Fluorescence Spectroscopy for the Diagnosis of Endometritis in the Mare

Abstract

By exploiting the PMN property to produce high quantities of oxygen peroxide to neutralize pathogens, the oxygen peroxide content of uterine cells was measured to diagnose endometritis. After preliminary in vitro studies in which endometrial cells from slaughtered mares were mixed with leukocytes from peripheral blood, endometrial samples were collected by uterine flushing from mares before insemination. Staining endometrial cells with H2DCF-DA was combined with hydroethidine to normalize the fluorescence intensity with the cellular content of the sample. Stained cell smears were assumed as the gold standard of endometritis, and based on this assay, the samples were considered positive (C+) and negative (C−) for endometritis. The amount and the turbidity of fluid recovered by uterine flushing were significantly (p < 0.01) higher in C+ than in C−. Moreover, the oxygen peroxide content of the endometrial cells was significantly higher in the C+ than in the C− group (6.31 ± 1.92 vs. 3.12 ± 1.26, p = 0.001). Using the value of 4.4 as the cutoff level of this fluorescence cytology assay, it was found that only one C− sample exceeded the cutoff level (false positives = 7.7%) while three C+ samples showed values below the cutoff level (false negative = 11.5%).

Serological and Uterine Biomarkers for Detecting Endometritis in Mares

“…A part of it was smeared on a slide to carry out the traditional cytological examination [20]. The remaining part was incubated with fluorescent dyes for fluorescence cytological assessment, as previously reported [14] with little modifications.…”

“…Uterine flushing samples were tested by conventional bacteriological detection, as previously reported [10,14,21]. In brief, the samples were inoculated in enrichment broth (Brain-Heart infusion) and incubated at 37 • C [21].…”

“…However, cytological preparations are not always easy to read and require a high level of experience. To facilitate the application of this technique, we have recently introduced the use of the fluorescence spectrometry technique [14], based on the possibility of marking polymorphonuclear leukocytes (PMNs), i.e., the main cells of the immune system involved in the pathogenesis of endometritis, with fluorescent dyes. PBE can be treated post-insemination; however, it is difficult to monitor the outcome of the treatment, considering that sampling the uterine material on >3 days post-breeding may have repercussions for subsequent embryonic development.…”

Relationship between Oxidative Stress and Endometritis: Exploiting Knowledge Gained in Mares and Cows

“…A high correlation was found between positivity to the microbiological test, the finding of endometritis and fertility [ 100 ]. The cytological examination is based on the collection, staining and microscopic observation [ 101 , 102 ] or fluorescence spectroscopy/microscopy [ 103 ] of uterine cells to identify the presence of immune cells; these are mainly represented by PMN both in mares and cows, whereas by plasma cells in humans [ 104 ]. This would suggest that the primary immune response in cattle and mare is cell-mediated while it is humoral in humans.…”

Electrophysiology and Fluorescence Spectroscopy Approaches for Evaluating Gamete and Embryo Functionality in Animals and Humans

“…This method does not permit a discrimination between individual cells; rather, it allows an average measurement of the fluorescence intensity representative of a cell population [ 162 ]. Weaknesses of this methodology may be attributable to the difficulty of excluding possible contaminants in the sample, such as cell debris and other components providing nonspecific fluorescence, as well as aggregates and cellular matrices potentially capable of retaining the fluorochrome and, nonspecifically, magnifying the fluorescence intensity of the sample [ 9 , 163 ]. This source of errors can be partially solved by combining, in the preliminary phase, this analysis with microscopic examinations of the fluorescent target and adopting appropriate positive controls.…”