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

The C-reactive protein promoter polymorphism of selected rabbits

The polymorphism of rabbit C-reactive protein promoter was investigated in two groups subjected to strict divergent selected rabbits according variation range of live-born kits at least after 3 litters. Difference in CRP plasma level between selected groups was observed on the basis of the sequence accession number NW_003159286 region: 13347320..13352360 available in GenBank, two oligonucleotide primers, rCRP_F and rCRP_R were designed and used to amplify a 501 bp-long fragment containing the CRP gene promoter, 5’UTR region and part of coding sequence was amplified by PCR and sequenced. A three single nucleotide polymorphism were detected. High resolution melting analysis was used for genotyping of P and F1 generation. The results showed that HRM curve analysis was capable of detecting variations of 3 bp in PCR products of 501 bp. Results suggests that identified SNPs of rabbit CRP gene promoter may be relevant in the divergent selection of appropriate parental genotypes

Beneficial effects of Enterococcus faecium EF9a administration in rabbit diet

[EN] Forty-eight rabbits aged five weeks (Hycole breed, both sexes) were divided into experimental (EG) and control (CG) groups, 24 animals in each, and fed a commercial diet with access to water ad libitum. Rabbits in EG had Enterococcus faecium EF9a probiotic strain added to their drinking water (1.0×109 colony forming units/mL 500 μL/d/animal) for 28 d (between 35 and 63 d). The experiment lasted for 42 d. The animals remained in good health condition throughout the experiment, and no morbidity and mortality was noted. There was a higher live weight at 63 d of age (+34 g; P&lt;0.0001), final live weight at 77 d of age (+158 g; P=0.0483), and average daily weight gain between 63 and 77 d of age in the EG group rabbits than in CG group rabbits (+8 g/d; P&lt;0.0001). No significant changes in caecal lactic acid and total volatile fatty acid concentrations, jejunal morphological parameters and phagocytic activity were noted during the treatment. The tested serum parameters were within the range of the reference values. EF9a strain sufficiently established itself in the rabbit’s gastrointestinal tract. At 63 d of age, a significant decrease in coliforms (P&lt;0.05), coagulase-positive staphylococci (P&lt;0.01), pseudomonads (P&lt;0.01) and coagulasenegative staphylococci (CoNS, P&lt;0.001) was noted in the faeces of the EG group rabbits compared to the CG rabbits. Antimicrobial effects of EF9a strain in the caecum against coliforms (P&lt;0.001), CoNS (P=0.0002) and pseudomonads (P=0.0603) and in the appendix (coliforms, P&lt;0.05) were detected.Slovak – Hungarian project APVV:SK-HU-0006-08 and the national VEGA project 2/0006/17 This work was financially supported by the bilateral Slovak – Hungarian project APVV:SK-HU-0006-08 and the national VEGA project 2/0006/17. Part of the preliminary results was presented in the Proceedings from the Conference in Kaposvár, Hungary, 30.05.2012, pp. 89-92. We are grateful to Mrs. M. Bodnárová and Mr. P. Jerga for their skilful technical assistance. We are also grateful to Dr. V. Párkányi and Dr. R. Jurčík, from the National Agricultural and Food Centre in Nitra for blood sampling and Mr. J. Pecho for slaughtering. All care and experimental procedures involving animals followed the guidelines stated in the Guide for the Care and Use of Laboratory Animals and the trials were accepted by the Ethic Commission of the Institute of Animal Physiology in Košice and by the Slovak Veterinary and Food Administration. We would like to thank to Mr. A. Billingham for English language correction.Pogány Simonová, M.; Lauková, A.; Chrastinová, Ľ.; Plachá, I.; Szabóová, R.; Kandričáková, A.; Žitňan, R.... (2020). Beneficial effects of Enterococcus faecium EF9a administration in rabbit diet. World Rabbit Science. 28(4):169-179. https://doi.org/10.4995/wrs.2020.11189OJS16917928

Effect of GnRH (Lecirelinum) on some quality parameters of rabbit ejaculate

The aim of this study was to evaluate the effect of two concentrations of GnRH in insemination doses on selected quality parameters of rabbits ejaculate in vitro. Insemination doses (ID) were diluted to a concentration of 50 x 106 spermatozoa in ID (0.5 ml). Subsequently ID was divided into 3 samples (control - C, experiment 1, experiment 2). Implementor GnRH (Lecirelinum – commercial product Supergestran, Ferring Pharmaceuticals, the Czech Republic) was added to experimental insemination dose samples at concentrations as follows: experiment 1 to 0.2 ml (5 mg) GnRH / ID and experiment 2 to 0.3 ml (7.5 mg) GnRH / ID. Experimental samples were compared with the control sample. For the assessment of spermatozoa motility the CASA (Computer-Assistend Sperm Analysis) system SpermVision (MiniTüb, Tiefenbach, FRG) with a microscope Olympus BX 51 (Olympus, Japan) was used. Monitored spermatozoa parameters were motility (%), progressive motility (%), velocity (μm/s), curvilinear velocity of motility (μm/s) and beat cross frequency. In experimental samples (experiment 1, 2) increase of the spermatozoa motility values was detected in time periods of 1 and 3 hours (1 hour – C: 47.30 ± 7.99%, experiment 1: 86.39 ± 5.60%, experiment 2: 72.48 ± 3.80%, 3 hours – C: 57.09 ± 23.36%, experiment 1: 89.42 ± 2.41%, experiment 2: 63.92 ± 12.65%) and decrease over a period of 6 hours (C: 64.65 ± 8.60%, experiment 1: 35.26 ± 5.22%, experiment 2: 50.08 ± 8.27%). Progressive spermatozoa motility within time periods of 1 and 3 hours showed a similar trend as spermatozoa motility (1 hour – C: 30.50 ± 7.35%, experiment 1: 79.18 ± 6.58%, experiment 2: 59.85 ± 6.03%; 3 hours – C: 42.06 ± 22.69%, experiment 1: 82.31 ± 3.64%, experiment 2: 44.45 ± 12.01%) and decreased over a period of 6 hours (C: 56.34 ± 8.88%, experiment 1: 23.36 ± 5.95%, experiment 2: 39.07 ± 11.17%). Spermatozoa curvilinear velocity in experiment 1 reached after 1 hour 82.26 ± 4.47 μm/s, after 3 hours 68.40 ± 3.20 μm/s, after 6 hours 58.21 ± 3.89 μm/s; in experiment 2 was after 1 hour 62.00 ± 4.33 μm/s, after 3 hours 44.37 ± 9.19 μm/s and after 6 hours 52.73 ± 9.10 μm/s, in control group after 1 hour 71.86 ± 8.19 μm/s, after 3 hours 62.35 ± 7.89 μm/s and after 6 hours 73.93 ± 8.18 μm/s. Lower concentration of the implementor (1 to 0.2 ml GnRH / ID) ​​increased level of motility, progressive motility, velocity and curvilinear velocity of motility in the time period 1 and 3 hours after GnRH implementor application compared with the control sample. In 6 hours after application only lower changes of monitored parameters has occurred. The effect of GnRH under in vivo conditions may vary significantly comparing with results obtained in vivo

