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Simple Summary Ram semen frequently presents with bacterial contamination, which may affect the resulting sperm vitality and fertilization ability. Since semen quality is of paramount importance for the successful artificial insemination of ewes, we focused on the description of bacterial profiles of ejaculates collected from three Slovak sheep breeds. Furthermore, we strived to unravel the effect of bacteriospermia on the immunological characteristics and oxidative profile of semen, both of which seem to play a role in bacteria-inflicted damage to male gametes. Abstract 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.

SummaryRabbits 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.

Fluorescence-activated cell sorting (FACS) of spermatozoa was mainly used for sex sorting. Recently, FACS has been used to eliminate DNA-damaged human spermatozoa using YO-PRO-1 staining. Fragmentation of sperm DNA is considered as one of the reasons of male infertility. Moreover, YO-PRO-1 can effectively mark apoptotic as well as dead spermatozoa. Till now, only two FACS instruments were used for the spermatozoa sorting. However, both of them are not more commercially available from different reasons. In this study, we used novel FACSMelody Cell Sorter for the elimination of apoptotic and dead cells from the rabbit and ram semen samples in order to improve their overall quality. Briefly, semen samples were stained using YO-PRO-1 dye (apoptotic and dead cells) and/or propidium iodide (PI; only dead cells). Three different sorting experiments were performed: E1 – YO-PRO-1 and PI stained rabbit sperm cells were sorted into the tubes containing 1 ml of PBS; E2 – PI stained rabbit sperm cells were sorted into tubes that were washed with FBS prior adding PBS; and E3 – YO-PRO-1 and PI stained ram sperm cells were sorted into tubes washed with FBS prior adding PBS. As a sheath fluid sterile PBS was used. All samples, control (before sorting), negatively and positively sorted fractions, were analysed using CASA for motility assessment. Moreover, all sorted samples were re-stained with PI for viability assessment. In conclusion, elimination of dead (PI+) sperm from rabbit samples might improve their quality, since their progressive motility increased significantly (P

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.

SummaryRam 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.

SummaryDual inhibition (2i) of Ras–MEK–ERK and GSK3β 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 TGFβ/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, TGFβ/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.

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 composition of seminal plasma of individual sires varies and so does the fertilizing ability. Micro and macro elements along with seminal enzymes, hormones, proteins, and lipids contained in seminal plasma are essential for the proper physiological function of spermatozoa. However, elevated levels against the normal physiological values, especially in the case of trace metals, result in the production of reactive oxygen species. The deficiency of antioxidants in the seminal plasma that could scavenge free radicals causes an impairment of spermatozoa quality. Ejaculates were obtained from 19 stallions. The fresh semen was analyzed to evaluate qualitative parameters of spermatozoa in terms of the motility, viability, and integrity of DNA. Separated seminal plasma underwent the assessment of the chemical and biochemical composition and RedOx markers. Based on the obtained concentrations of individual chemical elements, the correlation analysis suggested a negative impact of Cu in seminal plasma on the SOD, GPx, and LPO. Contrary, positive correlation was detected between FRAP and motility features. While Cu negatively correlated with sperm motion parameters, the adverse effect on viability was suggested for Cd. Our data suggest that seminal plasma has a potential due to its availability to become the potential biomarker of the reproductive health of farm animals.

Since semen quality is important for the successful artificial insemination (AI) or cryopreservation, we focused on the evaluation of Slovak national rabbit breeds, New Zealand rabbit and Liptov Bold-Spotted rabbit semen as a possible source of gene reserve. Besides standard sperm traits such as motility, viability, acrosome integrity, and oxidative damage (ROS), bacterial profiles were also assessed in semen samples of New Zealand rabbit (control; n=2; N1 and N2) and Liptov Bold-Spotted rabbit (experimental; n=2; L2 and L4). After initial analysis, samples were divided into good (>70%) and poor (

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.

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,...

