RNaseq-based whole transcriptome analysis in the jejunum of preweaned calves under different milk feeding regimes

An early dietary nutrition plan in calves is crucial for later appropriate heifer and cow performance. However, there is controversy about the appropriate milk supply during the first weeks of life. Currently, there is only limited knowledge about the genomewide expression pattern for the juvenile intestine. Therefore, we performed a next-generation-sequencing-based holistic whole transcriptome analysis of the jejunum in male German Holstein calves fed two different diets. Calves received colostrum for 3 d and then milk replacer (MR; 125 g powder/L). For one group of calves, MR was restricted to a volume of 6 L MR (n = 6), whereas the alternative group was fed on an ad libitum protocol for 8 wk (n = 6). Subsequently, MR intake for all calves was reduced to 2 L/d from wk 8 to 10 and maintained at this level until the end of the trial. During the entire experiment, both groups were provided hay and concentrate ad libitum. Eighty-one days after birth, the calves were euthanized, and epithelial sections of the jejunum were collected, snap frozen in liquid nitrogen, and stored at -80°C until further analyses. Total RNA was isolated with specific attention to avoid genomic DNA contamination. For each sample, an indexed, stranded sequencing library was prepared including a polyA bead-based selection step. Libraries were sequenced in a 2 x 80 bp paired-end protocol on an Illumina HiSeq2500. After demultiplexing, reads were trimmed for quality and adaptor sequences. The reads passing quality control were aligned to the bovine genome, and transcripts were assembled with an annotation-guided approach enabling discovery of yet-unannotated genes and transcripts. Differential expression between calf groups was analyzed based on expression normalized on total number of reads and length of the respective genes. On average, 56.8 million paired-end reads were obtained per sample, and the average percentage of reads mapping to the bovine genome was 88%. Between 18,395 and 20,904 genes per sample could be identified after applying a threshold of 10 read counts per gene. The number of novel transcripts amounted to 16,378. Applying a threshold of q < 0.1, which accounted for multiple testing, 99 genes showed a differential expression between groups. Among those, 22 were without previous annotation in the bovine genome. Our analyses provide the first comprehensive catalog of RNaseq-based whole transcriptome for the calf jejunum. Furthermore, our data prove persistent effects of