Cryopreservation of stallion semen induces oxidative stress that compromises sperm motility, membrane integrity, and mitochondrial function. Given the high mitochondrial reliance of equine spermatozoa and their susceptibility to oxidative damage, compartment-specific antioxidant strategies may improve post-thaw functional resilience. This study evaluated the effects of astaxanthin, a membrane-active carotenoid, and MitoTEMPO, a mitochondria-targeted superoxide scavenger, on the functional and oxidative status of frozen-thawed stallion spermatozoa. Ejaculates from nine stallions were cryopreserved in BotuCrio® extender supplemented with astaxanthin (10, 25, 50 µM) or MitoTEMPO (5, 10, 25 µM). Post-thaw quality was assessed using computer-assisted sperm analysis and multiparametric flow cytometry to evaluate motility, viability, apoptosis-like changes, acrosomal integrity, mitochondrial activity, reactive oxygen species (ROS) production, and intracellular calcium levels. Both antioxidants improved selected post-thaw functional parameters compared with controls (P < 0.05), including total and progressive motility, kinematic parameters, viability, and acrosomal integrity, and were associated with reduced intracellular and mitochondrial ROS levels. Astaxanthin was primarily associated with improved plasma membrane stability, whereas MitoTEMPO was linked to enhanced mitochondrial activity. The most consistent improvements were observed at intermediate concentrations (25 µM astaxanthin and 10 µM MitoTEMPO). These findings indicate that membrane- and mitochondria-targeted antioxidants exert complementary protective effects during stallion semen cryopreservation and may enhance post-thaw functional competence of spermatozoa. Further studies including fertility assessment are warranted to confirm their practical relevance in equine artificial insemination.

Local poultry breeds represent an essential component of agricultural biodiversity and play an important role in ensuring the resilience of livestock production systems under changing environmental conditions. The Slovak national chicken breed Oravka is a dual-purpose genotype adapted to mountain and sub-mountain production environments and classified as an endangered genetic resource. The conservation of this breed therefore requires integration of traditional breeding programmes with modern reproductive biotechnologies. The present literature review summarises the origin, breeding characteristics and adaptive potential of the Oravka breed and provides a comprehensive overview of current ex situ conservation strategies based on cryopreservation of reproductive and stem cells. Special attention is devoted to the cryopreservation of primordial germ cells, mesenchymal stem cells and rooster spermatozoa, which represent key biological material stored within national gene bank infrastructure. Differences in cryosurvival between blastodermal cells and germline stem cells are discussed together with methodological limitations of avian embryo cell preservation. The review highlights the importance of combining in situ and ex situ conservation approaches and emphasises the role of poultry gene banks as strategic tools for safeguarding genetic diversity under climate change conditions. The Oravka breed represents a valuable model for integrated conservation strategies linking biodiversity protection, reproductive biotechnology and sustainable poultry production in Europe.

PGCs offer a viable means for cryobanking both female and male avian genetic material. These early-migrating cells ultimately give rise to eggs and sperm cells, making them valuable for long-term conservation of poultry genetic resources. In this study, PGCs from the endangered Oravka chicken, a local breed native to Slovakia, were isolated, propagated in vitro, characterized, and cryopreserved. A total of 18 stable PGC lines were successfully established, comprising 12 female and 6 male lines. Characterization using immunohistochemistry, gene expression analysis, karyotyping, cell proliferation assays, TEM, and in vivo migration assays confirmed the identity and developmental competency of the in vitro cultured PGCs. A total of 108 cryopreserved samples were deposited into the gene bank. This study reports the first successful derivation, in vitro culture and cryobanking of stable PGC lines from the Oravka breed, providing a foundational resource for the conservation and future restoration of this distinctive local genetic lineage.

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.

Across one year of ageing, we characterized how semen quality deteriorates in Oravka roosters, an endangered Slovak chicken breed. Ejaculates were collected from the same mature males at approximately 62.5 weeks (Year 1) and 114.5 weeks (Year 2). We combined computer-assisted sperm analysis (CASA) of motility and kinematics with assessments of plasma membrane and acrosome integrity, phosphatidylserine externalization, membrane lipid organization, mitochondrial membrane potential, ATP metabolism, DNA integrity, oxidative status, seminal plasma biochemistry, antibacterial proteins, cytokines, immunoglobulins, Gram-negative and total aerobic bacterial loads, endotoxin and culturable seminal microbiota composition. Year 2 samples showed consistent declines in total and progressive motility, velocities and linearity, together with reduced membrane integrity and acrosome-intact fractions and higher Annexin V-, merocyanine- and propidium iodide-positive signals, indicating progressive membrane destabilization. Older ejaculates also exhibited mitochondrial depolarization, lower ATP, increased DNA fragmentation, higher reactive oxygen species production, increased lipid and protein oxidation and reduced total antioxidant capacity. Biochemically, semen from older roosters displayed lower pH and bicarbonate, increased osmolarity, sodium/potassium imbalance, and reduced calcium/magnesium, defining a less permissive extracellular milieu. Bacteriologically, Year 2 ejaculates showed higher Gram-negative and aerobic loads, elevated endotoxin and a shift from lactic acid-dominated communities to Escherichia coli- and Pseudomonas-rich profiles, together with reduced antibacterial proteins and a more pro-inflammatory cytokine pattern. Collectively, these data indicate that microbial and oxidative-inflammatory drift, membrane remodeling, mitochondrial dysfunction and biochemical imbalance jointly drive the age-linked decline in Oravka semen quality, and provide baseline evidence for selecting the age window and pre-collection management conditions for future conservation-oriented semen collection in this breed.