The Influence of Apple, Carrot and Red Beet Pomace Content on the Properties of Pellet from Barley Straw

Influence of wastes generated during juice production: apple, carrot and red beet, added to barley straw, on density of pellet mass, pellet hardness, ash content and calorific value was assessed. Dry mass content of additives in the substrate to pellet production was: 0, 10, 20 and 30% of the mixture weight. The relative humidity of the raw material was: 17.0, 19.5 and 22%. Higher percentages of additives and higher moisture content in the raw materials increased the hardness and density of the pellet. The contents of natural polymers such as lignin, hemicellulose and cellulose were determined in primary materials used to prepare substrate and in pellet. Changes in the determination of these substances was observed as a result of the granulation process

Possible stimulatory effect of quercetin on secretion of selected pituitary hormones

Quercetin is found in various types of foods such as apples, red onions, grapes, berries, citrus fruits, cherries, broccoli, tea etc. It is characterized by antioxidative, anti-carcinogenic, bacteriostatic and anti-inflammatory effects on the animal organism. The aim of our study was to examine its effect on endocrine system of the rabbit in vivo. Twenty healthy adult female rabbits were divided into four groups (control group and three experimental groups). Various concentrations of quercetin (10, 100 and 1000 µg/kg body weight) were intramuscularly administrated to rabbits in experimental groups during 30 days. A sensitive, biochemical method, ELISA was used to determine the concentrations of selected hormones (follicle-stimulating hormone - FSH, luteinizing hormone – LH, prolactin – PRL) after 30 days of administration. Non-significant differences between groups were found after application of different quercetin concentrations. Stimulatory effect was observed on FSH secretion by higher dose of quercetin. Similarly, LH and PRL increased at concentration 100 µg/kg and 1000 µg/kg. Our results indicate the possible effect of quercetin on secretion of selected pituitary hormones

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

Blood plasma levels of anterior pituitary hormones of rabbits after apricot seed exposure in vivo

The present study describes possible changes in plasma levels of anterior pituitary hormones induced by bitter apricot (Prunus armeniaca L.) seeds in young female rabbits in vivo. Prunus armeniaca L. is an important medicinal edible plant species commonly known as “apricot”. The apricot is a member of the Rosaceae and subfamily Prunoideae. It is one of the most delicious and commercially traded fruits in the world. Apricot kernel is the inner part of the seed of the apricot fruit. The kernel is used to produce oil and other chemicals used for medicinal purposes. The seeds are potentially useful in human nutrition and for treatment several diseases especially cancer. In the present study apricot seeds were mixed with feed at different doses 0, 60, 300, 420 mg*kg-1 of body weight. ELISA was used to determine the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL). 58-days application of apricot seeds did not affect the concentration (P≥0.05) of PRL, LH in blood plasma. Significant (P≤0.01) inhibition of FSH levels induced by the seeds was found at the dose of 420 mg*kg-1 but not at 60 and 300 mg*kg-1 of body weight. These results are suggesting that the natural substances present in apricot seeds may be involved in mechanisms of ovarian folliculogenesis

Influence of apricot kernels on blood plasma levels of selected anterior pituitary hormones in male and female rabbits in vivo

Amygdalin is represented in the family Rosacea more precisely in an apricot kernels and an almonds. There are a lot of components such as trace elements, vitamins, carbohydrates, organic acids, esters, phenols, terpenoids, except cyanogenic glycoside in the seeds. It is known that bioregulators can modulate the activity of specific enzymes and hormones very exactly at low levels and in a short time. The aim of our study was examine the effects of selected doses (0, 60, 300, 420 mg/kg b.w.) of apricot kernels in feed on the plasma levels of anterior pituitary hormones in young male and female rabbits in vivo. A sensitive, biochemical method, ELISA was used to determine the hormones prolactin (PRL), luteinizing hormone (LH) and follicle stimulating hormone (FSH). 28-day application of apricot kernels did not affect the concentration of PRL, LH, FSH in blood plasma of males. No significant (P≤0.05) differences in case of PRL and LH levels in the blood plasma of females were found. On the other hand a significant (P≤0.05) inhibition of FSH release induced by kernels at the doses 300, 420 mg/kg was found. Our results indicate that apricot kernels could affect secretion of anterior pituitary hormone FSH in female rabbits

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