Factors affecting rabbit sperm cryopreservation: a mini-review.

Rabbits are an important animal species for meeting the nutritional requirements of the world's growing population due to the high conversion rate of feed. In most countries, the rabbit industry currently relies on artificial insemination with fresh or chilled and frozen-thawed spermatozoa. Various factors during the freezing process, including diluents, sperm preparation and freezing techniques, antioxidants, sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for the poor sperm quality post thaw. Despite the extensive progress reached in the field of rabbit sperm cryopreservation, new methodological approaches that could overcome problems in sperm cryopreservation are necessary. The aim of this review was to describe the factors that affect the cryopreservation of rabbit sperm.

Rabbit Endothelial Progenitor Cells Derived From Peripheral Blood and Bone Marrow: An Ultrastructural Comparative Study.

The present study was designed to compare the ultrastructure of early endothelial progenitor cells (EPCs) derived from rabbit peripheral blood (PB-EPCs) and bone marrow (BM-EPCs). After the cells had been isolated and cultivated up to passage 3, microphotographs obtained from transmission electron microscope were evaluated from qualitative and quantitative (unbiased stereological approaches) points of view. Our results revealed that both cell populations displayed almost identical ultrastructural characteristics represented by abundant cellular organelles dispersed in the cytoplasm. Moreover, the presence of very occasionally occurring mature endothelial-specific Weibel­Palade bodies (WPBs) confirmed their endothelial lineage origin. The more advanced stage of their differentiation was also demonstrated by the relatively low nucleus/cytoplasm (N/C) ratios (0.41 ± 0.19 in PB-EPCs; 0.37 ± 0.25 in BM-EPCs). Between PB-EPCs and BM-EPCs, no differences in proportions of cells occupied by nucleus (28.13 ± 8.97 versus 25.10 ± 11.48%), mitochondria (3.71 ± 1.33 versus 4.23 ± 1.00%), and lipid droplets (0.65 ± 1.01 versus 0.36 ± 0.40%), as well as in estimations of the organelles surface densities were found. The data provide the first quantitative evaluation of the organelles of interest in PB-EPCs and BM-EPCs, and they can serve as a research framework for understanding cellular function.

Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers.

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.

Ram semen quality can be assessed by flow cytometry several hours after post-fixation.

Ram spermatozoa are very sensitive to any cold shock or oxidative damage, therefore making them unsuitable for prolonged storage or distant transport to specialized laboratories for flow-cytometric analysis. The aim of this study was to stain ram semen samples with several fluorescent markers and analyse their stability during formaldehyde fixation. Briefly, freshly collected semen samples were stained for apoptosis (annexin V-FITC, YO-PRO™-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX™ Green] and cell viability (live/dead fixable viability dyes). Next, samples were fixed in buffer containing formaldehyde and then washed. Stained sample were analyzed using flow cytometer before fixation, immediately after fixation, and at 5 h and 20 h post-fixation. Fluorescent signals and the proportion of positively stained spermatozoa were compared statistically in fresh and post-fixed samples. All examined markers, except YO-PRO-1 (decreased significantly, P < 0.05), retained their fluorescence intensities after fixation. In conclusion, several tested markers were able to withstand formaldehyde fixation of ram semen samples as follows: annexin V and FLICA for apoptosis; PNA for acrosomal status; MitoTracker Red CMXRos for mitochondrial activity; and CellROX Green for oxidative status in combination with a suitable live/dead fixable viability dye. This optimized methodology could help to comprehensively analyse the quality of ram semen from local farms countrywide.

Secretome Analysis of Rabbit and Human Mesenchymal Stem and Endothelial Progenitor Cells: A Comparative Study.

