Image pro premium

Photomicrographs of sections through the corpus callosum were taken at 5800 × magnification. Eight to twelve images per animal were quantified. To measure the myelin thickness, we used Image Pro Premium software (Media Cybernetics, Rockville MD). A binary mask was first created using the minimum variance “auto dark” algorithm to select areas of positive myelin staining. Then, using the line profile function, two horizontal lines and two vertical lines were created and applied onto the same locations of each image mask. Only axons that fell on one of the four lines were evaluated. A total of 824 ± 136 axons were counted per animal. With the edge function, the rising length and falling length were recorded separately and sequentially. Here, we created a macro to standardize and automate the measurement of each image. The thickness of a myelin sheath was then calculated as T_myelin = L_falling− L_rising. The numbers myelinated axons and demyelinated axons were counted manually. Demyelinated axons were identified as having a diameter larger than 0.3 μm with no detectable compact myelin [71 (link), 103 (link)]. Axons smaller than 0.3 μm were excluded from analysis as potentially being normal unmyelinated fibers.

The ability of neutrophils to move from the mucosal region of the endometrium through the epithelium in response to the external environment was assessed as follows:
Endometrial samples (~1 cm²), which included the surface epithelium, were harvested from three animals for each group (heifers exposed to LF semen, heifer exposed to HF, and CTRL heifers—no semen exposure) within 30 min of slaughter and fixed in 10% neutral buffered formalin for 48 h. Following processing into paraffin, tissue blocks were sectioned at 5 μm thickness using a Leica microtome and stained with hematoxylin and eosin. The sections were examined using an Olympus BX43 microscope fitted with an image analyser (Image—Pro Premium; Media Cybernetics, MD, USA). Using a 63x oil immersion objective, 20 and 40 fields of view (FOV) were selected per sample. Neutrophils within the columnar epithelium region only were quantified. The count was expressed as the average number of neutrophils per FOV per group. On average, 106 FOV were analyzed per treatment group.

Uterine biopsies from three heifers from each treatment were used for immunohistochemical localization IL-1 alpha. The formalin-fixed paraffin-embedded endometrial biopsies were sectioned at 5 μm thickness and stained using primary antibody Interleukin-1 alpha (#P420A, Thermo Fisher Scientific). Samples underwent antigen retrieval in 0.01 M citrate buffer and were blocked for endogenous peroxidases using 0.3% H₂O₂. A horseradish-peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody (Abcam, Cambridge, UK) was applied and developed with 3,3’diaminobenzidine substrate (DAB substrate, Abcam) before being mounted. Representative pictures were taken using an Olympus BX43 microscope fitted with an image analyser (Image—Pro Premium; Media Cybernetics). A negative control with no primary antibody was applied.