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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. In five bulls of the Pinzgau breed, the sperm fertilising ability was determined using the in vitro fertilisation. Bulls were divided according to the achieved blastocyst rate: bulls with good fertilising ability (NOB - 38.6%, KAZ - 28.9%, GAL - 29.3%) and bulls with low fertilising ability (LOH - 19.4%, NUS - 22.1%). In addition to the sperm motility and morphology, we monitored common physiological sperm characteristics: viability, apoptosis, acrosomal status, capacitation, mitochondrial activity and generation of reactive oxygen species using the flow cytometry procedure. Novel fertility-related biomarkers, such as ubiquitination, overexpression of MKRN1, SPTRX-3 and PAWP proteins or histone modification (H3K4me2), were also analysed by flow cytometry. From all monitored parameters, more proper characteristics of impaired in vitro fertilising ability proved to be high incidence of apoptotic markers (YO-PRO, Caspase 3/7) and higher counts of morphologically abnormal spermatozoa. Although the in vitro fertilisation (IVF) test can be an advantageous method for evaluating the sperm fertilising ability, there are still differences between the in vivo and in vitro fertilisation processes, which must be considered for example, when evaluating the sperm capacitation status.

Abstract Pou6f2 is a genetic connection between central corneal thickness (CCT) in the mouse and a risk factor for developing primary open-angle glaucoma. POU6F2 is also a risk factor for several conditions in humans, including glaucoma, myopia, and dyslexia. Recent findings demonstrate that POU6F2-positive retinal ganglion cells (RGCs) comprise a number of RGC subtypes in the mouse, some of which also co-stain for Cdh6 and Hoxd10. These POU6F2-positive RGCs appear to be novel of ON–OFF directionally selective ganglion cells (ooDSGCs) that do not co-stain with CART or SATB2 (typical ooDSGCs markers). These POU6F2-positive cells are sensitive to damage caused by elevated intraocular pressure. In the DBA/2J mouse glaucoma model, heavily-labeled POU6F2 RGCs decrease by 73% at 8 months of age compared to only 22% loss of total RGCs (labeled with RBPMS). Additionally, Pou6f2 −/− mice suffer a significant loss of acuity and spatial contrast sensitivity along with an 11.4% loss of total RGCs. In the rhesus macaque retina, POU6F2 labels the large parasol ganglion cells that form the magnocellular (M) pathway. The association of POU6F2 with the M-pathway may reveal in part its role in human glaucoma, myopia, and dyslexia.

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.

BackgroundThough intravenous (IV) ketamine and intranasal (IN) esketamine are noted to be efficacious for treatment-resistant depression (TRD), access to each of these treatments within healthcare systems is limited due to cost, availability, and/or monitoring requirements. IV ketamine has been offered at two public hospital sites in Edmonton, Canada since 2015. Since then, demand for maintenance ketamine treatments has grown. This has required creative solutions for safe, accessible, evidence-based patient care.ObjectivesAims of this paper are twofold. First, we will provide a synthesis of current knowledge with regards to the clinical use of ketamine for TRD. Consideration will be given regarding; off-label racemic ketamine uses versus FDA-approved intranasal esketamine, populations treated, inclusion/exclusion criteria, dosing, assessing clinical response, concomitant medications, and tolerability/safety. Second, this paper will describe our experience as a community case study in applying evidence-based treatment. We will describe application of the literature review to our clinical programming, and in particular focus on cost-effective maintenance treatments, long-term safety concerns, routes of ketamine administration other than via intravenous, and cautious prescribing of ketamine outside of clinically monitored settings.MethodologyWe conducted a literature review of the on the use of ketamine for TRD up to June 30, 2023. Key findings are reviewed, and we describe their application to our ketamine program.ConclusionEvidence for the use of ketamine in resistant depression has grown in recent years, with evolving data to support and direct its clinical use. There is an increasing body of evidence to guide judicious use of ketamine in various clinical circumstances, for a population of patients with a high burden of suffering and morbidity. While large-scale, randomized controlled trials, comparative studies, and longer-term treatment outcomes is lacking, this community case study illustrates that currently available evidence can be applied to real-world clinical settings with complex patients. As cost is often a significant barrier to accessing initial and/or maintenance IV or esketamine treatments, public ketamine programs may incorporate SL or IN ketamine to support a sustainable and accessible treatment model. Three of such models are described.