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) have been studied several years for their immunomodulatory effect through the paracrine mechanism and cytokine secretion. In combination with endothelial progenitor cells (EPCs), MSCs have great therapeutical potential for the repair of endothelium and wound healing. However, little is known about the cytokine profile of rabbit AT-MSCs or even EPCs. The aim of this study was to analyze the secretomes of these rabbit stem/progenitor cells. A large-scale human cytokine array (up to 80 cytokines) was used to identify and compare cytokines secreted into conditioned media of human and rabbit AT-MSCs as well as HUVECs and rabbit EPCs. Few cytokines were highly expressed by human AT-MSCs (TIMP-2, TIMP-1), HUVECs (MCP-1, TIMP-2, GRO, Angiogenin, IL-8, TIMP-1), or by rabbit EPCs (TIMP-2). Several cytokines have moderate expression by human (MCP-1, GRO, Angiogenin, TGF-ß 2, IL-8, LIF, IL-6, Osteopontin, Osteoprotegerin) and rabbit AT-MSCs (TIMP-2, TGF-ß 2, LIF, Osteopontin, IL-8, IL-5, IL-3) or by HUVECs (IL-6, MIF, TGF-ß 2, GCP-2, IGFBP-2, Osteoprotegerin, EGF, LIF, PDGF-BB, MCP-3, Osteopontin, Leptin, IL-5, ENA-78, TNF-ß) and rabbit EPCs (TGF-ß 2, Osteopontin, GRO, LIF, IL-8, IL-5, IL-3). In conclusion, the proposed method seems to be useful for the secretome analysis of rabbit stem/progenitor cells.

Isolation, Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem Cells.

Mesenchymal stem cells (MSCs) represent an attractive source within the field of tissue engineering. However, their harvesting often requires invasive medical procedures. Urine-derived stem cells (UDSCs) display similar properties to MSCs, and their obtention and further processing is non-invasive for the donors as well as low cost. Here, we offer a comprehensive analysis of their biological properties. The goal of this study was to analyze their morphology, stemness, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm2, they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed that two different types of the cell populations have been present. The first type had a rice-grain shape and the second one was characterized by a polyhedral shape. In several cell cultures, dome-shaped cells were observed as well. All examined UDSCs expressed typical MSC-like surface markers, CD73, CD90 and CD105. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL-8, IL-6 and chemokines MCP-1 and GM-CSF. We have also successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Our findings indicate that UDSCs might have immense potential in the regeneration of the damaged tissues.

The effect of dual inhibition of Ras-MEK-ERK and GSK3ß pathways on development of in vitro cultured rabbit embryos.

Dual inhibition (2i) of Ras-MEK-ERK and GSK3ß pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFß/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFß/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

Aldehyde dehydrogenase in fresh primordial germ cells as a marker of cell 'stemness'.

SummaryChicken primordial germ cells (PGCs) are the primary pluripotent stem cell types that will differentiate towards germ cells. High aldehyde dehydrogenase (ALDH) activity is considered as a functional marker for the detection of cell 'stemness'. In our study the ALDEFLUOR™ kit was used for determination of ALDH activity in PGCs. PGCs were co-stained with diethylaminobenzaldehyde (DEAB) and ALDH and analyzed by flow cytometry. Our results showed a small cell population (8.0 ± 3.3%) upon preincubation of the cells with the specific inhibitor DEAB, however cells without inhibitor staining showed a fluorescence shift as an ALDH-positive population (70.5 ± 1.6%). These findings indicate higher expression of ALDH in PGCs and ALDH activity can therefore be used as a new functional marker for the detection of cell 'stemness' in chicken PGCs. These results may have importance for characterization of PGCs as a potential genetic resource in poultry. Further research is necessary to elucidate the role of this functional marker in these cells.

Microscopic Assessment of Dead Cell Ratio in Cryopreserved Chicken Primordial Germ Cells.

