Development of a metabolomic- and immunological platform as a precision phenotyping tool for selection of pigs with improved resilience

For the international market for pig genetics, we have focused on disease resistance and robustness as a response to challenging environments. On a global scale, the Norwegian production conditions are very good, which indirectly might cause limited international growth potential for Norsvin in robustness and disease resistance traits. This is because there are few situations in Norwegian pig production where these traits can be measured as the production conditions and animal health status are good. Hence, it is difficult to measure traits that are related to stress tolerance.. Due to this, the project developed new sampling routines that are related to the existing phenotype recording at the boar station and stressful situations that the animals undergo. Then, new biomarkers related to stress response were identified from blood samples and analyzed. The project aimed to investigate the potential of performing in vitro (laboratory) disease challenge tests on cells from healthy young animals. Further, the project identified new methods to evaluate the animals' disease and stress responses through novel bio markers, and to investigate genetic variation between animals for future implementation in Norsvins breeding program. Blood samples from pigs were collected at two time points for 350 pigs, one at introduction to the boar testing station and one at CT scanning. Metabolome analyses of the blood were analyzed at SINTEF in Trondheim, to find bio markers for stress tolerance, disease resistance and robustness. The metabolome analysis was performed with a combination of targeted and non-targeted analysis. Targeted analysis included analysis of amino acids and neurotransmitters/tryptophan metabolites. Non-targeted analyses were carried out using LC-MS with ultra-high-resolution mass spectrometry. Ultra-high-resolution mass spectrometry provides fingerprints of the metabolome with thousands of metabolite signals. In total, over 900 blood samples were been collected. Multivariate data analysis was used to compare and cluster these complex spectra and to link metabolome analyzes with other information that may indicate robustness. The parameters that were chosen can give an indication of the pigs' robustness and tolerance to stress and disease, including stayability, variation in feed intake and weight gain over time. Pearson correlations between various blood metabolites and the animal's breeding values for different robustness traits were estimated. Overall, the correlations between breeding values and metabolites were low, ranging from -0.35 to 0.39. The highest positive correlation was between growth and alanine and osteochondrosis and alanine. Still, a high number of metabolites had significant relationship to breeding values of different robustness traits. These results provide several relevant bio markers that can be used in the breeding program to enhance robustness to disease and stress tolerance. Several amino acids (alanine, isoleucine, serine, and phenylalanine) had a significant correlation to the breeding value for longevity, indicating the ability to stay in production. Furthermore, results for tryptophan metabolism indicated that kynurenine is relevant bio marker linked to variation in feed intake and body condition score at weaning. For further investigation of between-animal variation, genetic analyses of the different target metabolites are initiated, but still running. Still, it is reasonable to expect that several of the metabolites are heritable and have potential for implementation. The dataset gathered in this project is unique in terms of number of animals and the complexity of the blood metabolites on these number of animals. Non-target metabolites have also been investigated and the results showed that metabolome data can be used to say something about the animal’s total breeding value, and that there exists a covariation between specific metabolite groups and the animal’s total breeding value. This is means that metabolome data can be used as an explanatory variable for robustness traits, and potentially be utilized in the genetic evaluation of animals. Overall, this project has provided a detailed dataset on potential biomarkers for robustness to disease and resilience. Several bio markers have been identified which can be related to breeding values for robustness traits. Amongst these are kynurenine, alanine, glutamine, isoleucine, serine and phenylalanine. Several indicator traits have been investigated, and the results suggest that they all play a role in the overall robustness of the animals. Non-target metabolome data has significant covariates to the total breeding value of the animal and can be used in the genetic evaluation of animals to enhance robustness to disease and improved stress tolerance. The knowledge from this project has provided us with several opportunities for further investigation of these types of traits.

Different potential indicator traits (metabolites) was identified and linked to relevant breeding values related to resilience and robustness. These results provides us with relevant information regarding resilience, stress tolerance and robustness of the animals, and can be used further for phenotyping in high health status herds. If genetic parameters are identified for these metabolites, they can be used as indicator traits for rescilience and selection for improved resilience can be acheived. This will increase the on-farm profitability in terms of reduced reqruitment costs and veterinary costs, both nationally and internationally. Further, it will increase our competetiveness with rival providers of genetic material and strengthen Norsvins brand worldwide. Further, to enhance resilience, stress tolerance and robustness would lower the usage of antibiotics in pork production. This is a common global goal to reduce the threat of antibiotic resistance in human and porcine pathogens. The results also have positive effect with regards to a more sustainable production of livestock; thus leading to increased animal welfare for the animals and reduce the medical treatment and loss of animals. This also has the potential to reduce the impact of the environment by improving efficiency and sustainability of food production.

The project is intended to lead to new phenotypes and models to create more resilient pigs, better adapted to the international market. Resilience is the ability of the animals to recover quickly from a wide range of disruptive challenges back to the original level of normal health, fertility, and production. According to this definition it is clear that in practice, measuring and then selecting for resilience in livestock is particularly challenging. This is even more challenging in the high health, and fortunate, environment present in Norway, where natural exposure to challenging conditions is limited. The underlying idea in this project is to fill main gaps that must be addressed to be able to breed properly for improved resilience. Norsvin has high ambitions for expansion on international markets. Sustainability and high animal welfare are fundamental elements of Norsvins breeding strategy that have helped to ensure that the ambitious international effort is well on the way to success. However, due to more incidences of diseases worldwide it is essential to develop breeding programs for selection of resilience in order to keep competiveness internationally and improve animal welfare in challenged environments. The main objective of this project is therefore to enhance the ability of pigs to resist and minimize the impact of disease by developing methods to select for disease and stress resilience in a high-health nucleus breeding populations. In this project our ambition is to address these issues by using novel state of the art metabolomic tools combined with immunologic methods and bioinformatics to examine blood plasma for potential novel and known metabolic and immunologic biomarkers, and their role related to resilience. Additionally, instead of direct disease challenge on pigs, the macrophages from blood and lung will be in-vitro challenged with the most important pathogens to obtain genetic variants useful for selection.