The primer pairs used to amplify the coding region of the TLR4 gene (Table 1 ) were designed using the Primer3 tool (http://bioinfo.ut.ee/primer3-0.4.0/ ), based on the Bos taurus sequence (GenBank accession number: AC_000165.1 ). The TLR4 gene consists of 3 exons. Exons 1 and 3 include coding regions and the non-coding regions 5'UTR and 3'UTR, respectively. One pair of primers was designed for exons 1 and 2 each, whereas three further pairs of primers were designed for exon 3.
The amplification of coding regions of the TLR4 gene was performed by polymerase chain reaction (PCR) assays, which were conducted in Bio-rad S1000 thermal cyclers (Bio-Rad, Hercules, CA, USA) in a final volume of 15 μL, consisting of 100 ng of genomic DNA, 15 pM of each primer and the GoTaq Colorless MasterMix kit (Promega, Madison, WI, USA). The thermal profile was expressed by initial denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 95°C for 30 s; annealing at the melting temperature of each primer (Table 1 ) pair for 30 s; and extension at 72°C for 30 s. A final extension at 72°C for 5 min was performed. Further, 2 μL of PCR products were visualized by electrophoresis on 2% agarose gels stained with the GelRed system (Biotium, Inc., Hayward, CA, USA).
The amplification of coding regions of the TLR4 gene was performed by polymerase chain reaction (PCR) assays, which were conducted in Bio-rad S1000 thermal cyclers (Bio-Rad, Hercules, CA, USA) in a final volume of 15 μL, consisting of 100 ng of genomic DNA, 15 pM of each primer and the GoTaq Colorless MasterMix kit (Promega, Madison, WI, USA). The thermal profile was expressed by initial denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 95°C for 30 s; annealing at the melting temperature of each primer (