Metabolic Capacity Comparison of Laying Hen and Broiler Line Isolated Peripheral Blood Mononucleated Cells

Cellular metabolism reflects the combined effects of environment, genetics, nutrition, and individual variation in an animal. Advanced metabolic analysis can be used to further describe phenotypic differences in immune cells from distinct populations. To generate foundational immune cellular metabolic data in layer and broiler strains, the Seahorse XFe Analyzer (Agilent) was used to determine preferred substrate, pathway, and optimal cell count. Because past work using poultry models has been done largely with immortal cell lines rather than primary cells, titrations were completed using fresh peripheral blood mononuclear cells (PBMCs). Blood samples were collected from a colony of singly-housed Bovans White hens and from a straight run flock of Ross 308s (age 5-7 weeks) housed in pens of 10. The optimal concentration for both strains was determined to be 3 million cells/well based on the necessity for a cell monolayer. The Cell Energy Phenotype test (Agilent) measured the baseline mitochondrial respiration of the cell (Oxygen Consumption Rate; OCR) and the baseline glycolytic activity (Extracellular Acidification Rate; ECAR), and then applied a challenge using metabolic pathway inhibitors (FCCP and Oligomycin) to determine metabolic capacity and preferred pathways. Baseline and peak minus baseline (metabolic capacity) OCR and ECAR values were compared between strains using the Welch’s t test on JMP Pro 13. The difference between baseline OCR values was not significant (p=0.4944), but baseline ECAR values were significantly different (p=0.0006) in laying hens (mean of 41.34 mpH/min) compared to broilers (mean of 62.98 mpH/min), implying that the different strains utilize the mitochondrial respiration pathway differently and that broiler PBMCs may be more inclined to utilize this pathway. Hen and broiler PBMCs were independently titrated for optimal FCCP concentration; optimal conditions were determined by calculating the difference between peak and baseline OCR and ECAR values. Optimal FCCP concentration was 0.5 mM in hens and a range of 0.25-0.5 mM was optimal for broilers. Ultimately, hen and broiler PBMCs were compared within the same test plate using 0.5 mM FCCP, resulting in a mean OCR of 505.62 pmol/min for hens and 413.11 pmol/min for broilers, and a mean ECAR of 64 mpH/min for hens and 56.23 mpH/min for broilers. There were no significant differences observed between broiler and laying hen OCR (p=0.6403) or ECAR (p=0.3573) means, leading us to believe that while the birds may be using different pathways to produce energy, their metabolic capacity/ability to respond to an immune challenge is not significantly different.