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.

The aim of this study was to enhance the quality of fresh ram semen by purification of spermatozoa using combination of annexin V and lectin (PNA and LCA) coated nanoparticles. Semen samples from two rams were collected regularly during the breeding season using electro-ejaculation. Samples (n=5) with poor quality (decreased sperm motility and viability) were chosen for the nanopurification procedure. Fresh (control) samples and purified (depleted from damaged spermatozoa) samples were analysed by CASA for total and progressive motility. Flow cytometry was used to check presence of apoptotic-like changes (annexin V-FITC) and integrity of acrosome (PNA-AF488 and rhodamine LCA) as well as proportion of dead cells (propidium iodide) in semen samples before and after nanopurification. Transmission electron microscopy (TEM) was performed to assess changes in sperm ultrastructure and interaction with nanoparticles. Purified ram spermatozoa exhibited increased viability (p<0.01) and both motility parameters (p<0.05) and decreased proportion of spermatozoa with damaged acrosome (p<0.05). On the other hand, no significant differences were observed for annexin V and LCA positive spermatozoa between fresh and purified samples. TEM analysis revealed occurrence of spermatozoa with swollen acrosome and damaged plasma membrane as well as abundance of nanoparticles surrounding the cells or bound to the membrane in positively stained spermatozoa. In conclusion, nanopurification process can improve the quality of ram semen, mainly in terms of sperm viability, motility and presence of damaged spermatozoa, which might result in better fertilization ability of insemination doses or even higher cryosurvival rates of stored spermatozoa.

This paper strove to characterize and compare ejaculates collected from two geese breeds over the period of two years in the context of selected traditional, biochemical, oxidative, bacteriological, and immunological properties. Slovak white and Suchovy ganders were selected for sperm quality evaluation, while bacteria were characterized using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Oxidative profile of semen was assessed using chemiluminescent and colorimetric protocols. Levels of pro-inflammatory cytokines and antibacterial proteins were evaluated with the enzyme-linked immunosorbent assay. Seminal plasma biochemistry was assessed with an automated clinical chemistry analyzer. Overall, lower semen quality was found in the Slovak white breed and during the first year of semen collection in both breeds. At the same time, the sperm motility was in a strong positive correlation with the plasma membrane integrity (P < 0.0001), whilst being negatively associated with the bacterial load (P = 0.0056), reactive oxygen species production (P < 0.0001), DNA fragmentation (P < 0.0001) and the levels of interleukin-1 (P < 0.0001) and C-reactive protein (P < 0.0001). Higher semen quality in ganders was accompanied by increased seminal plasma levels of lysozyme (P < 0.0001), cathelicidin (P < 0.0001), calcium (P < 0.01), magnesium (P < 0.01 in the case of the Suchovy breed), phosphorus (P < 0.001), total proteins (P < 0.0001 with respect to the Slovak white breed; P < 0.01 with regards to the Suchovy breed) and cholesterol (P < 0.05), and a decreased activity of aspartate transaminase (P < 0.0001). We may conclude that semen of overall better quality was obtained during the second year of semen collection as opposed to the first year in ganders of both selected goose breeds.

Commercial turkey breeding relies almost entirely on artificial insemination, yet avian sperm are unusually vulnerable to cooling and freezing injury. As a result, extender chemistry and processing steps, especially low-temperature equilibration, are pivotal for post-thaw performance. We evaluated how extender choice, paired with equilibration time, shapes the post-thaw quality of turkey semen. Ejaculates were diluted in Beltsville, Sperm Motility Medium (SMM), Botucrio, or Kobidil+, then equilibrated for 20 or 40 min before freezing; samples were cryostored for one month and assessed immediately after thawing. The outcomes included motility/kinematics, membrane integrity, mitochondrial activity and membrane potential, apoptosis/necrosis, reactive oxygen species (ROS), DNA fragmentation, and bacteriological load. Overall, 20 min equilibration improved post-thaw motility and membrane integrity, and reduced DNA fragmentation and ROS. Among extenders, Beltsville delivered the best overall sperm quality. Considering the extender × time interaction, Beltsville, Botucrio, and Kobidil+ performed best at 20 min, whereas SMM performed best at 40 min. Thus, Beltsville and SMM provide strong, time-specific options for turkey semen cryopreservation—Beltsville at 20 min and SMM at 40 min.