C-reactive protein (CRP) is an acute phase serum protein secreted by liver hepatocytes. Besides its presence in the serum, CRP was also found on the surface of human leukocytes. However, the binding ability of CRP to rabbit leukocytes has never been previously investigated. The objective of this study was to optimize the detection of rabbit CRP binding leukocytes in order to observe the acute phase of immune response by flow cytometry after Complete Freund´s Adjuvant (CFA) administration. Blood samples were analysed using ELISA assay and flow cytometry immediately before and 2 days after the CFA administration. Significant (P, C-reaktívny proteín (CRP) je sérový proteín akútnej fázy vylučovaný pečeňovými hepatocytmi. Popri jeho výskyte v sére bolo zistené, že CRP sa nachádza aj na povrchu ľudských leukocytov. Avšak, schopnosť CRP viazať sa na králičie leukocyty, nebola doteraz preskúmaná. Cieľom našej štúdie bolo optimalizovať detekciu králičích leukocytov viažucich CRP pomocou prietokovej cytometrie za účelom sledovania akútnej fázy imunitnej odpovede vyvolanej aplikáciou Kompletného Freundovho adjuvansu (CFA). Vzorky krvi boli analyzované pomocou ELISA metódy a prietokovou cytometriou ihneď pred a 2 dni po aplikácii CFA. Vo vzorkách po CFA imunizácii bol zaznamenaný signifikantný (P

Sperm cryopreservation is an important tool for storage of genetic material of animal species. Therefore, the aim of this study was to assess the association between extender and cryoprotective agents (CPAs) used in the term of sperm quality parameters. In our study, the comparison of two extenders (Kobidil+ (K+) and saline (S)) and their combinations with CPAs such as dimethyl sulfoxide (DMSO) or glycerol (GL) on the sperm motility and viability was analyzed. For detailed assessment of association between extenders with CPAs and sperm quality parameters of frozen/thawed semen, multivariate analysis was used. The obtained data showed that the both extenders with specific CPAs significantly affect the motility as well as the viability parameters. GL/K+ (p-value=0.0168) had effect on the progressive motility, whereas GL/S (p-value=0.0174) influenced the total motility and velocity curved line parameters. Moreover, DMSO/K+ (p-value=0.0346) had higher effect on the proportion of dead and apoptotic sperm compared to DMSO/S (p-value=0.036), which had higher impact on the proportion of acrosome damaged sperm. Future studies might benefit from these findings to establish the associations of these subpopulations with other rooster sperm quality parameters and thus improve the sperm quality. Association between the values can help us to better understand the effect of extenders and CPAs on the semen quality parameters.

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.

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 (&gt, 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.

Hematopoietic stem and progenitor cells (HSC/HPCs) of human or few animal species have been studied for over 30 years. However, there is no information about rabbit HSC/HPCs, although they might be a valuable animal model for studying human hematopoietic disorders or could serve as genetic resource for the preservation of animal biodiversity. CD34 marker is commonly used to isolate HSC/HPCs. Due to unavailability of specific anti-rabbit CD34 antibodies, a novel strategy for the isolation and enrichment of rabbit HSC/HPCs was used in this study. Briefly, rabbit bone marrow mononuclear cells (BMMCs) were sorted immunomagnetically in order to remove all mature (CD45+) cells. The cells were depleted with overall purity about 60–70% and then cultured in a special medium designed for the expansion of CD34+ cells. Quantitative Polymerase Chain Reaction (qPCR) analysis confirmed the enrichment of primitive hematopoietic cells, as the expression of CD34 and CD49f increased (p < 0.05) and CD45 decreased (p < 0.001) at the end of culture in comparison to fresh BMMCs. However, cell culture still exhibited the presence of CD45+ cells, as identified by flow cytometry. After gating on CD45− cells the MHCI+MHCII−CD38+CD49f+CD90−CD117− phenotype was observed. In conclusion, rabbit HSC/HPCs might be isolated and enriched by the presented method. However, further optimization is still required.

Dual inhibition (2i) of Ras-MEK-ERK and GSK3β 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 TGFβ/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, TGFβ/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.

