Effects of group versus individual housing on ovaries in house mice (Mus musculus)

These data are from an experiment investigating how group versus individual housing influences ovaries in house mice (Mus musculus). The data were collected using methods described below. Animals and Housing Treatment Female Swiss-Webster mice (Mus musculus; Taconic Biosciences, Germantown, NY) arrived at our facility after weaning at three weeks. Upon arrival, mice were placed into a five-week housing treatment of either individual housing (n = 9) or housing in groups of three (n = 9). Mice in both treatments were kept in standard laboratory rodent cages (30 cm x 20 cm x 15 cm) in a temperature-controlled room maintained at 20 - 22 °C with 30% humidity and a 12:12 light-dark cycle. Standard laboratory rodent chow and water were provided ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Utica University (protocol #2021-001). Tissue Processing and Analysis At the conclusion of the 5-week housing treatment, mice were euthanized by CO2 inhalation and decapitated (Pack and Slepecky, 1995). The ovaries were subsequently harvested, blind to treatment group, and fixed in 2% glutaraldehyde, in 0.4 M PO4 (pH 7.2) for 12h. Ovaries were washed in 0.1 M phosphate buffer (pH 7.2) for 24 h, then dehydrated in increasing concentrations of ethanol, cleared in 100% propylene oxide, and infiltrated with Durcupan ACM. Ovaries were oriented in molds transversely to the oviduct and cured at 50°C for 48h. Width (i.e., size perpendicular to oviduct) versus length (i.e., size parallel to oviduct) proportions were measured to ensure cross-sectional area variations reflected differences in ovary size and were not due to shape differences. Using a microtome (JB-4, Sorvall), we cut 2-µm sections transverse to the oviduct for 650 µm into the ovary. The sections were then stained with methylene blue/azure II. We analyzed the tissue sections at 50-µm intervals and photographed the sections utilizing a camera (Moticam 2500) attached to an Olympus CX31 microscope. A researcher, blind to the experimental conditions, hand-counted all follicles utilizing the follicular basal lamina as the indicator of a follicle. Ovary cross-sectional area was measured using ImageJ (Schneider et al., 2012) for free-form area measurements, tracing the ovarian germinal epithelium. Pixel to micron conversion was calibrated with a stage micrometer. An additional analysis of post-primary follicle stages was conducted on sections at depths of 200, 400, and 600 µm for each ovary. For these, we classified post-primary follicle stages, utilizing visualization of the zona pellucida to characterize follicular stages (Myers et al., 2004). See the "READ ME" file for more information regarding the organization of the images in the zipfile and the tabs in the Excel spreadsheet.