Oil red O staining is effective in detection and quantification of neutral lipid droplets in tissues such as the liver. However, converting images into testable data using ImageJ can be time-consuming and is prone to inaccuracy or bias. We describe a protocol for automated qualitative measurement of lipid droplets in the bird liver using a batch processing macro script. We explain steps for extracting tissue, cryosectioning, staining, and imaging, followed by script generation for quantification of lipid in the images.

The visual perception of birds is an incredibly exciting subject of research. Birds have significantly higher visual acuity than most other animals, which helps them stay safe in flight and detect their prey. Understanding how the eyes send information to the brain for additional processing is crucial. The brain has sections (nuclei) that accept input from the retina. The key areas where information is processed are the hyperpallium apicale (HA), hippocampus (HP), optic tectum (TeO), nucleus rotundus (RoT), and the geniculatus lateralis ventralis (Glv); among these, the RoT is one of the most investigated nuclei for vision. This study looked at how the visual centers of non-photoperiodic songbirds (Spotted Munia) adapt in different life history stages by looking at NPY expression. We immunohistochemically quantified NPY expression in four different seasons, including pre-breeding (June), breeding (September), post-breeding (December), and regressed (March) in the brain of Spotted Munia. We evaluated changes in the expression levels of the peptide throughout the year, by determining the expression at four different periods throughout the year. Peptide expression levels were projected to fluctuate within photoperiod-induced seasons. It was discovered that the parts of the brain related to vision (RoT, HA, and HP) have a higher number of immunoreactive cells during their mating season, i.e., during the summer. The appearance of NPY, a non-photic marker, in brain areas linked with light perception, was fascinating. Indirectly, NPY aids avian reproduction in a variety of ways. These findings demonstrate the importance of these nuclei in the process of reproduction, as well as the involvement of NPY in the visual brain areas of Spotted Munia.