Six female littermate piglets were used in an experiment to evaluate the mRNA expression in tissues from piglets given one or two 1 mL injections of iron dextran (200 mg Fe/mL). All piglets in the litter were administered the first 1 mL injection < 24 hours after birth. On d 7, piglets were paired by weight (mean BW = 1.72 ± 0.13 kg) and one piglet from each pair was randomly selected as control (CON) and the other received a second injection (+Fe). At weaning on d 22, each piglet was anesthetized, and samples of liver and duodenum were taken from the anesthetized piglets and preserved until mRNA extraction. Differential Gene Expression data were analyzed with a fold-change cutoff (FC) of |1.2| P < 0.05. Pathway analysis was conducted with Z-score cutoff of P < 0.05. In the duodenum 435 genes were significantly changed with a FC ≥ |1.2| P < 0.05. In the duodenum, Claudin 1 and Claudin 2 were inversely affected by +Fe. Claudin 1 (CLDN1) plays a key role in cell-to-cell adhesion in the epithelial cell sheets and was upregulated (FC = 4.48, P = 0.0423). Claudin 2 (CLDN2) is expressed in cation leaky epithelia, especially during disease or inflammation and was downregulated (FC = -1.41, P = 0.0097). In the liver, 362 genes were expressed with a FC ≥ |1.2| P < 0.05. The gene most affected by a second dose of 200 mg Fe was HAMP (Hepcidin Antimicrobial Peptide) with a FC of 40.8. HAMP is a liver-produced hormone that is the main circulating regulator of Fe absorption and distribution across tissues. It also controls the major flows of Fe into plasma by promoting endocytosis and degradation of ferroportin (SLC4A1). This leads to the retention of Fe in Fe-exporting cells and decreased flow of Fe into plasma. Metabolic pathway changes in the duodenum and liver provide evidence for the improved feed conversion and growth rates in piglets given two iron injections pre-weaning with contemporary pigs in a companion study. In the duodenum, there is a down regulation of gene clusters associated with gluconeogenesis (P < 0.05). Concurrently, there was a decrease in the mRNA expression of genes for enzymes required for urea production in the liver (P < 0.05). These observations suggest that there may be less need for gluconeogenesis, and possibly less urea production from deaminated amino acids. The genomic and pathway analyses provided empirical evidence linking gene expression with phenotypic observations of piglet health and growth improvements.

RNA Sequencing

Samples were submitted to Zymo Research (Irvine, CA, USA) for total mRNA extraction, cDNA library preparation and RNA sequencing. Total RNA-Seq libraries were constructed from 250 ng of total RNA. To remove rRNA, a method described by Bogdanova et al., 2011 was followed. Libraries were prepared using the Zymo-Seq RiboFree Total RNA Library Prep Kit (Cat # R3000) according to the manufacturer’s instructions (Zymo-Research, 2022). The RNA- Seq libraries were sequenced on an Illumina NovaSeq to a sequencing depth of at least 30 million read pairs (150 bp paired-end sequencing) per sample.

Detection of DGE and Bioinformatic Data Handling

Differentially expressed genes were detected using GeneSpring software (Agilent, Santa Clara, CA) using selection criteria that accepted a DGE threshold of greater than a |1.2|-FC in expression level and statistical probability levels of P < 0.05.

The filtered genes were then subjected to Ingenuity Pathway Analysis (IPA; QIAGEN Inc., Redwood City, CA; https://digitalinsights.qiagen.com/products/) to gain insights into canonical pathways, networks, and biological functions. Qiagen IPA uses algorithms, tools, and visualizations to combine the directional information from gene expression patterns (up- or down-regulation) with the expected causal effects of the genes, as reported in the published literature. A prediction for effects of a treatment on a particular biological pathway function or disease can then be made based upon the direction of change in gene expression and calculated Z-scores. Briefly, a Z-score is used to compare data that have different means and standard deviations. The Z-score is the distance of a point, such as a complete pathway, from the mean of the distribution in terms of the standard deviation (Corchete et al., 2020; Shao et al., 2020; Wieder, Lai and Ebbels, 2022).