This study evaluated the effect of astaxanthin (AX), a potent antioxidant carotenoid, on the post-thaw quality of cryopreserved rabbit sperm. Semen samples were frozen with AX supplementation at concentrations of 0 (CONTROL), 0.5, 1, or 2 μM. Post-thaw sperm quality was assessed by computer-assisted sperm analysis (CASA) for motility, and by flow cytometry to evaluate viability, early apoptosis, mitochondrial activity, reactive oxygen species (ROS) levels, and acrosomal membrane integrity. Supplementation with 0.5 and 1 μM AX significantly improved total motility, viability, and mitochondrial activity compared to the control group (p < 0.05). These concentrations also led to significantly reduced levels of apoptotic cells and ROS. Acrosomal damage was not significantly affected by AX supplementation. These findings demonstrate that low-dose AX addition during cryopreservation attenuates oxidative and apoptotic damage in rabbit spermatozoa and enhances several key post-thaw quality parameters. Astaxanthin may thus represent a promising additive for improving cryosurvival in rabbit sperm used for assisted reproduction.

This article strove to characterize and compare biochemical, oxidative, bacteriological and immunological semen properties in Zemplin, Liptov Baldspotted and New Zealand rabbits. Besides, bacteria were characterized using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Oxidative profile of semen was assessed using chemiluminescent and colorimetric protocols. Levels of pro-inflammatory cytokines were evaluated with the enzyme-linked immunosorbent assay. Seminal plasma biochemistry was assessed with an automated clinical chemistry analyser. The lowest sperm concentration and motility were found in the Liptov Baldspotted ejaculates which also presented with significantly (p < 0.01) elevated levels of tumour necrosis factor alpha (TNF-α) and interleukin 1, free radicals (p < 0.0001) and malondialdehyde (p < 0.0001) in comparison to New Zealand rabbits. The prevailing bacterial genera in semen were Stenotrophomonas spp., Staphylococcus spp., Micrococcus spp. and Acinetobacter spp. Significantly increased levels of alanine transaminase and creatinine (Crea) were found in New Zealand rabbits in comparison to the Liptov Baldspotted breed (p < 0.001). Overall, Liptov Baldspotted rabbits produced semen of lower quality than Zemplin and New Zealand rabbits, suggesting that this breed may be more predisposed to a higher susceptibility to internal and external stresses which may interfere with male fertility.

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.

Cryopreservation reduces the antioxidant activity of spermatozoa and makes them more sensitive to damage caused by reactive oxygen species (ROS). The addition of antioxidants to the freezing medium could prevent cryo-damage by mitigating the harmful effects of ROS and, thus, protecting the spermatozoa. This study aimed to evaluate the effect of ellagic acid (EA) on the rabbit sperm traits after freezing-thawing. Semen samples collected from New Zealand White rabbit males were cryopreserved in a BotuCrio freezing medium (Nidacon, Sweden) supplemented with different concentrations of ellagic acid (EA at 0, 0.5, 1.5 and 2.5 mM) using the manual slow freezing procedure. After thawing, sperm motility parameters were evaluated by CASA. The parameters of viability (DRAQ7), apoptosis (Yo-Pro-1), acrosome integrity (peanut agglutinin; PNA), intracellular ROS (CellROX) and mitochondrial activity (MitoTracker) were evaluated by flow cytometry. EA added to the freezing medium at all concentrations led to a significant reduction (P < 0.05) in intracellular ROS in frozen-thawed sperm cells. However, this effect was not reflected in motility parameters. Semen supplemented with 1.5 mM EA also yielded a lower proportion of apoptotic cells compared to the control group. In conclusion, EA supplementation of semen extender demonstrated its antioxidative properties protecting spermatozoa against oxidative damage during cryopreservation. Nevertheless, to draw a definitive conclusion regarding the effect of EA on spermatozoa functionality, additional research is necessary.

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.

