128 N-METHYL-D-ASPARTIC ACID CAN BE USED TO PARTIALLY REPLACE BOVINE SERUM ALBUMIN IN CULTURE MEDIUM

Abstract

One major obstacle in mammalian embryo culture has been unidentifiable biological contaminants in the media due to the inclusion of Bovine Serum Albumin (BSA) or Fetal Bovine Serum. The goal of this study was to remove BSA from culture media and develop chemically defined media based off the embryo’s biological and physiologic makeup. We evaluated the presence of message in various stages of porcine embryos and found that the message for the ionic glutamate receptor, N-Methyl-D-aspartic acid (NMDA) increased about 3-fold from oocyte to blastocyst. Thus, this study was conducted to determine if the addition of NMDA (0.5 mm) would improve development of embryos in an already chemically defined medium. Slaughterhouse derived ovaries were aspirated, cumulus–oocyte complexes were identified and then matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris buffered medium with 0.25 × 10⁶ mL^–1 frozen–thawed porcine semen for 5 h. Presumptive zygotes were then transferred to Porcine Zygote Medium with 0.3% BSA (PZM3) or 0.1% PVA (PZM4). After 28 h, cleaved embryos were selected and embryos were placed into treatment groups: (1) PZM3, (2) PZM4, or (3) PZM4 + 0.5 mm NMDA. Embryos were cultured in 5% CO₂, 5%O₂, 90% N₂ until Day 7. For this experiment the number of cleaved embryos cultured in each treatment group were 260 for group 1, 220 for group 2 and 300 for group 3. Percentage of development to blastocyst was determined and analyzed with SAS Proc GENMOD Procedure (a,b P < 0.05). The percentage developed to blastocyst was (1) 47.5% a, (2) 29.6% b, and (3) 36.1% a,b, respectively. Total cell number of the blastocysts was determined by using Hoechst nuclear stain and statistically analyzed by SAS Proc GENMOD Procedure. The average cell number for the treatment groups was (1) 25.8 a, (2) 19.6 b, and (3) 22.9 a,b, respectively. Culture without BSA significantly reduced development to blastocyst and total cell number; however, with the addition of 0.5 mm NMDA there was no significant difference from media containing BSA. This indicates that NMDA can be used to partially replace BSA to form a chemically defined media.

Funded by a grant from the USDA NRI 2006-35203-17282.