normal goat serum at room temperature to saturate any sites for nonspecific binding of proteins. Sections were incubated with anti-TOR3A or anti-GSTA1 primary antibodies overnight at 4C. TOR3A and GSTA1 immunolocalisation was established with the VECTASTAIN ABC kit. Finally, the sections were visualized using 3,3-diaminobenzidine hydrochloride, counterstained with haematoxylin, dehydrated, cleared through xylene twice for 5 min and mounted. Negative controls were performed following the same procedure without primary antibodies. Immunostained sections were examined using a Nikon Eclipse brightfield microscopy and captured by NIS-Elements Br Microscope Imaging Software 3.2. Immunohistochemical quantification was performed with Leica QWin Pro program. Each pixel from digital image consisted of a number between 0 and 255 representing the intensity of transmitted light or grey level at a point. Grey level was related with protein content. Oviductal fluid collection Oviducts from PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1970500 animals at the early luteal phase were obtained. The fluid was collected by mechanical aspiration using an automatic pipette. A mean volume of 40 l per oviduct was collected. The oviductal fluid was then centrifuged at 7,000 g for 10 min at 4C in order to remove cellular debris. Supernatant was kept at -80C until use. The oviductal fluid of a total of 6 animals per group was collected and pooled in to two batches of 3 animals each. SDS-PAGE and western blotting Protein concentrations of the samples were measured by the Bradford protein assay. Samples were re-suspended in Laemmli buffer, and boiled for 3 min. After centrifugation, 1 l of -mercaptoethanol was added to the samples, which were boiled again for 3 min. Finally, the samples were centrifuged. Eighteen g of total protein were loaded per line and subjected to SDS-PAGE and electro-transferred to PVDF membranes. The membranes were blocked with 5% BSA in TBS containing 0.1% Tween 20. The primary antibody was diluted 1:400 in T-TBS + 5%-BSA. TOR3A protein was detected by secondary antibody diluted 1:10,000 in T-TBS + 5%-BSA and developed using an enhanced chemiluminescence detection kit according to the manufacturer’s instructions. Results and Discussion The oviduct plays an important role during the reproductive process. In many mammals PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19704080 but even more so in pigs, it provides of a series of natural barriers against polyspermy. Fertilization is a highly synchronized process between male and female gametes, which takes place in a very specific area of the oviduct. In pigs, it occurs in the ampullary-isthmic junction. Understanding the mechanisms that act during this time would be of great importance to advance assisted reproduction techniques. In this study, we offer a new physiological perspective and provide new insights in to local changes in gene expression that take place in the porcine oviduct around the time of fertilization. When microarray analysis was performed, the data analysis revealed a total of 126 genes differentially expressed, between the inseminated and buy JW-55 non-inseminated sows. Setting the fold change to 2, 26 identified differentially expressed genes were selected. Only one gene with a fold change lower than 2, was included in our analysis given its importance in reproduction. To integrate Fig 2. Scheme of the protocol 
followed to process and analysed the data obtained from array hybridization. doi:10.1371/journal.pone.0130128.g002 7 / 18 Insemination Influences Oviductal Transcriptome in Pigs doi:
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