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Sensitivity to freezing remains a critical issue in stallion semen cryopreservation procedures. To explore this topic in-depth, semen was collected from ten stallions, diluted with three different extenders, transported to the laboratory, and then centrifuged and frozen with four different extenders. We conducted analyses of sperm kinetics, mitochondrial membrane potential (MMP), and hydrogen peroxide content both before and after freezing. Additionally, we assessed antioxidant activity using the ABTS and FRAP methods and measured nitric oxide stable metabolites (NOx) in the blank extenders, seminal plasma, and extenders conditioned by spermatozoa before and after freezing. We found significant variability in the antioxidant activity and NOx content of the blank extenders and the seminal plasma. In the seminal plasma, ABTS-based antioxidant activity and NOx values were correlated with some sperm kinematic parameters and MMP in refrigerated semen, while no correlation was observed in frozen sperm parameters. Sperm function varied significantly between stallions but not between extenders, either before or after freezing. However, significant differences in antioxidant activities and NOx values were found among extenders conditioned following freezing. These results provide new insights into the factors contributing to the variability in individual stallions’ tolerance to sperm freezing.

Ceruloplasmin (Cp) assessment in biological samples exploits the oxidase activity of this enzyme against several substrates, such as p-phenylenediamine (p-P), o-dianisidine (o-D) and, most recently, ammonium iron(II) sulfate (AIS). Once developed in humans, these assays are often used in veterinary medicine without appropriately optimizing in the animal species of interest. In this study, two assays using AIS and o-D as substrates have been compared and validated for Cp oxidase activity assessment in horse’s plasma. The optimization of the assays was performed mainly by varying the buffer pH as well as the buffer and the substrate molar concentration. Under the best analytical conditions obtained, the horse blood serum samples were treated with sodium azide, a potent Cp inhibitor. In the o-D assay, 500 µM sodium azide treatment completely inhibits the enzymatic activity of Cp, whereas, using the AIS assay, a residual analytical signal was still present even at the highest (2000 µM) sodium azide concentration. Even though the analytical values obtained from these methods are well correlated, the enzymatic activity values significantly differ when expressed in Units L−1. A disagreement between these assays has also been detected with the Bland–Altman plot, showing a progressive discrepancy between methods with increasing analytical values.

Due to global warming, animals are experiencing heat stress (HS), affecting many organic functions and species’ survival. In this line, some characteristics of immune cells in sea urchins subjected to short-term HS were evaluated. Paracentrotus lividus adult females were randomly divided into three groups and housed in tanks at 17 °C. In two of these tanks, the temperatures were gradually increased up to 23 and 28 °C. Celomatic fluid was collected after 3 and 7 days. The coelomocytes were morphologically typed and evaluated for their mitochondrial membrane potential (MMP), lipoperoxidation extent (LPO), and hydrogen peroxide content (H2O2). Respiratory burst was induced by treatment with phorbol 12-myristate 13-acetate (PMA). HS caused a significant change in the coelomocytes’ type distribution. MMP increased in the 23 °C-group and decreased in the 28 °C-group at both 3 and 7 days. LPO only increased in the 28 °C-group at 7 days. H2O2 progressively decreased together with the temperature increase. Respiratory burst was detected in all groups, but it was higher in the 17 °C group. In conclusion, the increase in temperature above the comfort zone for this animal species affects their immune cells with possible impairment of their functions.

Serological analysis may provide relevant information on endometritis diagnostics. Therefore, mares scheduled for AI with refrigerated semen, at the time of heat signs, underwent blood and uterine fluid samplings using a swab, uterine lavage for culture analysis, and treatment with human chorionic gonadotropin to induce ovulation. After 24–28 h, the mares were inseminated and, if positive at the culture test, treated with antibiotics chosen based on the susceptibility test. Uterine cells obtained by swabs were used for cytological examination with both classical and fluorescence techniques. Blood serum and uterine fluid samples were analyzed for assessing parameters related to redox balance, inflammation, and protease regulator potential. In blood serum, total antioxidant capacity, measured as the ferric reducing ability of plasma (FRAP), was significantly lower in cytologically endometritis-positive than -negative mares. In the uterine fluid, total thiol levels (TTL), nitric oxide metabolites (NOx), protease activity and total protein content varied significantly between groups. Although the cytological examination was more capable of discriminating between endometritis-positive and -negative mares in relation to the parameters examined, no statistically significant differences emerged in terms of pregnancy rate in relation to cytological and culture diagnosis as well as in mares diagnosed as positive and negative for endometritis.

The etiopathogenesis of endometritis in mares and cows differs significantly; this could depend on a different sensitivity and reactivity of the uterus but also on endocrine and rearing factors and different stress sources. In both species, microorganisms and the immune system play a primary role in the generation of this pathology. Microbiological and cytological tests support clinical examination and significantly improve diagnostic accuracy. For both species, during the inflammation, immune cells invade the endometrium and release bioactive substances to contrast primary or secondary pathogen contamination. These molecules are traceable to cytokines, chemokines, and prostaglandins as well as reactive oxygen and nitrogen species (ROS and RNS), collectively known as RONS. The RONS-mediated oxidation causes morphological and functional alterations of macromolecules, such as proteins, lipids, and nucleic acids, with the consequent production of derivative compounds capable of playing harmful effects. These bioactive molecules and by-products, which have recently become increasingly popular as diagnostic biomarkers, enter the bloodstream, influencing the functionality of organs and tissues. This review has collected and compared information obtained in cows and mares related to the diagnostic potential of these biomarkers that are assessed by using different methods in samples from either blood plasma or uterine fluid.

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%).