This study aimed to compare three methods of cell death assessment [trypan blue exclusion (TBE), propidium iodide viability assay (PIVA), and transmission electron microscopy] to evaluate fresh and frozen-thawed chicken primordial germ cells (PGCs). For this study, chicken PGCs were collected from ROSS 908 and Oravka breed hens, cryopreserved-thawed according to the protocol, and submitted for different cell death assessments. We observed significant differences between TBE and PIVA techniques in the detectable proportion of dead cells in fresh (14.14 ± 1.27 versus 7.16 ± 1.02%, respectively) and frozen-thawed (44.00 ± 2.11 versus 33.33 ± 1.67%, respectively) samples of the Oravka breed. Moreover, significant differences (p < 0.05) between TBE and PIVA techniques in the detectable proportion of dead cells in fresh (9.20 ± 0.60 versus 5.37 ± 0.51%) samples of ROSS 908 breed were recorded. Differences may be due to methodological, sensitivity, and toxicity features of each technique tested, where TB stains cell cytoplasm of dead cells and PI penetrates and intercalates into DNA of dead cells. Therefore, we suggest using a more precise and sensitive PIVA for viability evaluation of PGCs. Further research is needed to apply various fluorochromes for more detailed cell viability evaluation.

Low dose exposure of patulin and protective effect of epicatechin on blood cells in vitro.

In the present study, we aimed to assess antioxidant status in erythrocytes in vitro after patulin (PAT) and epicatechin exposure by measuring antioxidant enzymes (superoxide-dismutase - SOD, glutathione peroxidase - GPx and catalase - CAT) and parameters associated with oxidative stress (malondialdehyde - MDA and ROS). We also investigated the effect of PAT on viability and count of lymphocytes and lymphocyte subpopulations in rabbit blood in vitro. Whole blood of rabbits was used for analysis of antioxidant changes in rabbit erythrocytes after epicatechin and PAT treatment (separately or in combination, at concentrations of 0.2; 2; 20; 200 µg mL-1 of epicatechin and 0.5; 5; 10 µg mL-1 of PAT). Whole blood of rabbits was also used for analysis of count and viability of lymphocytes after PAT treatment at concentrations of 10; 25 and 50 µg mL-1. Results from our experiment confirmed the ability of epicatechin to protect cells against oxidative stress and lipoperoxidation. Our findings indicate that mycotoxin PAT in low concentrations did not affect the activity of antioxidant enzymes in erythrocytes of rabbits significantly. Only slight non-significant changes in lymphocytes count after treatment with low doses of PAT in rabbit blood were observed.

In vitro response of human ovarian cancer cells to dietary bioflavonoid isoquercitrin.

Isoquercitrin is a dietary bioflavonoid used as a food supplement. We studied the mechanism underlying its effect in human ovarian cancer cells using OVCAR-3 cell line. Viability, survival, apoptosis, release of human transforming growth factor-ß1 (TGF-ß1) and TGF-ß1 receptor, and intracellular reactive oxygen species (ROS) generation by OVCAR-3 cells were examined after isoquercitrin treatment at concentrations 5, 10, 25, 50, and 100 µg mL-1. AlamarBlue assay revealed that isoquercitrin did not cause any significant change (P > 0.05) in cell viability as compared to control. Apoptotic assay using flow cytometry did not find any significant change (P > 0.05) in the proportion of live, dead and apoptotic cells as compared to control. ELISA also showed that the release of human TGF-ß1 and TGF-ß1 receptor were not significantly (P > 0.05) affected by isoquercitrin as compared to control. Chemiluminescence assay demonstrated that lower concentrations (5, 10, and 25 µg mL-1) were able to exhibit beneficial effects by inhibiting the generation of intracellular ROS. In contrast, elevated concentrations of 50 and 100 µg mL-1 led to oxidative stress (P < 0.05). We concluded that the beneficial effect of isoquercitrin on ovarian cancer cells may be mediated by an antioxidative pathway that involves inhibition of intracellular ROS generation, thereby limiting oxidative stress.

In vitro effect of various cryoprotectants on the semen quality of endangered Oravka chicken.