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.

ABSTRACT: Mesenchymal stem cells (MSCs) are multilineage cells able to differentiate into other cell types. MSCs derived from bone marrow or compact bones are the most accessible stem cells used in tissue engineering. Therefore, the aim of this study was to isolate, characterize and cryopreserve MSCs of endangered Oravka chicken breed. MSCs were obtained from compact bones of the femur and tibiotarsus. MSCs were spindle-shaped and were able to differentiate into osteo-, adipo-, and chondrocytes under the specific differentiation conditions. Furthermore, MSCs were positive for surface markers such as CD29, CD44, CD73, CD90, CD105, CD146 and negative for CD34CD45 by flow cytometry. Moreover, MSCs demonstrated high positivity of “stemness” markers aldehyde dehydrogenase, alkaline phosphatase as well as for intracellular markers vimentin, desmin, α-SMA. Subsequently, MSCs were cryopreserved using 10% dimethyl sulfoxide in liquid nitrogen. Based on the results from the viability, phenotype, and ultrastructure assessment we can concluded that the MSCs were not negatively affected by the cryopreservation. Finally, MSCs of endangered Oravka chicken breed were successfully stored in animal gene bank, thus making them a valuable genetic resource.

In the process of cryopreservation, the semen of farm animals can be threatened by many factors, such as oxidative damage, which affect the motility and plasma membrane function of the sperm. As an endogenous antioxidant in animals, glutathione (GSH) can significantly improve the quality of thawed sperm when added to the diluent of the semen. This study was aimed at examining the effect of GSH (given at 0.5 mM, 1.5 mM and 5 mM) on the motility and viability of ram sperm following storage under cooling conditions for several days. GSH reduced the negative effect of the cooling conditions on the total and progressive motility (CASA), stability of the membranes (annexin V/PI) and incidence of the apoptosis/necrosis (Yo-Pro-1/PI) in the sperm maintained at 5 °C for 24 h and 48 hours. However, this effect was neither reflected in the penetrating nor in the fertilising ability (zygotic cleavage rate) of the sperm examined in a heterologous (bovine oocytes and ram sperm) in vitro fertilisation test with GSH applied at 1.5 mM for 48 h of cooling storage. These results indicate that the addition of GSH to the sperm extender can maintain the quality of the ram sperm stored at cooling conditions for at least two days. To make a definite conclusion about the GSH effect on the sperm functionality (fertilising ability), other GSH concentrations should be tested.

PurposeThe purpose of this study was to investigate the role of Lysine specific demethylase 1 (Lsd1) in murine retinal development. LSD1 is a histone demethylase that can demethylate mono- and di-methyl groups on H3K4 and H3K9. Using Chx10-Cre and Rho-iCre75 driver lines, we generated novel transgenic mouse lines to delete Lsd1 in most retinal progenitor cells or specifically in rod photoreceptors. We hypothesize that Lsd1 deletion will cause global morphological and functional defects due to its importance in neuronal development.MethodsWe tested the retinal function of young adult mice by electroretinogram (ERG) and assessed retinal morphology by in vivo imaging by fundus photography and SD-OCT. Afterward, eyes were enucleated, fixed, and sectioned for subsequent hematoxylin and eosin (H&E) or immunofluorescence staining. Other eyes were plastic fixed and sectioned for electron microscopy.ResultsIn adult Chx10-Cre Lsd1fl/fl mice, we observed a marked reduction in a-, b-, and c-wave amplitudes in scotopic conditions compared to age-matched control mice. Photopic and flicker ERG waveforms were even more sharply reduced. Modest reductions in total retinal thickness and outer nuclear layer (ONL) thickness were observed in SD-OCT and H&E images. Lastly, electron microscopy revealed significantly shorter inner and outer segments and immunofluorescence showed modest reductions in specific cell type populations. We did not observe any obvious functional or morphological defects in the adult Rho-iCre75 Lsd1fl/fl animals.ConclusionLsd1 is necessary for neuronal development in the retina. Adult Chx10-Cre Lsd1fl/fl mice show impaired retinal function and morphology. These effects were fully manifested in young adults (P30), suggesting that Lsd1 affects early retinal development in mice.