Magnetic-activated cell sorting (MACS) has become a standard method for the isolation of hematopoietic stem/progenitor cells (HSC/HPC) in human or mouse model using CD34 antibodies. However, at the present there is no useable CD34 antibody that could be successfully used for the selection of rabbit HSC/HPC. Therefore, the aim of this preliminary study was to remove all mature cells (CD45+) from the heterogeneous mixture of rabbit peripheral blood and bone marrow mononuclear cells (PBMCs and BMMCs) in order to enrich these cell populations for the CD34+ cells. Briefly, cells were incubated with a CD45 antibody and proper magnetic microbeads. Three different MACS sorting strategies were used in the experiment that differed mainly in the sample loading rate and the number of used magnetic columns. Control (unsorted) and sorted cells were assessed for the sorting efficiency (% of double positive cells for CD45 and Labelling Check Reagent – LCR) by flow cytometry and for the relative expression of CD34 antigen by qPCR. According to flow cytometry, Depl025 mode showed the best sorting efficiency in terms of the lowest percentages of CD45+LCR+ cells for rabbit PBMCs as well as BMMCs. qPCR analysis confirmed this mode as the best in terms of the relative CD34 expression for rabbit PBMCs. However, higher relative expression of CD34 in BMMCs was obtained by other mode – Posselds. In conclusion, this study demonstrates a possible enrichment of rabbit (CD34+) HSC/HPC by the magnetic depletion of mature hematopoietic (CD45+) cells.

Aim of this study was to optimize the methodology of isolation and cultivation of rabbit adipose derived stem cells. Visceral fat and subcutaneous fat from interscapular region of the New Zealand White Line rabbit were used for experiments. Stem cells were cultured in αMEM and DMEM medium supplemented with 20% FBS and 1% antibiotics. We created 4 experimental groups based on source of fat tissue and cultivation medium: Interscapular_αMEM, Interscapular_DMEM, Visceral_αMEM, Visceral_DMEM. Using flow cytometry we analyzed viability of stem cells and intracellular and surface markers: vimentin, desmin, αSMA, CD29, CD34, CD44 and CD45. Morphology analyses revealed typical fibroblastic-like spindle shape cells in all cultured groups after few days of culture. Results of our study confirmed positive expression of CD29 and CD44 in all experimental groups thereby we confirmed that adipose derived stem cells have mesenchymal origin. Cells also showed positive expression of intracellular markers. Typical hematopoietic markers CD34 and CD45 were not expressed. Statistical analysis did not show significant differences between individual experimental groups, but based on tendency to fasten cell growth (recorded by regular observation and population doubling time analysis), we selected Interscapular_αMEM as a group for further experiments.

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581, and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34+ cells labelled by AC136 in comparison to the clone 581 and pCD34 (P < 0.01). A higher percentage of rabbit CD133+ cells were also detected by 293C3 compared to the AC133 clone (P < 0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34+ cells using magnetic-activated cell sorting (MACS). The enrichment of the rabbit CD34+ cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34+ cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34+ HSCs and new specific anti-rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1278-1289, 2018.

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-β 2, IL-8, LIF, IL-6, Osteopontin, Osteoprotegerin) and rabbit AT-MSCs (TIMP-2, TGF-β 2, LIF, Osteopontin, IL-8, IL-5, IL-3) or by HUVECs (IL-6, MIF, TGF-β 2, GCP-2, IGFBP-2, Osteoprotegerin, EGF, LIF, PDGF-BB, MCP-3, Osteopontin, Leptin, IL-5, ENA-78, TNF-β) and rabbit EPCs (TGF-β 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.

SummaryWe 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 inKobidil+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 assessedin vitroafter 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 evaluationin vivois needed in order to evaluate the possible contribution for the bank of animal genetic resources.

