o pieces ranging from 35 to 200 bases in size, and confirmed using Agilent 2100 Bioanalyzer technology. Fragmented cRNA samples were hybridized onto chips by incubating for 17 h at 65.8C with constant rotation, 
followed by a LY3039478 web two-step microarray wash of 1 min in two buffers. Hybridized microarrays were scanned in a GenePix 4100A scanner. The Gene Pix Pro 6.0. software was used for array image analysis and for the calculation of spot intensity measurements, which were considered to be raw data. Statistical analysis was performed using BioConductor for R with the packages Linear Models for Microarray data 4 / 18 Insemination Influences Oviductal Transcriptome in Pigs , Marray, pcaMethods, EMA and RankProd. The data were analysed by subtracting the background using the “Normexp” method, from the LIMMA package, with an offset of 10. Finally they were normalized by the “quantile” method form the same LIMMA package. Cutoff values were set to a two-fold difference in expression values and a false discovery rate of 1%. Identified probe sets were compared with published sequence databases using the basic local alignment search tool at the National Centre for Biotechnology Information. If a gene was identified in the results by more than one probe set, the mean fold change was calculated. All results from microarray analysis were submitted to Gene Expression Omnibus database with the accession number GSE68148. Ingenuity pathway analysis To integrate our results into a more general model, differentially expressed transcripts classified according to Gene Ontology and functional associations were investigated using the Ingenuity Pathway Analysis software. As indicated, only genes with a fold change >2 were included. Moreover, Osteopontin with a fold change PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19734939 of 1.47 was also included in the Ingenuity analysis because previous studies had shown its specific role in porcine fertilization. Microarray validation by Real-time RT-PCR The relative expression levels of eight selected genes in the total RNA from the oviduct were determined by real-time RT-PCR PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19735043 using specific primers for each gene: torsin 3A, Ras-related protein Rab-1B, arachidonate 12-lipoxygenase, glutathione S-transferase alpha 1, complement component 3, inter-alpha-trypsin inhibitor heavy chain family, member 4, Osteopontin and tropomodulin 3. -actine and GADPH genes were used as control for the data normalization. The qPCR experiment was performed as follows. First, the retrotranscription reaction was made using MultiScribe Reverse Transcriptase and 1 g of complete RNA. After this, the qPCR was completed in a StepOne Thermal Cycler using the 5x HOT FIREPolEvaGreenqPCR Mix Plus, with SYBR Green and normalizing with ROX. The Cts values were obtained from the OneStep Software. The genomic DNA contamination was abolished using a DNAse treatment and oligonucleotides designed with their sequence interrupted by intronic regions. The Cts values doi:10.1371/journal.pone.0130128.t001 5 / 18 Insemination Influences Oviductal Transcriptome in Pigs were analysed by statistical software, comparing the average between inseminated and non-inseminated samples, and obtaining the fold change values. The p-value was calculated between both groups and in all cases the significance was statistically relevant. Immunohistochemistry Torsin family 3, member A and GSTA1 protein expression were analysed immunohistochemically in the oviduct. The oviductal tissue samples were fixed immediately after collection in Bouin’s