Ketamine has gained rapid popularity as a treatment option for treatment resistant depression (TRD). Though seen only in limited contexts, ketamine is a potential drug of abuse, addiction and diversion. Clinical ketamine studies to date have not systematically evaluated factors relevant to addiction risk in patients with TRD, but in treating patients with ketamine, risks of potential harms related to addiction must be considered. As clinical access to intravenous ketamine programs is limited in much of Canada, these considerations become even more important for clinicians who elect to offer patients less supervised, non-parenteral forms of ketamine treatment. This study explores factors relevant to addiction risk in a real-world sample of 33 patients with TRD currently or previously treated with sublingual (SL) or intranasal (IN) ketamine in the community. First, patients were surveyed using a Drug Liking and Craving Questionnaire (DLCQ) to assess their level of drug liking and craving for ketamine, and to screen for symptoms of a ketamine use disorder. Second, the pharmacy records of these patients were reviewed for red flags for addiction such as dose escalation or early refills. Third, surveys were administered to the treating psychiatrists of patients who had discontinued ketamine to determine if abuse concerns contributed to reason for discontinuation. Though limited to a small sample, results indicate that ketamine is not a universally liked or craved substance in patients with TRD. Prescribers of non-parenteral ketamine should monitor patients and prescribe cautiously. Factors related to addiction (as in the DLCQ) should be explored for clinicians to consider individual risk/benefit for judicious use of ketamine in patients with TRD.

This article deals with the legal basis of due managerial care, whose nature determines the rules applied in case of a breach of due managerial care concerning damages. The article summarizes recent professional discussions on the topic and deals with three major theoretical approaches defining due managerial care either as an obligation from law, or an obligation from contract, or an obligation sui generis. The article discuses arguments for and against each approach, puts forward the author’s critics regarding the mentioned discourses, and brings the author’s own look at the problem, the key point of which is the necessary consistency of a conclusion not only for a statutory body, but also for other elected bodies of a legal person.

Summary: 3D bioprinting—a type of additive manufacturing—can create 3D tissue constructs resembling in vivo tissues. Here, we present a protocol for 3D printing neural tissues using Axolotl Biosciences’ fibrin-based bioink and the CELLINK BIO X bioprinter with a pneumatic printhead. This workflow can be applied to printing 3D tissue models using a variety of cell lines and any chemically crosslinked bioink. These 3D-printed tissue models can be used for applications such as drug screening and disease modeling in vitro.

PurposeIn vivo methods for detecting oxidative stress in the eye would improve screening and monitoring of the leading causes of blindness: diabetic retinopathy, glaucoma, and age-related macular degeneration.MethodsTo develop an in vivo biomarker for oxidative stress in the eye, we tested the efficacy of a reactive oxygen species (ROS)-activated, near-infrared hydrocyanine-800CW (H-800CW) fluorescent probe in light-induced retinal degeneration (LIRD) mouse models. After intravitreal delivery in LIRD rats, fluorescent microscopy was used to confirm that the oxidized H-800CW appeared in the same retinal layers as an established ROS marker (dichlorofluorescein).ResultsDose-response curves of increasing concentrations of intravenously injected H-800CW demonstrated linear increases in both intensity and total area of fundus hyperfluorescence in LIRD mice, as detected by scanning laser ophthalmoscopy. Fundus hyperfluorescence also correlated with the duration of light damage and functional deficits in vision after LIRD. In LIRD rats with intravitreal injections of H-800CW, fluorescent labeling was localized to photoreceptor inner segments, similar to dichlorofluorescein.ConclusionsHydrocyanine-800CW detects retinal ROS in vivo and shows potential as a novel biomarker for ROS levels in ophthalmic diseases.

Vitrification of bovine oocytes can impair subsequent embryo development mostly due to elevated oxidative stress. This study was aimed at examining whether glutathione, a known antioxidant, can improve further embryo development when added to devitrified oocytes for a short recovery period. Bovine in vitro matured oocytes were vitrified using an ultra-rapid cooling technique on electron microscopy grids. Following warming, the oocytes were incubated in the recovery medium containing glutathione (0, 1.5, or 5 mmol L−1) for 3 h (post-warm recovery). Afterwards, the oocytes were lysed for measuring the total antioxidant capacity (TAC), activity of peroxidase, catalase and glutathione reductase, and ROS formation. The impact of vitrification on mitochondrial and lysosomal activities was also examined. Since glutathione, added at 5 mmol L−1, significantly increased the TAC of warmed oocytes, in the next set of experiments this dose was applied for post–warm recovery of oocytes used for IVF. Glutathione in the recovery culture did not change the total blastocyst rate, while increased the proportion of faster developing blastocysts (Day 6–7), reduced the apoptotic cell ratio and reversed the harmful impact of vitrification on the actin cytoskeleton. These results suggest that even a short recovery culture with antioxidant(s) can improve the development of bovine devitrified oocytes.