This study examines the impact of taurine on the viability, morphology and acrosome integrity of rabbit spermatozoa in vitro. Semen samples, obtained from four to five sexually mature and healthy New Zealand White rabbits, were pooled in heterospermic semen sample. This was divided and treated with taurine in a concentration of 0 (control), 1.5, 7, 12.5, 50 mM to a final concentration of 108 spermatozoa/ml. The samples were then incubated at 37°C for 4 hr. A combination of fluorescent probes SYBR-14/propidium iodide/PNA-Alexa Fluor 647 was used to assess spermatozoa viability and acrosome integrity on a flow cytometer. The sperm morphology was evaluated under a light microscope following fixation in 1.5% paraformaldehyde. The experiment was repeated three times. According to the obtained results, the spermatozoa neither could have benefit from immediate taurine treatment, nor had they after 4-hr incubation with respect to viability and acrosome integrity. Taurine did not initially alter the total and acrosome morphology of treated spermatozoa nor has it by 4 hr upon treatment. In conclusion, taurine may have no protective effect on the viability, morphology and acrosome integrity of short-term stored rabbit spermatozoa.

Over the years, there has been much confusion in defining molecular markers of mesenchymal stem cells (MSCs) for other than human species due to a lack of species-specific antibodies. Therefore, the aim of our study was to define rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs) and to reflect upon the current identification of AF-MSCs by providing a summary of detected surface markers in different species. The expression of rAF-MSC surface markers was analyzed at the protein and mRNA level. Flow cytometry analyses showed that rAF-MSCs were positive for CD29 and CD44, low positive for CD90, but negative for CD73, CD105, and CD166. Interestingly, RT-PCR (reverse transcription-polymerase chain reaction) exposed a discprepancy between transcribed mRNA and protein expression, as the cells expressed mRNA of all MSC markers: CD29, CD44, CD73, CD90, CD105, and CD166. Our results also confirmed the mesenchymal nature of isolated cells by morphology, ultrastructure, and intracellular marker expression profile. In addition, the expression of few pluripotency markers was also detected. We also found that passaging did not affect apoptosis and viability. Similarly, changes in karyotype were not observed during passaging. In conclusion, the provided characteristics may be used as a comprehensive set of criteria to define and characterize rAF-MSCs, required for the identification of these cells in preclinical investigations. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1601-1613, 2017.

We aimed at the evaluating the occurrence of macrophages in rabbit semen and finding possible relationship between macrophage concentration and spermatozoa quality. The concentration of macrophages in semen samples from broiler rabbit males of lines M91 and P91 (n = 30) without overt evidence of genital tract infections was determined using monocyte/macrophage lineage antigen CD14 and flow cytometry. Then the rabbits were assigned into three groups according to the macrophage concentration in semen (MΦ1 group with less than 1 × 10 6 macrophages/mL, MΦ2 group with 1.5–3.5 × 10 6 macrophages/mL and MΦ3 group with more than 8 × 10 6 macrophages/mL). Spermatozoa viability parameters such as occurrence of apoptotic (Yo-Pro-1) and dead/necrotic (propidium iodide) spermatozoa and plasma membrane integrity (PNA-Fluos) were evaluated using flow cytometry. Sperm motility parameters were determined by CASA (Computer Assisted Semen Analysis). Ultrastructural detection of macrophages was performed using transmission electron microscopy. Spermatozoa fertility potential was examined after intravaginal artificial insemination of rabbit doses. Significantly higher proportions of the apoptotic and necrotic spermatozoa and spermatozoa with lower plasma membrane integrity were revealed in the MΦ3 group compared to MΦ1 and MΦ2 groups. The percentage value of total motility and progressive movement was significantly highest in the MΦ1 group, whilst lowest in the MΦ3 group. The conception rate and the kindling rate were significantly decreased in the group with the highest macrophage concentration (MΦ3). Based on our results we can conclude that the abundance of seminal macrophages in the rabbit semen may be closely associated with poor spermatozoa quality.

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...

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.

