cal data for inseminated and non-inseminated animals. doi:10.1371/journal.pone.0130128.g001 3 / 18 Insemination Influences Oviductal Transcriptome in Pigs ensure the presence of growing PF-562271 cost follicles in both ovaries. A diagram of the experimental design is shown in Fig 1A and animal data sheet is shown in Fig 1B. Inseminations were performed with seminal doses from mature hybrid boars of proven fertility. Fresh semen diluted in 100 ml of Beltsville Thawing solution at a concentration of 3 x 107 spermatozoa/ml was used. Animals were diagnosed as post-ovulatory when no large follicles were observed by ultrasonography, ensuring that ovulation had occurred before removing the oviducts. Then, animals were subjected to sedation and anaesthesia in order to perform a mid-ventral incision and expose the genital tract. The ovaries were photographed, the number of recentlyovulated follicles was recorded, and the oviducts were removed no more than 20 minutes after sedation. No differences between inseminated and control groups were found for the number of previous farrowings, the number of ovulated follicles nor for progesterone concentrations. These data are similar to those detected in previous studies by other authors. Complete oviductal tissue samples from the ampullary-isthmic junction were taken and immediately frozen in liquid nitrogen for mRNA extraction, cDNA reverse transcription and further study by microarray. Oviductal flushing was not performed to avoid transcriptome alteration. Tissue samples were fixed immediately after collection in Bouin’s solution for the immunohistochemical assay. Blood samples were also taken in order to determine the systemic oestradiol and progesterone concentrations for each animal, using a chemoluminescence immunoassay of microparticles. RNA isolation Total RNA was extracted using the `TRIzol method’ according to the protocol recommended by the manufacturer. The quantity and quality of the RNA samples were analysed with ARN 6.000 NanoLabChip kit and the Agilent 2100 Bionalyzer respectively. Values of RNA integrity number in analysed samples ranged from 6.5 and 8.5, and only those samples with RIN > 7.0 were used for microarray analysis. Microarray analysis Equal amounts of total RNA PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19702757 from samples of the oviducts of each animal were used. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19704080 All the samples were hybridized in the Porcine Gene Expression Microarray which encompassed 43,803 probes. Protocols for 
sample preparation and hybridization of the oviductal samples were adaptations of those in the Agilent Technical Manual. In brief, first strand cDNA was transcribed from 300 ng of total RNA using T7-Oligo Promoter Primer. Samples were transcribed in vitro and Cy-3-labelled using a Quick-AMP labelling kit. The synthesis yielded 10 15 g of cRNA. Following a further clean-up round, the cRNA was fragmented into 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 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
