Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H₂DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

The aim of our research was to compare three Slovak sheep breeds in the quality parameters of cryopreserved sperm. The ejaculates of Slovak Dairy (SD), Native Wallachian (NW), and Improved Wallachian (IW) sheep rams (n = 12) were collected by electro-ejaculation. Heterospermic samples were created from suitable ejaculates, separately for each breed (at least 90% of total and 80% of progressive motility). Samples were equilibrated in a Triladyl^® diluent and frozen by automated freezing. Sperm samples were subjected to the motility, morphology, (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), fertilizing capability (penetration/fertilization test (P/F) in vitro) and acrosomal status (transmission electron microscopy) assays before freezing and after thawing. It was found that there were no significant differences (p < 0.05) between the evaluated breeds in motility, viability, apoptosis, morphological properties, and fertilizing ability of cryopreserved sperm. Significant differences occurred in acrosomal status. Our results demonstrate that the use of the selected cryopreservation protocol is suitable for at least three different sheep breeds, which can greatly benefit the biodiversity protection and simplifies the creation of an animal genetic resources gene bank.

Although the rabbit is a frequently used biological model, the phenotype of rabbit adipose-derived mesenchymal stem cells (rAT-MSCs) is not well characterized. One of the reasons is the absence of specific anti-rabbit antibodies. The study aimed to characterize rAT-MSCs using flow cytometry and PCR methods, especially digital droplet PCR, which confirmed the expression of selected markers at the mRNA level. A combination of these methods validated the expression of MSCs markers (CD29, CD44, CD73, CD90 and CD105). In addition, cells were also positive for CD49f, vimentin, desmin, α-SMA, ALDH and also for the pluripotent markers: NANOG, OCT4 and SOX2. Moreover, the present study proved the ability of rAT-MSCs to differentiate into a neurogenic lineage based on the confirmed expression of neuronal markers ENO2 and MAP2. Obtained results suggest that rAT-MSCs have, despite the slight differences in marker expression, the similar phenotype as human AT-MSCs and possess the neurodifferentiation ability. Accordingly, rAT-MSCs should be subjected to further studies with potential application in veterinary medicine but also, in case of their cryopreservation, as a source of genetic information of endangered species stored in the gene bank.

Bacterial contamination of semen is an often overlooked, yet important, factor contributing to decreased sperm vitality. Understanding the impact of bacterial presence on sperm structural integrity and functional activity may assist the development of effective strategies to prevent, or manage, bacteriospermia in the breeding practice. The aim of this study was to describe the bacterial profiles of ram semen (n = 35), and we also focused on the associations between bacteriospermia, sperm structure, and function, as well as oxidative and inflammatory characteristics of semen. For a better insight, the samples were divided into three groups, according to the breeds used in the study: native Wallachian (NW), improved Wallachian (IW), and Slovak dairy (SD) breeds. The results showed a significantly lower motility and membrane integrity in the NW group in comparison to the IW and SD groups, which was accompanied by a significantly higher concentration of leukocytes, increased reactive oxygen species (ROS) generation, and subsequent oxidative insults to the sperm lipids and proteins. Accordingly, the NW group presented with the highest bacterial load, in which Staphylococcus and Escherichia were the predominant representatives. The Pearson correlation analysis uncovered positive relationships amongst the bacterial load and leukocytospermia (r = 0.613), the extent of lipid peroxidation (r = 0.598), protein oxidation (r = 0.514), and DNA fragmentation (r = 0.638). Furthermore, positive correlations were found between the bacterial load and pro-inflammatory molecules, such as the C-reactive protein (r = 0.592), interleukin 1 (r = 0.709), and interleukin 6 (r = 0.474), indicating a possible involvement of the immune response in the process of bacteriospermia. Overall, our data indicate that ram semen quality may be equally affected by the bacterial load and diversity. Furthermore, we can assume that the presence of bacteria in ejaculates triggers inflammatory processes, causes ROS overproduction, and, thereby, contributes to alterations in the sperm structure, while at the same time compromising the fertilization ability of male gametes.

Cryopreservation plays a critical role in animal breeding and the conservation of endangered species, but it often compromises sperm characteristics such as morphology, motility, and viability due to oxidative stress. This study explores the antioxidative effect of Mito-Tempo (MT) and Berberine (BER) to enhance post-thaw sperm quality in rabbits. Pooled rabbit sperm samples were supplemented with different concentrations (0.0, 0.5, 5, 10, 50 µmol/L) of MT and BER. Sperm motility was evaluated using computer-assisted semen analysis, while viability, apoptosis, reactive oxygen species (ROS) levels, acrosome integrity, and mitochondrial function were assessed through flow cytometry. The results revealed that MT at 5 and 10 µmol/L and BER at 10 µmol/L significantly improved total and progressive motility, mitochondrial activity, and sperm viability compared to the control group. Furthermore, 10 µmol/L BER enhanced acrosome integrity, while both 5 µmol/L MT and 10 µmol/L BER effectively reduced ROS levels and apoptosis. This study is the first to demonstrate the protective effects of MT and BER on rabbit sperm during cryopreservation. By mitigating oxidative stress and reducing apoptosis, these antioxidants markedly improved post-thaw sperm quality, positioning MT and BER as promising agents for improving sperm cryosurvival.

Endothelial progenitor cells (EPCs) have been broadly studied for several years due to their outstanding regenerative potential. Moreover, these cells might be a valuable source of genetic information for the preservation of endangered animal species. However, a controversy regarding their characterization still exists. The aim of this study was to isolate and compare the rabbit peripheral blood- and bone marrow-derived EPCs with human umbilical vein endothelial cells (HUVECs) in terms of their phenotype and morphology that could be affected by the passage number or cryopreservation as well as to assess their possible neuro-differentiation potential. Briefly, cells were isolated and cultured under standard endothelial conditions until passage 3. The morphological changes during the culture were monitored and each passage was analyzed for the typical phenotype using flow cytometry, quantitative real–time polymerase chain reaction (qPCR) and novel digital droplet PCR (ddPCR), and compared to HUVECs. The neurogenic differentiation was induced using a commercial kit. Rabbit cells were also cryopreserved for at least 3 months and then analyzed after thawing. According to the obtained results, both rabbit EPCs exhibit a spindle-shaped morphology and high proliferation rate. The both cell lines possess same stable phenotype: CD14⁻CD29⁺CD31⁻CD34⁻CD44⁺CD45⁻CD49f⁺CD73⁺CD90⁺CD105⁺CD133⁻CD146⁻CD166⁺VE-cadherin⁺VEGFR-2⁺SSEA-4⁺MSCA-1⁻vWF⁺eNOS⁺AcLDL⁺ALDH⁺vimentin⁺desmin⁺α-SMA⁺, slightly different from HUVECs. Moreover, both induced rabbit EPCs exhibit neuron-like morphological changes and expression of neuronal markers ENO2 and MAP2. In addition, cryopreserved rabbit cells maintained high viability (>85%) and endothelial phenotype after thawing. In conclusion, our findings suggest that cells expanded from the rabbit peripheral blood and bone marrow are of the endothelial origin with a stable marker expression and interesting proliferation and differentiation capacity.