Novel assay for the detection of CRP protein in rabbit leukocytes using flow cytometry

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 our 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<0.01) increase in the proportion of CRP+ leukocytes (up to 10%) and in their subsets (lymphocytes up to 14% and granulocytes up to 8%) were observed in samples after CFA immunization. ELISA also revealed significantly (P<0.01) higher CRP concentration in rabbit blood plasma after CFA immunization (about 3 mg/L) compared to control samples (about 1.5 mg/L) before immunization. In conclusion, the increase in level of CRP protein during the immune response in rabbits could be measured beside the ELISA also using flow cytometry via CRP binding leukocytes. This novel assay could be therefore successfully applied in further biomedical and veterinary research

Effect of Green Tea on Weight Gain and Semen Quality of Rabbit Males

The goal of the current study was to evaluate the action of the green tea plant (Camellia sinensis, L) on male rabbit reproduction and some non-reproductive indexes. Male rabbits were fed either a standard diet (control group) or a diet enriched with green tea powder (experimental groups; E): 5 g (E1) or 20 g (E2) per 100 kg of the milled complete feed mixture. Weight gain, sperm concentration, total and progressive motility, as well as haematological, and biochemical parameters and changes in testicular tissue histomorphology were evaluated. Feeding with green tea, at both tested concentrations, decreased weight gain per week and the total average weight gain compared to the control group (p &lt; 0.05). Furthermore, green tea decreased sperm concentration, motility and progressive motility in the group fed with a lower dose (5 g) of green tea powder (p &lt; 0.05), whilst a higher dose (20 g) was neutral. Some haematological and biochemical indexes, like medium-size cell count (MID), mean corpuscular haemoglobin concentration (MCHC), platelet percentage (PCT), levels of phosphorus (P) and total proteins (TP) were decreased in one or both experimental groups (p &lt; 0.05), whilst the triglyceride level (TG) was increased in the E2 group (p &lt; 0.05). The thicknesses of the testicular seminiferous tubules and epithelial layer were not affected by any concentration of green tea powder (p &gt; 0.05). These observations suggest that green tea in the diet may have an adverse effect on rabbit growth and sperm quality, but their effect may be potentially dose-dependent

Different MACS sorting strategies for the enrichment of Lin– (CD34+ CD45–) hematopoietic progenitor cells: preliminary study

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

Phenotype and ultrastructure of stem cells derived from amniotic fluid of Nitra rabbit

The isolation of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) has been already shown in human and several other species including rabbit. However, prior to the preclinical research on various animal models it is desirable to define AF-MSCs by a panel of surface protein markers. Therefore, the aim of this preliminary study was to detect the expression of several protein markers on the surface of AF-MSCs isolated from local breed of Nitra rabbit. Amniotic fluid was collected from humanely sacrificed rabbits (n = 3) and AF-MSCs were cultured to a third passage. Flow cytometry was used to detect surface protein marker expression and for viability testing. Rabbit AF-MSCs (rAF-MSCs) were also analyzed by transmission electron microscopy to define the ultrastructure. rAF-MSCs showed both sufficient viability (more than 80%) and low apoptosis rates at third passage and highly expressed CD29 (88.17 ± 7.17%) and CD44 (80.00 ± 2.28%). However, a dim expression of CD90 (17.24 ± 1.31%) and negative expression of CD73 (1.21 ± 0.56% and 4.41 ± 1.46%), CD105 (1.67 ± 0.37%) and CD166 (0.96 ± 2.26%) was observed. Additionally, ultrastructure analysis revealed eccentrically located nucleoli, an abundance of thin pseudopodia on cells’ surfaces and proved the presence of typical mesenchymal stem cell features. In conclusion, this set of data contributes to more detailed information on rAF-MSCs, which were previously proposed feasible for preclinical stem cell research and as a suitable source for the cryopreservation of animal genetic resources in gene bank

Sperm quality of Zemplin Rabbit and Liptov Bold-Spotted Rabbit breeds

Zemplin Rabbit and Liptov Bold-Spotted Rabbit are Slovak national breeds. Our aim was to characterize the sperm quality of these two breeds, as these characteristics are important for artificial insemination and cryopreservation as biodiversity conservation tools. For this evaluation, sperm samples of sexually mature Zemplin Rabbit (ZR) males (n = 6) and Liptov Bold-Spotted Rabbits (LR) (n = 4) were used. According to progressive motility (PM) data (CASA), samples were divided into two groups: A (>30% PM) and B (<30% PM). In addition to PM (ZR-A: 48.6±3.8%, ZR- B: 16±3.2%, LR-A: 35.38±2.6%, LR-B: 4±2.2%), total motility (TM) (ZR-A: 69.1±4.1%, ZR-B: 35.5±4.1%, LR-A: 59.1±3%, LR-B: 26.9±4.1%) and morphological abnormalities (ZR-A: 29.7±1.2%, ZR-B: 40±4%, LR-A: 34.3±4.3%, LR-B: 48.3±4.7%) were also assessed using the CASA system. The proportion of dead/live, apoptotic and oxidative damaged spermatozoa (Spz) was assessed by flow cytometry using fluorescent dyes: SYBR-14, Sytox Green, Yo-Pro-1 and CellROX Green, respectively. The results of flow cytometry correspond to the values of motility and morphometry. Sperm with PM less than 30% does not show proper quality values, while the sperm with PM higher than 30% is suitable for further analysis and use

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

Phenotypical Characterization and Neurogenic Differentiation of Rabbit Adipose Tissue-Derived Mesenchymal Stem Cells

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

Enrichment of Rabbit Primitive Hematopoietic Cells via MACS Depletion of CD45+ Bone Marrow Cells

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&ndash;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 &lt; 0.05) and CD45 decreased (p &lt; 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&minus; cells the MHCI+MHCII&minus;CD38+CD49f+CD90&minus;CD117&minus; phenotype was observed. In conclusion, rabbit HSC/HPCs might be isolated and enriched by the presented method. However, further optimization is still required