We aimed to compare the effect of three different permeating cryoprotectants on the post-thaw spermatozoa quality. Pooled semen from Oravka cock line (n = 6) was diluted in Kobidil+ extender and frozen in cryoprotectant solutions containing 8% dimethylsulfoxide (DMSO), 8% ethylene glycol (EG) or 8% glycerol (GL) in liquid nitrogen vapours before being plunged into the liquid nitrogen. Spermatozoa motility parameters were assessed in vitro after freezing-thawing by a computer-assisted semen analysis (CASA) system and viability status was examined using fluorescent probes. The lower percentage (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing were obtained in all experimental groups (DMSO, EG, GL) compared with the control. Significant (P < 0.05) differences in total motility and progressive movement between GL and DMSO, EG groups were observed. However, the higher number (P < 0.05) of acrosome damaged spermatozoa was found in the DMSO and EG groups and no significant differences were observed in the GL group compared with the control. Differences (P < 0.05) between experimental groups and the control in the results of spermatozoa necrosis were observed. No significant differences in the percentage of apoptotic spermatozoa were found between control and experimental groups. However, significant differences (P < 0.05) in number of live and necrotic spermatozoa between GL and DMSO, EG groups were examined. The findings of the present study indicate that glycerol seems to be suitable for semen cryopreservation in the gene banks. In addition, fertility evaluation in vivo is needed in order to evaluate the possible contribution for the bank of animal genetic resources.

Reproductive performance of New Zealand White rabbits after depletion of apoptotic spermatozoa.

The objective of this study was to assess the utility of the magnetic-activated cell sorting (MACS) technique used for improving characteristics and quality of insemination doses by the elimination ofapoptotic rabbit spermatozoa from a heterospermic pool (Experiment 1) as well as from the ejaculates of individual bucks (Experiment 2). Superparamagnetic microbeads conjugated with annexin V eliminated spermatozoa with externalized phosphatidylserine via MACS. The control (untreated) and magnetically separated spermatozoa (in both E1 and E2) were used for artificial insemination of hormonally treated rabbit does. MACS separation of spermatozoa yielded two fractions: annexin V-negative (AnV) and annexin V-positive (AnV+). The CASA analysis after MACS sperm sorting revealed that the proportion of apoptotic spermatozoa in the semen of New Zealand White bucks varied from 7 to 20%. Transmission electron microscopy revealed that MACS treatment might eliminate spermatozoa with membrane damages and released acrosomal matter. However, the MACS separation (in both E1 and E2) did not affect the reproductive parameters of rabbit does.

Modulatory effect of pomegranate peel extract on key regulators of ovarian cellular processes in vitro.

In this study, response of ovarian cells (human granulosa cell line HGL5, and human adenocarcinoma cell line OVCAR-3) to short-term pomegranate peel extract (PPE) treatment (for 24 hours in cell culture) was evaluated in vitro. Quantitative and qualitative screening of polyphenols revealed punicalagins α and ß as major polyphenolic components. Total phenolic content (TPC) was 93.76 mg GAE/g d.w. with a high antioxidant activity of 95.30 mg TEAC/g d.w. In OVCAR-3, PPE treatment inhibited the metabolic activity, and increased cyclin-dependent kinase 1 (CDKN1A, p21) level at the highest dose, but not in HGL5. Flow cytometry analysis could not detect any significant difference between proportions of live, dead, and apoptotic cells in both cell lines. Reactive oxygen species (ROS) revealed an antioxidant effect on HGL5, and a prooxidant effect by stimulating ROS generation in OVCAR-3 cells at the higher doses of PPE. However, in contrast to HGL5, PPE treatment decreased release of growth factors - TGF-ß2 and EGF at the highest dose, as well as their receptors TGFBR2 and EGFR in OVCAR-3 cells. PPE also influenced steroidogenesis in granulosa cells HGL5 by stimulating 17ß-estradiol secretion at higher doses. In conclusion, the present study highlighted the bioactive compounds in pomegranate peels and the possible mechanisms of action of PPE, shedding light on its promising role in ovarian cancer (chemo)prevention and/or management.

Mesenchymal stem cells of Oravka chicken breed: promising path to biodiversity conservation.

