Descriptive histological analysis of the upper, lower, and third eyelids and the conjunctiva-associated lymphoid tissue in birds of prey

Abstract Backgroud In this study, we present data obtained using light microscopy for the histological analysis of the upper eyelid (palpebra dorsalis), lower eyelid (palpebra ventralis), and third eyelid (palpebra tertia) also known as the nictitating membrane. We characterized the organized conjunctiva-associated lymphoid tissue (CALT) in selected raptor species. The aim of this study is to compare the histological structures of these eyelids in owls and diurnal raptors to identify potential evolutionary links or independent adaptations to their environments. Materials and methods We examined 34 individuals from 18 species representing Accipitriformes, Falconiformes, and Strigiformes, sourced from the Wrocław Zoological Garden (Poland), private bird collections (Poland), and wild birds found dead in the natural environment (Poland). The study involved morphometric analysis of the length and thickness of the tarsal plate of the lower eyelid. Microscopic evaluation included histological staining, using Masson-Goldner trichrome, Mayer’s hematoxylin & eosin, Movat pentachrome (modified Russell-Movat), and picro-Mallory trichrome. Results The structure of the eyelids in the analyzed bird orders proved to be highly diverse in terms of the presence of common features. The third eyelid, as well as CALT, exhibited the most variations morphological structures among the analyzed species. Strigiformes emerged as the most distinctive group of raptors, characterized by the greatest differences in eyelid morphology. This group of birds is not only distinct from other raptors but also internally diverse, with many significant differences observed between individual owl species. Conclusion Despite some common features, the upper, lower, and third eyelids of raptors from the orders Accipitriformes, Falconiformes, and Strigiformes exhibit significant morphological variation. The third eyelid and conjunctiva-associated lymphoid tissue (CALT) display the most diverse structures among the analyzed species. Owls stand out as a group of raptors with the most distinct eyelid morphologies, both compared to other raptors and within their own group. The small number of birds may lead to difficulties in distinguishing individual variation from species-specific traits, as we cannot be certain whether the observed differences result from genetic or environmental factors specific to the individual birds or if they are traits typical for the species. To address this issue, further studies involving a larger number of individuals from the same species are necessary to more accurately determine whether the morphological traits described in this study are consistent within the species or if significant variation exists among individuals.