Purpose: To visualize and analyze ex vivo flatmounted human RPE morphology from patients with age-related macular degeneration (AMD), and to compare the morphology with histologic findings. To establish whether the sub-RPE structures identified en face in RPE flatmount preparations are drusen with histopathological registration in serial sections. To detect characteristic patterns found en face in RPE with the same structures in histological cross sections from eyes from cadavers of patients with AMD. Methods: Twenty-eight postmortem eyes from 14 patients (16 eyes with AMD and 12 age-matched control eyes) were oriented and microdissected yielding a RPE-choroid preparation. The tissues were flatmounted, stained with Alexa Fluor 635 Phalloidin (AF635-phalloidin) for f-actin and propidium iodide for DNA, and imaged using confocal microscopy. Portions of tissue from macular regions were processed for electron microscopic examination. After confocal imaging, the samples were remounted for histologic processing, embedded in paraffin, and serially sectioned perpendicular to the plane of the RPE-choroid sheet. Scaled two-dimensional (2D) maps of drusen locations found with the histological cross sections were constructed and correlated with the en face confocal microscopic images. Results: Twenty-eight postmortem eyes with a mean time of death to tissue preservation of 23.7 h (range 8.0–51 h) from 14 donors (seven women and seven men) with an average age of 78 years (range 60–93 years) were evaluated. Eight donors had AMD, and six served as controls. Scattered small, hard drusen were present in the periphery of the eyes with AMD and the healthy eyes. The macular region of the eyes with AMD contained small (

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 < 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 < 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 < 0.05), whilst the triglyceride level (TG) was increased in the E2 group (p < 0.05). The thicknesses of the testicular seminiferous tubules and epithelial layer were not affected by any concentration of green tea powder (p > 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.

The aim of our research was to compare three Slovak sheep breeds in the quality parameters of cryopreserved sperm. The ejaculates of Slovak Dairy (SD), Native Wallachian (NW), and Improved Wallachian (IW) sheep rams (n = 12) were collected by electro-ejaculation. Heterospermic samples were created from suitable ejaculates, separately for each breed (at least 90% of total and 80% of progressive motility). Samples were equilibrated in a Triladyl® diluent and frozen by automated freezing. Sperm samples were subjected to the motility, morphology, (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), fertilizing capability (penetration/fertilization test (P/F) in vitro) and acrosomal status (transmission electron microscopy) assays before freezing and after thawing. It was found that there were no significant differences (p < 0.05) between the evaluated breeds in motility, viability, apoptosis, morphological properties, and fertilizing ability of cryopreserved sperm. Significant differences occurred in acrosomal status. Our results demonstrate that the use of the selected cryopreservation protocol is suitable for at least three different sheep breeds, which can greatly benefit the biodiversity protection and simplifies the creation of an animal genetic resources gene bank.

