Andrej Baláži

Sperm cryopreservation is the most effective method for the long-term preservation of genetic resources. However, rabbit sperm cryopreservation remains challenging. One of the key factors influencing freezing success is the choice of semen diluent; however, no specific diluent has been established for rabbit semen. In this study, we compared the effectiveness of modified Lake’s diluent and evaluated the cryosurvival of semen from the native Hungarian breed and a native Slovak rabbit breed. Both breeds were subjected to the same static freezing procedure. The following parameters were evaluated: motility, viability, plasma membrane integrity, mitochondrial activity, acrosomal status, and intracellular reactive oxygen species. In fresh samples, viability (93.5% vs. 77.4%), total motility (84.6% vs. 67.0%), and progressive motility (76.1% vs. 47.0%) were significantly higher for the Hungarian Giant than in the Chrabrany rabbit samples. Following cryopreservation, viability (56.4% vs. 43.4%), total (39.2% vs. 20.5%) and progressive (20.6% vs. 8.5%) motility, acrosomal status (72.2% vs. 44.9%) and ROS activity (33.8% vs. 24.6%) were higher in samples from the Chrabrany breed. The results suggest that the modified Lake’s diluent may also be suitable for other rabbit breeds and highlight the importance of developing breed-specific cryopreservation protocols.

Cryopreservation of rabbit semen represents an important tool for the conservation of genetic resources; however, reduced post-thaw sperm motility may limit its practical application in artificial insemination (AI) programmes. The present stud...

The continual spermatogenesis throughout adulthood is ensured by a rare and unique cell group named spermatogonial stem cells (SSCs), which undergo self-renewal and/or differentiate into sperm cells. SSCs also become a promising genetic source for the protection of animal biodiversity. However, the isolation and culture of SSCs in vitro is still a big challenge and poorly explored in rabbits. The main objective of this study was to isolate, culture, and deeply characterize SSCs obtained from rabbit testes. Briefly, rabbit testicular tissue was mechanically and enzymatically dissociated, and obtained testicular somatic and germ cells were cultured for a short term in culture media supplemented with specific molecular factors maintaining SSC self-renewal and proliferation (GDNF, GFRα-1, FGF2, etc.). Immunofluorescent and PCR techniques were used for molecular profiling of cultured SSCs, while TEM analysis revealed their ultrastructure. After a few weeks, round and grape-like SSC colonies emerged, growing on the feeder cell layer. Rabbit SSCs showed positive staining for DBA, GFRA1, PLZF, RET, PGP9.5, DAZL, and DDX4. Increased expression of additional SSC markers was noticed using RT-qPCR and dd PCR (RET, PLZF, PGP9.5, DAZL, DDX4, CDH1, CD9, CD14, CD90, c-kit, ALDH, SSEA-4, SALL4, OCT4, and SOX2), while ultrastructure typical for primitive undifferentiated cells was observed under TEM. In conclusion, we successfully established a method for rabbit SSC isolation, culture, and phenotyping, which might facilitate their collection for further cryopreservation. However, the self-renewal, proliferative, and differentiation capacities of cultured SSCs still need to be confirmed through an in vivo SSC transplantation experiment.

This study aimed to evaluate the suitability of proAKAP4 —a structural protein associated with flagellar integrity— as a potential biomarker for predicting sperm quality after thawing, in the context of establishing a gene bank for Slovak national rabbit breeds. A total of 48 samples from 8 males of the Chrabran rabbit breed were collected and analysed. ProAKAP4 concentration was measured using ELISA (Rabbit 4MID Kit) in fresh ejaculates prior to cryopreservation. Native and post-thaw sperm quality was assessed via computer-assisted sperm analysis, focusing on total (TM) and progressive (PM) motility, as well as secondary motility parameters. To support motility-based findings, flow cytometry was additionally applied, employing DRAQ7, YO-PRO-1, Caspase 3/7, and FITC-PNA to evaluate sperm viability, apoptotic activity and acrosomal integrity. ProAKAP4 concentration in fresh semen showed a moderate negative correlation with post-thaw motility traits (r=–0.67 for % TM after freezing and thawing (F/T); r=–0.59 for % PM F/T). These findings suggest that proAKAP4 may hold potential as a predictor of sperm freezability in rabbits, but its relationship to post-thaw quality appears to differ from that observed in other species. The recorded moderate negative correlation implies a species-specific mechanism, possibly linked to differences in sperm structure or response to cryostress. Further research is needed to clarify the functional role of proAKAP4 in rabbit sperm preservation.

The aim of this study was to investigate the effect of phyto-additive mixture supplementation on semen quality and on some reproductive parameters after artificial insemination in rabbits. The trial run 120 days on 20 adult New Zealand white rabbit bucks that were allocated into two different groups, first was control (CON; n = 10) fed with commercial pelleted-feed and second was considered experimental group (EXP; n = 10) which received in feed a natural feed additive mixture (0.1% of dried Chlorella vulgaris powder and 0.1% of dried Laurus nobilis leaves powder). Consequently, the quality assessment of semen by the Computer Assisted Semen Analyzer (CASA) system, samples were instrumentally inseminated on rabbit does for two consecutive reproductive cycles, and productive and reproductive indexes were evaluated. Results demonstrate that while spermatozoa concentration and ejaculate volume did not differ significantly among experimental groups or between reproduction cycles, spermatozoa motility parameters were significantly enhanced in rabbits receiving the phyto-additive mixture, as evidenced by increased total motility (87.83% vs. 70.63%) and progressive motility (75.68% vs. 50.10%) compared with the control group (p < 0.01). No differences were observed in prolificacy traits during the first reproductive cycle, whereas in the second cycle the phyto-additive treatment increased the number of kits born alive per litter (12.29 vs. 10.19; p < 0.05) and improved kit growth performance at birth (79.17 vs. 66.75 g), at weaning (1085.28 vs. 963.15 g), and in average daily gain (28.75 vs. 25.61 g/day). The study provides evidence of alternative practises based on feeding programme to enhance reproductive traits in rabbit production. The goal is to provide farmers with examples of good farming practise (such as precision farming), focused on sustainability and efficiency, and a certain transfer of knowledge.

