Whole mount preparation and analysis of rabbit mammary gland

The mammary gland undergoes dynamic structural and compositional changes throughout life, influenced significantly by hormonal fluctuations and environmental factors. From embryonic development through menopause, this tissue adapts to accommodate phases such as postnatal expansion, pregnancy-induced lactation, and post-weaning involution. Hormones, growth factors, cytokines, and exogenous factors regulate these innate processes, affecting mammary epithelial cell proliferation and sensitivity, particularly in terminal end buds (TEB) and lobules, which are highly susceptible to endocrine disruption. Rodent models have provided invaluable insights into mammary gland biology, yet differences exist compared to human development, prompting the exploration of alternative models like rabbits. Additionally, there is momentum to move away from the use of nonhuman primates in safety assessments, increasing the need for evaluation tools for all tissues in the rabbit model. Rabbit mammary glands exhibit similarities to humans, making them promising for studying breast biology and pathology. However, protocols for whole-mount analysis of rabbit mammary glands are lacking due to the technical challenges of working with thicker tissue than rodent mammary glands. Here, we developed a methodology modified from rodent studies for preparing and analyzing rabbit mammary gland whole mounts, which is essential for advancing research in mammary gland biology and understanding the effects of hormonal and toxicant-induced disruption of mammary gland growth and function.