The present study investigated whether dietary turmeric (Curcuma longa L.) can improve rabbit reproduction, ovarian function, growth, or viability. Female New Zealand White rabbits were either fed a standard diet (n=15) or a diet enriched with 5 g (group E1) or 20 g (group E2) turmeric powder per 100 kg feed mixture (n=16 or 15, respectively). After 295 days, weight gain, conception and kindling rates, pup and mother viability, ovarian macro- and micro-morphometric indices, release of leptin in response to the addition LH, and the release of progesterone, testosterone and leptin by isolated ovarian fragments were analyzed. Dietary turmeric failed to affect ovarian length and weight but did increase the number of primary follicles (E2: 32.5% greater than control group), as well as the diameter of primary (E1: +19.4%, E2: +21.1%), secondary (E2: +41.4%), and tertiary (E1: +97.1%, E2: +205.1%) follicles. Turmeric also increased the number of liveborn (E1: +21.0%) and weaned (E1: +25.0%) pups and decreased the number of stillborn pups (E2: −87.5%) but did not affect weight gain, conception, or kindling rate. Furthermore, dietary turmeric decreased doe mortality during the first reproductive cycle (13.3% in control; 0% in E1; and 6.7% in E2) but not during the second cycle. In vitro, the ovaries of the turmeric-treated rabbits released more progesterone (E1: +85.7%, E2: +90.0%) and less testosterone (E2: −87.0%) and leptin (E2: −29.0%) than the ovaries of control rabbits. Moreover, LH decreased the leptin output of control rabbits but increased that of experimental rabbits. Therefore, it is likely that dietary turmeric improves pup viability and that it could promote rabbit fecundity by either (1) promoting the production of primary ovarian follicles or (2) stimulating the growth of follicles at all stages of folliculogenesis.

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.

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.

Tumor cells can evade chemotherapy by acquiring resistance to apoptosis. We investigated the molecular mechanism whereby malignant and nonmalignant mammary epithelial cells become insensitive to apoptosis. We show that regardless of growth status formation of polarized, three-dimensional structures driven by basement membrane confers protection to apoptosis in both nonmalignant and malignant mammary epithelial cells. By contrast, irrespective of their malignant status, nonpolarized structures are sensitive to induction of apoptosis. Resistance to apoptosis requires ligation of beta4 integrins, which regulates tissue polarity, hemidesmosome formation and NFkB activation. Expression of beta4 integrin that lacks the hemidesmosome targeting domain interferes with tissue polarity and NFkB activation and permits apoptosis. These results indicate that integrin-induced polarity may drive tumor cell resistance to apoptosis-inducing agents via effects on NFkB.

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.

Stromal-epithelial interactions modulate mammary epithelial cell (MEC) growth and apoptosis by influencing cell adhesion and tissue organization. Perturbations in the mammary stroma and cell adhesion characterize breast tumors and underlie the altered tissue organization, disrupted tissue homeostasis and enhanced survival phenotype of the disease. Apoptosis resistance likely arises during malignant transformation via genetic and epigenetic modification of cell adhesion pathways induced by a changing tissue microenvironment. Acquisition of adhesion-linked survival networks that enhance MEC viability in the absence of basement membrane interactions probably promote malignant transformation, and may render breast tumors sufficiently resistant to exogenous apoptotic stimuli to generate multidrug resistance.

SSEA-4 antigen can be mainly found in embryos and embryonic stem cells. However, its expression has been observed also in adult stem and progenitor cells, or even in some differentiated cells. Moreover, we found a considerable number of SSEA-4 positive (SSEA-4+) cells within the rabbit peripheral blood and bone marrow mononuclear cells (PBMCs and BMMCs) in our previous study. Since no information about such cells can be found anywhere in the literature, the aim of this study was to identify their origin. At first, phenotypic analyses of fresh rabbit PBMCs and BMMCs were performed using flow cytometry and specific antibodies against SSEA-4 and leukocyte subsets. Then, SSEA-4+ were enriched using magnetic activated cell sorting (MACS) and analyzed for their phenotype using qPCR. We found significant SSEA-4+ cell population in PBMCs (~50%) and BMMCs (~20%). All those cells co-expressed CD45 and a majority of them also expressed B-cell marker (IgM; 50% of SSEA-4+ PBMCs and 60% of SSEA-4+ BMMCs). Increased (p < 0.05) expression of SSEA-4, CD45 and B-cell markers (IgM, CD79α and MHCII) were also noticed by qPCR in SSEA-4+ cells enriched via MACS (with efficiency over 80%). Both methods did not detect significant expression of monocyte or T-cell markers. In conclusion, SSEA-4+ cells in rabbit blood and bone marrow are of hematopoietic origin and probably belong to B-lineage cells as possessing the phenotype of B lymphocytes. However, the true function of SSEA-4 antigen in these cells should be explored by further studies.

