Martin Peter Rydal

Background: Understanding the genetic regulation of metabolism is essential for improving animal health and productivity and for strengthening the use of pigs as translational models for human metabolic diseases. Metabolomics, when integrated with genomic data, provides a powerful framework for identifying genetic factors that influence intermediate molecular phenotypes closely linked to physiology. While large-scale metabolomics genome‑wide association studies (GWAS) have been extensively performed in humans, comparable studies in pigs remain limited. Among omics disciplines, metabolomics, the systematic study of metabolites central to physiology, represents the omics layer most closely linked to end-point phenotypes in mammals, such as health and production traits (e.g. growth rate). Metabolite levels are influenced by genetics, diet, and microbiome composition, with human studies showing high heritability and identifying numerous metabolite quantitative trait loci (mQTLs). While such findings have advanced our understanding of disease biology in humans, comparable studies in pigs remain limited. Results: We performed GWAS of the serum metabolome in 213 three‑way crossbred pigs [(Landrace × Yorkshire) × Duroc], analyzing 248 metabolites and 30,628 metabolite ratios. We identified 41 significant metabolite–variant associations, which were consolidated into 18 mQTLs based on linkage disequilibrium (LD). Several regions were associated with multiple, biochemically related metabolites, while others showed highly specific effects. Cross-species comparison with the human GWAS catalog revealed candidate genes within seven mQTLs. Through an integrative prioritization strategy, putative causal genes—predominantly enzymes and transporters—were identified for 14 of 18 mQTLs. Conclusions: Our results expand the catalog of genetic loci influencing serum metabolite variation in pigs and highlight both conserved and pig specific components of metabolic regulation. These findings provide a foundation for incorporating metabolomics into pig breeding programs and reinforce the value of pigs as a translational model for studying metabolite-associated traits and diseases in humans.

Hematological traits are essential indicators of an animal's immune status and overall health, reflecting both physiological and pathological conditions. The complete blood count (CBC), a commonly used clinical test, evaluates the concentrations, proportions, and characteristics of various blood cell parameters, providing insights into an animal's current health status. CBC phenotypes are dynamic, influenced not only by health status but also by factors such as physiology, nutrition, environmental conditions, age, and genetic makeup. Previous studies have estimated heritability for specific blood parameters and genome‐wide association studies have identified loci associated with CBC phenotypes. While some loci seem to have similar effects across age and breeds and even across species, other loci seem to have a more breed and/or age‐specific effect. This study extends previous research by conducting genome‐wide association studies (GWAS) of 24 hematological traits measured in the same pig cohort at two ages (25 and 46 days). The GWAS included 1884 pigs at 25 days and 1615 pigs at 46 days, with 1499 pigs sampled at both time points. This analysis identified 57 QTLs for complete blood count (CBC) traits, including 11 lead SNPs associated with more than one trait. All animals were (Landrace × Yorkshire) × Duroc crossbred pigs. Thirty QTLs overlapped with previously reported CBC QTLs in different pig breeds, providing further evidence for shared regulatory regions for CBC traits across pig breeds. Genes in 29 QTLs were associated with comparable CBC phenotypes in humans.

Enterotoxigenic Escherichia coli (ETEC) of fimbriae types F4 and F18 causes post-weaning diarrhea (PWD) in pigs and susceptibility relates to host genotype. We examined if CHCF1/FUT1 genotypes were associated with ETEC-mediated PWD in seven Danish herds. Diarrhea was assessed in 1,919 pigs by evaluating perianal fecal staining in the first week after weaning. From each herd, twelve diarrheic and twelve non-diarrheic pigs were selected for genotyping, fecal dry matter (FDM) and microbiological testing. ETEC F4 diarrhea occurred only in CHCF1 heterozygous (RS) pigs. Furthermore, CHCF1 RS pigs had higher odds of diarrhea (Odds ratio (OR) 2.09; p = 0.026) and lower mean FDM (13.9%) than homozygous resistant (RR) pigs (18.6%; p = 0.0052). ETEC F4 was more frequently isolated from CHCF1 RS pigs compared to RR pigs (OR 8.88, p = 0.00005). Additionally, hemolytic E. coli shedding was higher in CHCF1 RS pigs (mean 47.4% vs. 24.1%; p = 0.00001). Too few FUT1 RR pigs were found for analyzing association between FUT1 genotypes and ETEC F18-mediated diarrhea. In conclusion, CHCF1 genotype was strongly associated with ETEC F4-mediated PWD under production settings. ETEC F4 resistance in pigs could help to reduce PWD occurrence and antibiotic use.

Introduction: Universal biomarkers in the fecal microbiota that predict susceptibility to post-weaning diarrhea (PWD) would be valuable for future intervention strategies. Genetic susceptibility to enterotoxigenic E. coli (ETEC) infection in pigs is a major determinant of PWD and may unfavourably alter early-life gut microbiota composition. We investigated whether pigs genetically susceptible to ETEC F4ab/ac had different fecal microbiota composition and diversity pre- and post-weaning compared to genetically resistant pigs. Method: Fecal microbiotas were characterized using long-read sequencing of the 16S rRNA gene in 24 CHCF1 heterozygous susceptible (RS) and 24 CHCF1 homozygous resistant (RR) pigs. These pigs were tested at early lactation (post-natal day (PND) 8) and late lactation (PND 22), which are critical periods for microbiota development and immune maturation. Twelve pigs from each group were weaned and transported to an experimental facility at PND 23, and were tested again at PND 24, prior to an ETEC F4ab challenge. This enabled studying immediate fecal microbiota changes after weaning and investigating whether CHCF1 RS pigs had compromised microbiotas compared to CHCF1 RR pigs preceding infection. Results: Across time, CHCF1 RS pigs had a higher number of observed OTUs (coef: 103, 95% CI [18.90; 192.76], p = 0.01) compared to CHCF1 RR pigs. There were no significant differences in the overall bacterial communities or differentially abundant taxa between genotypes. Littermates had bacterial communities more similar to each other compared to non-littermates during lactation (PND 8: R² = 0.2, p = 0.001 and PND 22: R² = 0.23, p = 0.001) and the litter effect persisted after weaning (PND 24: R² = 0.4, p = 0.001). Conclusion: We did not find major differences in the fecal microbiota between CHCF1 genotypes, pre- or post-weaning, that could help explain subsequent susceptibility to ETEC F4ab-mediated PWD. Litter explained a major part of the variation in the overall fecal bacterial community between pigs in the study.

