Research note: Turkey (Meleagris gallopavo) semen CD14+ macrophages – management, key features, and classification – a pilot study with optimized protocols

Semen macrophages (Mф) play a role in mediating innate immune responses and supporting adaptive immunity within the male reproductive tract. Knowledge about the semen Mф in avian species remains limited. This study aimed to isolate turkey semen Mф and optimize protocols for their characterization based on specific Mф markers, specifically cluster of differentiation 14 (CD14), inducible nitric oxide synthase (iNOS), and interleukin 10 (IL10), for further classification into CD14⁺ type 1 macrophages (CD14⁺iNOS⁺, akin CD14⁺Mф1) and CD14⁺ type 2 macrophages (CD14⁺IL10⁺, akin CD14⁺Mф2) subpopulations. In the current study, immunofluorescence (IF) staining and fluorescence-activated cell sorting (FACS) were employed to identify these markers, and the morphological features of CD14⁺Mф were examined. Our results demonstrated that turkey semen CD14⁺Mф are spherical, may contain one or more nuclei, and often exhibit large vacuoles. Based on FACS results, CD14⁺iNOS⁺(akin CD14⁺Mф1) accounted for 48.5 ± 12.45 % of CD14⁺ cells, whereas CD14⁺IL10⁺ (akin CD14⁺Mф2) represented 42.48 ± 14.62 % of them. This study provides a foundational framework for future investigations into the functional roles of turkey CD14⁺Mф in semen.