Effects of Metabolizable Protein Level on Mammary Gland Vascularity, Proliferation, and Alveoli Size during Late Gestation in Sheep

The majority of mammary gland growth, specifically alveolar proliferation takes place during late gestation in sheep. We hypothesized that inadequate metabolizable protein (MP) levels in diet would result in decreased mammary gland growth. The objective was to determine the impact of MP level on mammary gland vascularity, proliferation, and alveoli size. Multiparous Rambouillet pregnant ewes (n = 17) carrying singletons were randomly assigned at d 100 of gestation to one of the three nutritional treatments: 60% MP (MP60; n = 5), 80% MP (MP80; n = 8), or 100% MP (n = 4) of NRC requirements. Mammary gland tissues were collected on day 130 ± 1 of gestation, perfusion fixed in Carnoy’s fixative (70% ethanol, 30% glacial acetic acid, and 10% chloroform), processed, and embedded in paraffin. Mammary tissue samples were sectioned at 3 µm for immunofluorescent detection of Ki67 (a marker of cell proliferation) and CD31 (a marker of endothelial cells and thus vascularity). Cell nuclei were stained using DAPI. Percentage of proliferating alveolar cells, alveoli size (µm²), and vascularity expressed as a percentage of total tissue area occupied by blood vessels was determined by image analysis using Image-Pro Premier software (Media Cybernetics, Inc., Rockville, MD). For each image (n = 5 images/tissue section/ewe), at least 26 alveoli were analyzed. Results show that cell proliferation in alveoli was affected by MP level (P = 0.04); and was greater in MP60 than MP80 group (8.08% ± 1.63 vs. 2.46% ± 1.15), with MP100 being intermediate in (5.53% ± 1.63). There was no MP level effect on alveoli size (P = 0.24) and vascularity (P = 0.93). Perhaps consumption of a diet with inadequate MP results in altered growth rate of the mammary gland, with continued proliferation occurring during late gestation in sheep. We obtained samples at one specific time point during gestation on day 130 when alveolar size was similar in all ewes. However, we cannot exclude that the rate of alveolar proliferation across treatments was different during earlier gestation. In addition, further study is needed to determine if dietary MP levels impact milk production.