Introduction: The association between the porcine pre-weaning gut microbiota composition and diversity, and subsequent post-weaning diarrhea (PWD) susceptibility is currently being studied. In this longitudinal study, we examined the association between pre-weaning fecal microbiome composition and diversity, and PWD development in a Danish sow herd.Methods: Forty-five pigs were followed from birth until 7 days after weaning (post-natal day (PND) 33). At PND 33, the pigs were categorized as PWD cases or healthy controls based on fecal consistency. We compared their fecal microbiomes at PND 8, late lactation (PND 27) and 7 days post weaning (PND 33) using 16S rRNA V3 region high-throughput sequencing. At PND 27 and 33, we also weighed the pigs, assessed fecal shedding of hemolytic Escherichia coli by culture and characterized hemolytic isolates by ETEC virulence factors with PCR and by whole genome sequencing.Results: A total of 25 out of 45 pigs developed PWD and one Enterotoxigenic E. coli strain with F18:LT:EAST1 virotype was isolated from most pigs. At PND 33, we found differences in beta diversity between PWD and healthy pigs (R² = 0.027, p = 0.009) and that body weight was associated with both alpha and beta diversity. Pre-weaning fecal microbiome diversity did not differ between PWD and healthy pigs and we found no significant, differentially abundant bacteria between them.Conclusion: In the production herd under study, pre-weaning fecal microbiome diversity and composition were not useful indicators of PWD susceptibility.

Host genotype is important for enterotoxigenic E. coli (ETEC) susceptibility. We conducted two trials to evaluate the effect of CHCF1 genotype on incidence of ETEC diarrhea. In trial 1 (n = 15 pigs), pigs were inoculated with 10⁸ CFU or 10¹⁰ CFU doses of an ETEC F4ac strain. In trial 2 (n = 33 pigs), pigs were inoculated with ETEC F4ab or F4ac. Across trials, all inoculated pigs that developed ETEC diarrhea were CHCF1 heterozygous susceptible (6/6). No inoculated CHCF1 homozygous resistant pigs developed ETEC diarrhea (0/26). Susceptibility towards ETEC F4ac/ab infection might correspond with CHCF1 genotype.

Anemia is a common condition in sow herds. We aimed to study the effects of severe iron deficiency during gestation on sow and piglet health outcomes with an experimental model for blood-removal-induced iron deficiency anemia. In total, 18 multiparous sows (8 in trial I and 10 in trial II) were allocated to either a blood removal group or a control group. Hematologic parameters were monitored at regular intervals and the tissue iron concentrations were measured for the sows and newborn piglets after farrowing. In trial I, the mean liver iron content was reduced to 46.7 µg/g in the blood removal sows compared to 252.6 µg/g in the controls (p < 0.001). In trial II, sows in the blood removal group had lower iron content in the liver (147.8 µg/g), kidney (46.3 µg/g) and spleen (326.5 µg/g) compared to the control sows (323.2 µg/g, 81.3 µg/g and 728.9 µg/g, respectively; p = 0.009, 0.016, 0.01, respectively). In trial I, piglets from sows in the blood removal group had significantly decreased hematocrit (Hct), red blood cells (RBC) and a tendency for reduced hemoglobin (Hb) compared to the control piglets. We established a blood removal model that resulted in mild- to severe degrees of sow anemia and reduced tissue iron stores at farrowing.

Optimal nutrition is important after preterm birth to facilitate normal brain development. Human milk is rich in sialic acid and preterm infants may benefit from supplementing formula with sialyllactose to support neurodevelopment. Using pigs as models, we hypothesized that sialyllactose supplementation improves brain development after preterm birth. Pigs (of either sex) were delivered by cesarean section at 90% gestation and fed a milk diet supplemented with either an oligosaccharide-enriched whey with sialyllactose (n = 20) or lactose (n = 20) for 19 days. Cognitive performance was tested in a spatial T-maze. Brains were collected for ex vivo magnetic resonance imaging (MRI), gene expression, and sialic acid measurements. For reference, term piglets (n = 14) were artificially reared under identical conditions and compared with vaginally born piglets naturally reared by the sow (n = 12). A higher proportion of sialyllactose supplemented preterm pigs reached the T-maze learning criteria relative to control preterm pigs (p < 0.05), and approximated the cognition level of term reference pigs (p < 0.01). Furthermore, supplemented pigs had upregulated genes related to sialic acid metabolism, myelination, and ganglioside biosynthesis in hippocampus. Sialyllactose supplementation did not lead to higher levels of sialic acid in the hippocampus or change MRI endpoints. Contrary, these parameters were strongly influenced by postconceptional age and postnatal rearing conditions. In conclusion, oligosaccharide-enriched whey with sialyllactose improved spatial cognition, with effects on hippocampal genes related to sialic acid metabolism, myelination, and ganglioside biosynthesis in preterm pigs. Dietary sialic acid enrichment may improve brain development in infants.