Mitochondria are highly dynamic intracellular organelles with ultrastructural heterogeneity reflecting the behaviour and functions of the cells. The ultrastructural remodelling, performed by the counteracting active processes of mitochondrial fusion and fission, enables the organelles to respond to diverse cellular requirements and cues. It is also an important part of mechanisms underlying adaptation of mitochondria to pathophysiological conditions that challenge the cell homeostasis. However, if the stressor is constantly acting, the adaptive capacity of the cell can be exceeded and defective changes in mitochondrial morphology (indicating the insufficient functionality of mitochondria or development of mitochondrial disorders) may appear. Beside qualitative description of mitochondrial ultrastructure, stereological principles concerning the estimation of alterations in mitochondrial volume density or surface density are invaluable approaches for unbiased quantification of cells under physiological or pathophysiological conditions. In order to improve our understanding of cellular functions and dysfunctions, transmission electron microscopy (TEM) still remains a gold standard for qualitative and quantitative ultrastructural examination of mitochondria from various cell types, as well as from those experienced to different stimuli or toxicity-inducing factors. In the current study, general morphological and functional features of mitochondria, and their ultrastructural heterogeneity related to physiological and pathophysiological states of the cells are reviewed. Moreover, stereological approaches for accurate quantification of mitochondrial ultrastructure from electron micrographs taken from TEM are described in detail.

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48–72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.

Rabbit mesenchymal stem cells (rMSCs) are promising agents for the preservation of genetic biodiversity in domestic rabbit breeds. However, rMSCs must meet certain requirements to be used for cryopreservation in animal gene banks. Currently, there are numerous discrepancies in the published data regarding the rMSC phenotype, which may complicate efforts to evaluate their purity and suitability for reuse after cryopreservation in gene and tissue banks. We propose a combined approach (flow cytometry, PCR, differentiation and ultrastructure studies) for the characterization and recovery of rMSCs after cryopreservation. Flow cytometric analyses of rMSCs confirmed the expression of CD29, CD44, vimentin, desmin and α-SMA. RT-PCR revealed the expression of other markers at the mRNA level (SSEA-4, CD73, CD90, CD105, CD146 and CD166). rMSCs showed efficient multilineage differentiation into adipo-, chondro- and osteogenic lineages, SOX2 expression (pluripotency) and typical MSC morphology and ultrastructure. The confirmed rMSCs were subsequently used for cryopreservation. Efficient recovery of rMSCs after cryogenic freezing was demonstrated by high cell viability, normal ultrastructure of reseeded rMSCs, high expression of CD29 and CD44 and lineage differentiation capacity. The proposed combined approach could be used for characterization, cryopreservation and recovery of rMSCs as genetic resources for native rabbit breeds.

There is need for standardization of freezing-thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post-thaw motility and to analyse combined effect of the best permeating cryoprotectant (P-CPA) with one of four non-permeating cryoprotectants (N-CPA) on post-thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N-methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N-CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing-thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen-thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post-thaw motility. Moreover, ficoll addition to EG-based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N-CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.

Contents The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low-density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer-assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2–3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10–15% of macrophages. Males from macrophage group had lower (p

The objective of this study was to assess the effect of different diluent type and storage time on the quality of rooster insemination doses in terms of the spermatozoa motility and apoptosis (in vitro) and spermatozoa fertility potential (in vivo). The motility parameters were significantly ( P < 0.001) higher in the spermatozoa diluted in saline solution than those diluted in avian diluent at each storage time (0.5, 1 and 2 hours). The proportion of apoptotic spermatozoa was significantly ( P < 0.05) higher in the semen samples diluted in the avian diluent in comparison to the semen samples diluted in the saline solution after 1 hour of storage. The proportion of dead spermatozoa was significantly ( P < 0.05) higher in avian diluent in comparison to the semen samples diluted in the saline solution after 2 hours of storage. The fertility of spermatozoa diluted in the avian diluent significantly ( P < 0.01) decreased 1 hour after the ejaculate collection. In conclusion, saline solution maintained better quality of rooster spermatozoa in terms of their motility values and the proportion of apoptotic and dead cells ( in vitro) than the commercial avian diluent when stored at 4 °C for 2 hours. According to in vivo results, the saline solution seems to be suitable for semen dilution at low temperature, since similar fertility results were obtained over the whole storage time. Thus the commercial avian diluent could be replaced by the saline solution under certain conditions. Furthermore, the saline solution is cheaper and more accessible for practical use than the commercial avian diluent.