Replacement of bovine serum albumin with N‐methyl‐D‐aspartic acid and homocysteine improves development, but not live birth

We mined microarray data and found that message for the glutamate receptor, ionotropic N-methyl-D-aspartate 3A (GRIN3A) was higher in both in vitroand in vivo-produced fourcell and blastocyst stage embryos compared to germinal vesicle oocytes (Whitworth et al., 2005). Since GRIN3A is part of the Nmethyl-D-aspartic acid (NMDA) receptor, we inferred that the embryo would benefit fromNMDA and proposed to replace bovine serum albumin (BSA) in the culturemediumwith NMDA to develop more defined in vitro culture conditions. Preliminary data showed removal of BSA reduced DNA methylation. Since homocysteine (HC) hypermethylates DNA in bovine embryos (Ikeda et al., 2010), we also decided to add HC to the culture medium to restore DNA methylation levels. Twoto four-cell stage embryos produced in vitro (according to Whitworth et al., 2005) were cultured for 6 days in Porcine Zygote Medium 3 (PZM3, which contains 3mg/mL BSA), PZM4 (PZM3 with 0.1% polyvinyl alcohol replacing BSA), PZM4þ 0.5mM NMDA, PZM4þ 0.5mM NMDAþ 10mM HC, or PZM4þ 10mM HC. The percent of embryos that developed to the blastocyst stage was 63.3%, 29.7%, 46.1%, 55.6%, and 49.2%, respectively (P< 0.05, N1⁄4 630). Both NMDA and HC resulted in an increase in the percent blastocyst above PZM4. There was no difference in cell number among treatments, with a mean cell number of 31.4 1.3 (P> 0.10; Fig. 1). To further investigate the quality of these embryos, the surface area of the blastocysts was measured. Culture in the presence of HC resulted in larger blastocysts than culture in NMDAþHC (296,180 15,042, 295,114 16,228, 303,451 14,810, 271,913 14,884, 316,773 14,632 arbitrary units (P< 0.05), respectively N1⁄4 159). As HC has been shown to affect global DNA methylation of bovine embryos (Ikeda et al., 2010), we stained our embryos for 5-methylcytidine. The relative level of methylation (26.7 4.1, 16.9 3.2, 19.3 3.5, 13.6 2.9, 19.4 2.9 arbitrary units (P< 0.05), respectively) was higher in PZM3 than PZM4 or PZM4þNMDAþHC. Homocysteine may not have the same effect on DNA methylation in the absence of BSA as shown for bovine embryos in the presence of BSA (Ikeda et al., 2010). We performed real-time PCR for adenosylhomocysteinase and methionine adenosyltransferase II on embryos cultured in PZM3 or PZM4þNMDAþHC to evaluate message level for two genes whose products regulate DNA methylation, but found no difference. Six embryo transfers performed with blastocysts produced in PZM4þNMDA and 10 with blastocysts produced in PZM4þ NMDAþHC to synchronized surrogates did not produce any piglets (25–50 embryos were transferred per surrogate), whereas control embryo transfers to 12 gilts produced 5 litters of piglets. Development of NMDA-treated embryos is also in contrast with another contemporary embryo transfer in conditions where BSA was replaced with low-density lipoprotein (Spate et al., 2010), as a single transfer of 30 blastocysts produced 9 piglets. When embryos were cultured without BSA, development is reduced. Supplementing with NMDA and/or HC increases development, in some cases to control levels, as measured by percent blastocyst, surface area, and global DNA methylation. Yet, we were unable to produce piglets after embryo transfer.We conclude that even if a treatment increases the apparent quality of the blastocyst stage embryo, the embryos are not necessarily developmentally competent.