In ovo MRI of the chicken spleen: feasibility of a new tool for basic research

The avian spleen, as a major secondary lymphoid organ, plays a key role in immune regulation in chickens lacking structured encapsulated lymph nodes. As a secondary lymphoid organ, it provides microenvironment for immune interactions, antigen recognition, and cytokine signaling. Despite its importance, in vivo and in ovo assessments remain limited, with most data derived from postmortem studies. Magnetic resonance imaging (MRI) offers a non-invasive, high-resolution method for evaluating organ morphology and volume, enabling assessment of morphological changes intra vitam. This study evaluated in ovo MRI for spleen visualization and volumetry in chicken embryos as a potential tool for immunological and developmental research. Ten SPF chicken eggs were incubated under standardized conditions and imaged on day 18 using a 3 T MRI scanner. Volumetric analysis was conducted on T2W-CISS images using Amira software following manual segmentation of spleen and embryo. Mean spleen volume was 17.72 mm³ (SD 1.78; range 14.20-19.37) and mean embryo volume 21,439.27 mm³ (SD 944.16; range 19,823.24-23,127.17). Spleen-to-embryo volume ratio was 0.0825 ± 0.0060%. Mean spleen weight was 0.0154 g (SD 0.0046; range 0.008-0.022) and embryo weight 23.5 g (SD 3.37; range 15-25), with weight ratio 0.068 ± 0.025%. MRI volumetry strongly correlated with weight measurements (r = 0.96, p < 0.01). These findings demonstrate in ovo MRI as a feasible, non-invasive tool for splenic morphology and volumetry. It could support studies on infection dynamics, inflammation, and disease processes in avian models, and enable longitudinal evaluation beyond hatching, tracking splenic development and disease progression from embryo to postnatal life in the same individuals over time.