Digital Pathology-Based Morphometric Analysis of Saccharomyces cerevisiae Effects on Splenic Cell Populations in Broiler Chickens

Saccharomyces cerevisiae (SC) is a beneficial probiotic for poultry, serving as a natural alternative to antibiotics by promoting biological synergies that enhance animal health and productivity. This study aimed to evaluate the effects of SC on splenic immunomodulation in broiler chickens by characterizing and quantifying immune cell populations-including T lymphocytes (CD3⁺, CD4⁺, CD8⁺, and Treg CD25⁺), macrophages (CD163⁺), and B lymphocytes (CD20⁺)-per mm² of spleen tissue using immunohistochemistry (IHC). Two treatment groups were compared: a control group (CG; n = 8) without SC and an experimental group (PG; n = 8) supplemented with SC at 10⁷ CFU·g^-1. Splenic tissue sections were digitized at 20 × magnification using a MoticEasyScan Infinity 60 slide scanner. Immune cell density and positivity percentages (CD3⁺, CD4⁺, CD8⁺, CD20⁺, CD25⁺, and CD163⁺) were quantified using QuPath digital pathology software. SC supplementation significantly reduced cell density and positivity percentage of CD3⁺ and CD4⁺ T lymphocytes vs. CG and positivity percentage of CD25 cells in PG vs. CG. Supplementation with SC in broiler chickens significantly altered splenic immune cell morphometry, particularly in regions containing CD3⁺, CD4⁺, and positivity in CD25⁺, while no effects were observed on cell density or positivity in CD8⁺, CD20⁺, and CD163⁺ macrophages. Additionally, digital pathology proved effective in enabling precise morphometric quantification of immunohistochemical expression in digitized whole slides.