Glaucoma etiology often includes retinal ganglion cell (RGC) death associated with elevated intraocular pressure (IOP). However, even when IOP is managed well, disease can progress. It is thus important to develop therapeutic approaches that directly protect RGCs in an IOP-independent manner. Compromised nicotinamide adenine dinucleotide (NAD+) metabolism occurs in neurodegenerative diseases, including models of glaucoma. Here we report testing the protective effects of prophylactically systemically administered nicotinamide riboside (NR), a NAD+ precursor, in a mouse model of acute RGC damage (optic nerve crush (ONC)), and in a chronic model of RGC degeneration (ocular hypertension induced by intracameral injection of microbeads). For both models, treatment enhanced RGC survival, assessed by counting cells in retinal flatmounts immunostained for Brn3a+. In the ONC model, treatment preserved RGC function, as assessed by pattern electroretinogram, and suppressed retinal inflammation, as assessed by immunofluorescence staining of retinal fixed sections for glial fibrillary acidic protein (GFAP). This is the first study to demonstrate that systemic treatment with NR is protective in acute and chronic models of RGC damage. The protection is significant and, considering that NR is highly bioavailable in and well-tolerated by humans, may support the proposition of prospective human subject studies.

The most prevalent form of bioprinting—extrusion bioprinting—can generate structures from a diverse range of materials and viscosities. It can create personalized tissues that aid in drug testing and cancer research when used in combination with natural bioinks. This paper reviews natural bioinks and their properties and functions in hard and soft tissue engineering applications. It discusses agarose, alginate, cellulose, chitosan, collagen, decellularized extracellular matrix, dextran, fibrin, gelatin, gellan gum, hyaluronic acid, Matrigel, and silk. Multi-component bioinks are considered as a way to address the shortfalls of individual biomaterials. The mechanical, rheological, and cross-linking properties along with the cytocompatibility, cell viability, and printability of the bioinks are detailed as well. Future avenues for research into natural bioinks are then presented.

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.

The aim of the study was to define ultrastructural changes in the ovarian cyclic corpus luteum (CL) of dairy cows with different body condition score (BCS, a 5-point scale). The ovaries were recovered from slaughtered Holstein cows of BCS1 (emaciation; n= 4), BCS2 (tendency to emaciation; n=4), BCS3 (average body condition; n= 4), BCS4 (tendency to fattening; n=4) and BCS5 (fattening; n=4). Average ages of cows were: 6.2, 4.1, 5.7, 5.5 and 6.36 years for BCS1, BCS2, BCS3, BCS4 and BCS5, respectively. For electron microscopy, functioning CL at the mid-luteal phase were fixed in the aldehyde solution and post-fixed in 1% OsO4 solution. For lipid visualization the samples were soaked in 1% p-phenylenediamine solution in acetone. Durcupan ultrathin sections (90 nm) were contrasted with uranyl acetate and lead citrate and viewed under Jeol transmission electron microscope. Occurrence of cell organelles was determined by counting the relative volume of individual cellular components using the modified universal test network. Luteal regression of cyclic CL was characterized by atrophy of lutein cells, condensation of organelles and cytoplasmic inclusions, increase in relative volumes of vacuoles, lysosomes, peroxisomes, autophagosomes and lipids. There were significant differences (p

The aim of our study was to examine the influence of administration of N6,2’-dibutyryladenosine 3’5’-cyclic monophosphate (dbcAMP), a cAMP agonist, on ovarian folliculogenesis and atresia, as well as on reproductive efficiency in rabbits, whose ovarian cycle and ovulation was induced by gonadotropins. Ovarian cycle and ovulation of control rabbits were induced by 20 IU/kg PMSG followed by 35 IU/kg hCG administration. Experimental animals received PMSG and hCG together with dbcAMP (at 5, 25 or 50 μg/animal). After ovulation and insemination, the animals were sacrificed. Ovaries were weighted, histological sections of ovaries were prepared, and the presence of ovulated and not ovulated follicles and different stages of atresia was evaluated by light microscopy. The eggs were flushed from the oviducts after insemination and cultured up to blastocyst cell stage. Numbers of ovarian Corpora lutea, ovulated oocytes and oocyte-derived zygotes and embryos reaching hatched blastocyst stage were determined. Administration of dbcAMP (at doses 25 or 50 μg/animal, but not at 5 μg/animal) was able to increase the proportion of follicles with cystic and luteinization-related atresia. Furthermore, dbcAMP (50 μg/animal, but not lower doses) increased the ovarian mass, number of Corpora lutea, number of harvested oocytes, zygotes and embryos at blastocyst stage derived from these zygotes after culture. These data demonstrate that dbcAMP can stimulate rabbit ovarian follicle atresia, ovulation, oocyte, zygote and embryo yield and development. Furthermore, they confirm in the involvement of cyclic nucleotide-dependent intracellular mechanisms in the control of rabbit reproductive functions and potential practical usefulness of dbcAMP in improving animal reproduction and fertility.

