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Degenerative changes in tendons are common in human and land-dwelling vertebrates of all species, and are associated with both age and use. Tendon rupture and inflammation are common causes of lameness in broilers and breeders at multiple ages and has become a very important issue in the poultry industry attempts to analyze tendon health have relied primarily on visual assessment. The ability to conduct meaningful experiments on causes and interventions would be much improved by a direct assay of tendon structural integrity. The objective of this study is to test whether we can use Fourier transform-second harmonic generation (FT-SHG) imaging technique to quantify the differences in collagen fiber organization and nuclei morphology between normal and injured chicken tendons. Tendon injury/degeneration was induced by rearing chickens in wire-floor pens and feeding them an oxidized fat diet. Injured tendons had greater collagen fiber degradation/degeneration; more randomly organized collagen fibers, higher cellularity and more circular-shaped nuclei than normal tendons, indicating loss of collagen tertiary structure and lateral compression in injured tendons. Therefore, analysis of collagen fiber organization and nuclear morphology using FT-SHG imaging is a potential diagnostic tool to evaluate tendon structural health and to test the effects of nutrients and intervention strategies on the structural development of tendons in chickens.