A high protein model alters the endometrial transcriptome of mares

We developed an experimental model to elevate BUN during diestrus. There were both urea and control treatments (7 mares/treatment), done in a crossover design. Urea treatment consisted of a loading dose of urea (0.03 g/kg of urea) and urea injections over 6 hours (0.03 g/kg/hr). Control mares received the same volume of saline solution. Blood samples were collected to measure BUN. Uterine and vaginal pH were evaluated after the last intravenous infusion, then endometrial biopsies were collected for RNA-sequencing Overall design: A uterine biopsy was collected from the base of the uterine horn six hours after the start of the treatment (n= 7 per treatment). Total cellular RNA was extracted from endometrial samples using TRIzol Reagent (Thermo Fisher Scientific) following the manufacturer's recommendations. A total of 1 µg of RNA was treated with DNase I (Ambion Inc., Austin, TX) for 30 minutes at 37? to remove genomic DNA according to manufacturer's instructions. Paired-end reads with 150 nucleotides in length were produced. The RNAseq libraries were prepared with Illumina's TruSeq Stranded mRNAseq Sample Prep kit (Illumina, San Diego, CA). Read 1 aligns to the antisense strand and Read 2 aligns to the sense strand. The libraries were quantitated by qPCR and sequenced on one lane for 101 cycles from each end of the fragments on a HiSeq 4000 using a HiSeq 4000 sequencing kit version 1. The lane produced a total of 700 million reads. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 Conversion Software (Illumina). The Fastq files were evaluated for read quality using FastQC 0.11.4. Subsequently, Trim Galore 0.4.1 was used for adapter and read quality trimming (Phred score threshold of 30). Reads were mapped to the Equus caballus reference genome (EquCab 3.0) using the software STAR 2.5.3a, then annotated with the equine reference annotation from NCBI using Cufflinks 2.2.1. Fragments per kilobase per million (FPKM) were used to determine the expression level of genes. Lastly, we used Cuffdiff 2.2.1 to calculate differentially expressed genes (DEG) between samples from the control and urea groups. Significance level was set at FDR-adjusted p-value of the test statistic < 0.1 using a Benjamini-Hochberg correction.