The rabbit is a widely used experimental model for human translational research and stem cell therapy. Many studies have focused on rabbit mesenchymal stem cells from different biological sources for their possible application in regenerative medicine. However, a minimal number of studies have been published aimed at rabbit hematopoietic stem/progenitor cells, mainly due to the lack of specific anti-rabbit CD34 antibodies. In general, CD34 antigen is commonly used to identify and isolate hematopoietic stem/progenitor cells in humans and other animal species. The aim of this study was to develop novel monoclonal antibodies highly specific to rabbit CD34 antigen. We used hybridoma technology, two synthetic peptides derived from predicted rabbit CD34 protein, and a recombinant rabbit CD34 protein as immunogens to produce monoclonal antibodies (mAbs) specific to rabbit CD34. The produced antibodies were screened for their binding activity and specificity using ELISA, flow cytometry, and Western blot analysis. Finally, four mAbs (58/47/26, 58/47/34, 182/7/80, and 575/36/8) were selected for the final purification process. The purified mAbs recognized up to 2–3% of total rabbit bone marrow cells, while about 2% of those cells exhibited CD45 expression, which are likely rabbit primitive hematopoietic stem cells and their hematopoietic progenitors, respectively. The newly generated and purified mAbs specifically recognize CD34 antigen in rabbit bone marrow or peripheral blood and can be therefore used for further immunological applications, to study rabbit hematopoiesis or to establish a new animal model for hematopoietic stem cell transplantation studies.

Cryopreservation is a widely used method for the long-term preservation of reproductive or somatic cells. It is known that this storage method may negatively affect cell viability, proliferation, differentiation, etc. However, there is a lack of information about whether cryostorage can alter the content of intracellular minerals. Therefore, we focused this study on the analysis of the mineral composition of living cells before and after long-term cold storage. Briefly, three different primary cell lines were established from rabbits as follows: endothelial progenitor cells from peripheral blood (EPCs), endothelial progenitor cells from bone marrow (BEPCs), and mesenchymal stem cells from adipose tissue (AT-MSCs), which were cultured until passage 3 prior to cryopreservation in liquid nitrogen. Samples from freshly cultured and frozen–thawed cells were mineralized and analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES) for the content of minerals (macro: Ca, Na, K, and Mg, and micro: Zn, Fe, Cu, Al, Co, Mn, Sr, and Ni). After cryopreservation, we found significantly decreased content of K in frozen–thawed EPCs (p < 0.01) and BEPCs (p < 0.0001) and Ca in AT-MSCs (p < 0.05), while Na was increased in frozen–thawed BEPCs (p < 0.05). Concentrations of Fe and Al were reduced significantly in frozen–thawed EPCs (both p < 0.0001) and AT-MSCs (p < 0.001 and p < 0.0001, respectively). On the contrary, Fe and Al were elevated in frozen–thawed BEPCs (p < 0.0001 and p < 0.01, respectively) together with Ni (p < 0.0001). In addition, decreased Zn (p < 0.05) was observed in cryopreserved AT-MSCs. In conclusion, the ICP-OES technique might be used to analyze the basic elemental composition of animal cells in fresh or frozen–thawed conditions. Nevertheless, additional studies are needed to reveal the possible impact of cryopreservation on cell fate by changing the content of intracellular minerals.

Applicability of?cryopreserved insemination doses is?dependent on?the quality of?the semen after thawing. The aim of?the study was to?check the options of?new markers for the assessment of?sperm quality to?predict their fertilisation ability. ...

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

Incorporating of agro-industrial co-products into animal nutrition could represent an opportunity to lessen the environmental impact of the food production chain. One such co-product is a hempseed cake originating from cold pressing hemp seeds to extract oil for human consumption. The aim of the present study was to evaluate the action of hempseed cake in the diet on male rabbit reproductive and some non-reproductive indexes. Male rabbits were fed either a standard diet (control group; C; n = 10) or a diet enriched with hempseed cake (experimental group E5 with 5% of a hempseed cake; n = 10, and experimental group E10 with 10% of a hempseed cake; n = 10) in 100 kg of the milled complete feed mixture. Rabbit weight gain, sperm concentration, motility, progressive motility, and sperm quality were evaluated using CASA and flow cytometry. Feeding with a hempseed cake, given at both tested concentrations, had no effect on weight gain per week and the total average weight gain compared to the control group (p > 0.05). Hempseed cake addition had no effect on sperm concentration in ejaculate, sperm motility, and progressive motility (p > 0.05). Selected haematological and biochemical indexes were examined. The E5 group showed positive tendencies in hepatic profile parameters, while in the E10 group the tendencies were opposite, though within the reference values. Based on our results, no negative effects of hempseed cake feeding on rabbit reproduction and health status were found, and we can recommend the use of hempseed cake at doses up to 10% in the nutrition and feeding of rabbits. Therefore, agro-industrial co-products can decrease the feeding cost.