Studies that involved testing monoclonal antibodies (mAbs) for cross-species reactivity proved to be efficient for the identification of previously unrecognized antigens in a number of different species. Twenty-six mAbs specific to different bovine CD (cluster defined) antigens (CD9, CD18, CD45R, CD41/61, CD62L, MHC class I and bovine IgG light chain molecule) were assayed for reactivity with rabbit peripheral blood leukocytes. Four of the mAbs recognizing CD9 and CD41/61 were reactive with rabbit platelets or granulocytes. These were investigated further by immunoblotting and immunohistochemical staining. The study identified CD9 and CD41/61 molecules on rabbit cells by mAbs IVA-50 and IVA-38. It showed that IVA-50 is a new valuable CD9 reagent for rabbit immunology which could be used for immunofluorescence staining or ELISA assay, immunohistological and molecular studies of rabbit CD9 antigen. IVA-38 recognizes the CD41/61 on rabbit platelets in indirect immunofluorescence and ELISA assay.

Transgenic animals play a vital role in basic research, agriculture and pharmaceutical industries. Rabbits have the advantage of other large laboratory species in that they have a short gestation period and yield large numbers of embryos. Production of transgenic rabbits has been directed towards using the rabbit as a model for large domestic animals or as a basic biological model for studying the mammalian gene regulation. In connection with their use, developmental and health disorders have also been reported in genetically modified animals. Random integration of a transgene can disrupt the function or regulation of an endogenous gene, resulting in insertion mutations or chromosomal aneuploidy. Chromosomal abnormalities affect the developmental potential of early embryos and serve as potential predictors of developmental outcome. This study was aimed at analyzing the cytogenetic profile of transgenic rabbit embryos, which is necessary for selecting optimal lines for dissemination in order to eliminate animals with chromosomal aberrations. Conventional Giemsa stained c-metaphase spreads obtained from blastomeres of intact as well as microinjected transgenic (EGFP and hFVIII) and non-transgenic embryos revealed a significantly higher (P < 0.01) rate of aneuploid cells in transgenic rabbits compared to non-transgenic animals. However, microinjection did not seem to influence the rate of aneuploidy, as the incidence of aneuploidy in non-transgenic blastomeres was significantly lower (P < 0.01) in comparison with intact ones (14.3 vs. 73.33%). The findings suggest hypodiploidy as the prominent attributor to the occurrence of aneuploidy. This is the first report of 100% chromosomal aneuploidy in the embryos of both EGFP and hFVIII transgenic rabbits.

This study was designed to compare Cortalap® extender with tris-citric acid-glucose (TCG), Lepus® and Merk III®, to assess the in vitro preservability of rabbit spermatozoa stored for 72 h at 5°C. The best extender identified in vitro was then compared with fresh semen to determine fertility and prolificacy rates in vivo. Eight semen pools were split into four subsamples and each of them was diluted to a ratio of 1:10 (volume:volume) with four different extenders (Lepus®, Cortalap®, TCG and Merk III®) and stored at 5°C for 72 h. Sperm motility, viability, sperm membrane and acrosome integrity were evaluated in both fresh and chilled semen at 4, 24, 48 and 72 h of storage. The results showed that Cortalap® was the best extender to preserve the quality of rabbit semen in vitro over 72 h in comparison with the other extenders (P® was used for an artificial insemination (AI) trial to be compared with fresh semen. Two groups of does (n=30 each) were inseminated with fresh and chilled semen. The fertility and prolificacy rates for the does inseminated with chilled semen were significantly lower compared with those inseminated with fresh semen (P® proved to be the best extender to preserve semen quality in vitro after 72 h at 5°C, the in vivo results showed that its use is not recommended in AI programmes.