Cryopreservation of oocytes is an important tool for preserving genetic resources and for farm animals breeding. Processes taking place during vitrification affect oocytes and result in their reduced developmental capacity and lower fertilisation rates of cryopreserved oocytes. Further improvement in cryopreservation techniques is still required. Several authors already summarized the actual state and perspectives of oocyte cryopreservation as well as potential approaches to improve their development after thawing. The aim of this review is to specify factors affecting cryotolerance of mammalian oocytes, especially bovine in vitro matured oocytes, and to identify the areas, where more efforts were made to improve the success of oocyte cryopreservation. These factors include oocyte lipid content, membrane composition, mRNA protection, cytoskeleton stabilization and application of such potential stimulators of cell cryotolerance as antioxidants, growth factors or antifreeze proteins.

The objective of our research was to conduct a comparative analysis of cryopreserved sperm quality parameters of Tsigai (TS) sheep breed. Ejaculates (n=12) from TS sheep rams (n = 2) were collected using electro-ejaculation. Samples were prepared from suitable ejaculates, ensuring at least 70% progressive motility. These samples were then equilibrated in Triladyl® diluent and subjected to automated freezing. Before freezing and after thawing, the sperm samples underwent assessments including motility (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), mitochondrial activity (MitoTracker), capacitation status (FLUO4), and acrosomal status (PNA). The results revealed significant differences (P < 0.05) in total motility between fresh (91.25±0.80 %) and frozen/thawed (F/T) (41.14±12.04 %) samples, as well as progressive motility between fresh (89.83±1.85 %) and frozen/thawed (33.57±8.25 %) groups. Moreover, significant differences (P<0.05) were also observed in the proportion of apoptotic spermatozoa (2.26±0.60 %), dead (7.40±1.72 %), and mitochondrial active spermatozoa (84.52±5.60 %) in fresh samples compared to F/T samples (12.34±0.80 %, 46.59±2.82 %, 47.71±4.04 %). Our findings indicate that the selected cryopreservation protocol is relatively sufficient for the cryoconservation of sperm from Tsigai sheep breeds, since up to 50% of F/T sperm were motile and live. This has significant implications for biodiversity protection and simplifies the establishment of an animal genetic resources gene bank.

This research investigates the high prevalence of morphological defects in the sperm of a particular rabbit breed, namely the Zemplin rabbit (ZR). We tried to uncover the possible causes and consequences of the observed abnormalities. Ejaculate samples from ten adult males were divided into two qualitative groups according to the motility parameters revealed by CASA (computer-assisted sperm analysis). Group A contained samples with total motility (TM) above 50% and progressive motility (PM) above 30%. Group B contained samples with TM and PM below these values. Viability (SYBR-14/DRAQ5 (TM) and SYTOX (TM) Green/DRAQ5 (TM) staining) and apoptosis (YO-PRO TM -1/ DRAQ5 TM stainig) analyses through flow cytometry served as an additional insight into the characteristics of the qualitative groups. In both cases, we noted significant differences ( P < 0.05) between groups which corresponded to motility values. Both groups showed an increased incidence of abnormal spermatozoa, and we did not observe any significant differences between them. Simultaneously, we assessed the practical implications of these sperm defects on artificial insemination (AI), and also, we did not notice any significant differences in these parameters. The use of transmission electron microscopy enhanced our understanding of the morphological defects at the ultrastructural level. In summary, the study provides an understanding of the qualitative differences in rabbit semen, highlighting variations in quality parameters. While these differences exist, they may not necessarily translate into significant variations in AI outcomes. Our study shows higher values of cytoplasmic droplets in ZR semen, but it also shows that these conditions do not necessarily mean a fundamental obstacle to fertility in the case of AI.

Various antioxidants are tested to improve the viability and development of cryopreserved oocytes, due to their known positive health effects. The aim of this study was to find whether astaxanthin (AX), a xanthophyll carotenoid, could mitigate deteriorations that occurred during the vitrification/warming process in bovine oocytes. Astaxanthin (2.5 µM) was added to the maturation medium during the post-warm recovery period of vitrified oocytes for 3 h. Afterward, the oocytes were fertilized in vitro using frozen bull semen and presumptive zygotes were cultured in the B2 Menezo medium in a co-culture with BRL-1 cells at 38.5 °C and 5% CO2 until the blastocyst stage. AX addition significantly reduced ROS formation, lipid peroxidation, and lysosomal activity, while increasing mitochondrial activity in vitrified oocytes. Although the effect of AX on embryo development was not observed, it stimulated cell proliferation in the blastocysts derived from vitrified oocytes and improved their quality by upregulation or downregulation of some genes related to apoptosis (BCL2, CAS9), oxidative stress (GPX4, CDX2), and development (GJB5) compared to the vitrified group without AX. Therefore, the antioxidant properties of astaxanthin even during short exposure to bovine vitrified/warmed oocytes resulted in improved blastocyst quality comparable to those from fresh oocytes.