Analysis of Differential Messenger RNA Expression Between Bovine Blastocysts Produced in Different Culture Systems: Implications for Blastocyst Quality1

Using reverse transcriptase-amplified fragment length poly-morphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain re-action, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their abil-ity to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with gran-ulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, ar-tificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse tran-scription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were re-corded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7 % in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differ-entiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine em-bryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation. early development, environmen