7^a, 19.4^a, 13.6^b, 17.0^a, and 19.4^a arbitrary units (^a,bP < 0.05)] compared with the other treatments. When embryos were cultured without BSA development decreases, but adding NMDA and HC returns development to control levels as measured by percentage of blastocysts, surface area, and global DNA methylation. We conclude that PZM4 supplemented with 0.5 mM NMDA and 10 μM HC may replace PZM3 as a chemically defined culture media for early porcine embryos. Embryo transfer experiments will be necessary to confirm that these embryos have equal developmental competence.