Mesenchymal stem cells (MSCs) are multilineage cells able to differentiate into other cell types. MSCs derived from bone marrow or compact bones are the most accessible stem cells used in tissue engineering. Therefore, the aim of this study was to isolate, characterize and cryopreserve MSCs of endangered Oravka chicken breed. MSCs were obtained from compact bones of the femur and tibiotarsus. MSCs were spindle-shaped and were able to differentiate into osteo-, adipo-, and chondrocytes under the specific differentiation conditions. Furthermore, MSCs were positive for surface markers such as CD29, CD44, CD73, CD90, CD105, CD146 and negative for CD34CD45 by flow cytometry. Moreover, MSCs demonstrated high positivity of "stemness" markers aldehyde dehydrogenase, alkaline phosphatase as well as for intracellular markers vimentin, desmin, α-SMA. Subsequently, MSCs were cryopreserved using 10% dimethyl sulfoxide in liquid nitrogen. Based on the results from the viability, phenotype, and ultrastructure assessment we can concluded that the MSCs were not negatively affected by the cryopreservation. Finally, MSCs of endangered Oravka chicken breed were successfully stored in animal gene bank, thus making them a valuable genetic resource.

The Cryopreserved Sperm Traits of Various Ram Breeds: Towards Biodiversity Conservation.

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.

Elimination of apoptotic spermatozoa from rabbit insemination dose using annexin V associated with the MACS technique. A preliminary study.

The aim of this study was to verify whether the separation and elimination of the apoptotic fraction in rabbit semen using a MACS technique may improve sperm fertility potential and consequently rabbit kindling rate. Semen samples from 25 New Zealand White (NZW) rabbit males were collected using an artificial vagina and evaluated using the CASA system for concentration and motility. For artificial insemination the best 11 bucks were chosen based on motility parameters. Their ejaculates were mixed to make a heterospermic pool and routinely diluted in a commercial insemination diluent (MiniTüb, Tiefenbach, Germany) at a ratio of 1:6. Diluted heterospermic spermatozoa were filtered through a Sartorius filter to wash out seminal plasma, re-diluted in binding buffer (Annexin V Microbead Kit, Miltenyi Biotec, Germany) at a ratio of 1:3.66 and divided into two groups: an experimental group intended for MACS separation and control group without MACS separation. Then hormonally treated females of NZW rabbits were inseminated with fresh doses of filtered heterospermic semen (n = 27; 0.5 ml I.D. per female) and MACS separated semen (n=28; 0.5 ml I.D. per female). Separation and subsequent elimination of apoptotic spermatozoa (positive selection) from the insemination dose (after negative MACS selection) was verified under in vivo conditions on the basis of increased kindling rate in the experimental group in comparison with kindling rate in the control group (81.3% vs. 73.8%). In conclusion, elimination of apoptotic spermatozoa by the use of the MACS technique results in a slight improvement in kindling rate of rabbit does.

Cryopreservation of ram semen: Manual versus programmable freezing and different lengths of equilibration.

The aim of our study was to examine effects of the length of semen equilibration as well as two freezing techniques on ram sperm post-thaw quality. The ejaculates of Wallachian sheep rams (n = 12) were collected by an electro-ejaculation, equilibrated in a Triladyl® (0, 2, 4, 6, and 8 h) containing glycerol and egg yolk and frozen by programmable freezing (PF) or manual freezing (MF). After thawing, sperm samples were subjected to the motility (computer-assisted sperm analysis [CASA]), viability (SYBR-14/PI), and fertilizing ability (FA) (in vitro penetration/fertilization test on bovine oocytes) assays. It was found that the equilibration of 6 h (E-6) ensured higher post-thaw sperm motility and progressive movement compared with other lengths tested, irrespective of a freezing technique. The E-6 sperm viability did not differ between PF and MF but was lower (P < 0.05) than control. Sperm FA (E-6) was similar in PF (60.44%) and MF (62%) but slightly lower than in fresh (72.8%). Our data demonstrate that the use of MF was comparable with PF, which can be applied in the field conditions without need in a piece of cost-expensive equipment, which can greatly benefit the gene bank of animal genetic resources.

Molecular Profiling and Gene Banking of Rabbit EPCs Derived from Two Biological Sources.

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.

The Impact of Bacteriocenoses on Sperm Vitality, Immunological and Oxidative Characteristics of Ram Ejaculates: Does the Breed Play a Role?

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.