Neural stem cells (NSCs) are undifferentiated cells in the central nervous system (CNS) that are capable of self-renewal and can be induced to differentiate into neurons and glia. Current sources of mammalian NSCs are confined to regions of the CNS that are critical to normal function and surgically difficult to access, which limits their therapeutic potential in human disease. We have found that the filum terminale (FT), a previously unexplored, expendable, and easily accessible tissue at the caudal end of the spinal cord, is a source of multipotent cells in postnatal rats and humans. In this study, we used a rat model to isolate and characterize the potential of these cells. Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors. These proliferating, FT-derived cells formed neurospheres that could be induced to differentiate into neural progenitor cells, neurons, astrocytes, and oligodendrocytes by exposure to serum and/or adhesive substrates. Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro. In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo.

The aim of our study is to examine the influence of administration of 3-isobutyl-l-methyl-xanthine (IBMX), inhibitor of cAMP and cGMP phosphodiesterases on ovarian functions (folliculogenesis, atresia and luteogenesis), as well as on some reproductive parameters in rabbits whose ovarian cycle and ovulation was induced by gonadotropins. Ovarian cycle and ovulation of control rabbits were induced by PMSG followed by hCG administration. Experimental animals received 20IU/kg PMSG and 35IU/kg hCG together with IBMX (at 5, 25 or 50 μg/animal). After ovulation and mating, the animals were sacrificed. Histological slides of ovaries were prepared, and the presence of follicles and different stages of luteinisation and atresia were evaluated by light microscopy. The pronuclear stage eggs were flushed out from the oviducts and cultured up to blastocyst cell stage. Numbers of ovarian Corpora lutea , ovulated oocytes and oocyte-derived embryos reaching blastocyst stage were determined. Administration of IBMX was able to increase the proportion of luteinised follicles. Furthermore, IBMX treatment promoted occurrence of atresia in the remaining follicles after the gonadotropin treatment. Finally, IBMX increased the number of Corpora lutea , number of harvested zygotes and embryos at blastocyst stage derived from these zygotes after culture. These data demonstrate that IBMX can enhance the stimulatory effect of gonadotropins on the rabbit ovarian follicle luteinisation, atresia, ovulation, zygote and embryo yield and development. Furthermore, they confirm the involvement of cyclic nucleotide-dependent intracellular mechanisms in the control of rabbit reproductive functions and potential practical usefulness of IBMX in improvement of farm animal reproduction and fertility.

This study aimed to examine the effect of dietary flavonoid isoquercitrin on ovarian granulosa cells using the immortalized human cell line HGL5. Cell viability, survival, apoptosis, release of steroid hormones 17ß-estradiol and progesterone, and human transforming growth factor-ß2 (TGF-ß2) and TGF-ß2 receptor as well as intracellular reactive oxygen species (ROS) generation were investigated after isoquercitrin treatment at the concentration range of 5-100 µg.ml-1. It did not cause any significant change (p>0.05) in cell viability as studied by AlamarBlue assay in comparison to control. No significant change was observed (p>0.05) in the proportion of live, dead and apoptotic cells as revealed by apoptotic assay using flow cytometry. Similarly, the release of 17ß-estradiol, progesterone, TGF-ß2 and its receptor were not affected significantly (p>0.05) by isoquercitrin as detected by ELISA, in comparison to control. Except for the highest concentration of 100 µg.ml-1, which led to oxidative stress, isoquercitrin exhibited antioxidative activity at lower concentration used in the study (5, 10, 25, and 50 µg.ml-1) by hampering the production of intracellular ROS, in comparison to control, as detected by chemiluminescence assay (p<0.05). Findings of the present study indicate an existence of the antioxidative pathway that involves inhibition of intracellular ROS generation by isoquercitrin in human ovarian granulosa cells. [ABSTRACT FROM AUTHOR]

For the digital 1D-image processing we go out from the so-called photometric method. For the concrete applications of the introduced method its accuracy lowers, owing to that the form of an output video signal of linear CCD sensor is not smooth and not symmetrical. In some cases the output video signal of linear CCD sensor is devalued by the certain disperse of pixel sensitivity, respectively also that a measured object and its background are not uniform. Therefore we introduce a new modification of photometric method. The obtained results show, that introduced method gives a possibility to raise the measurement accuracy of the object dimension considerably.

The conception of system for digital 1D-images processing with digital CCD camera is presented. The system is created from these three basic parts: the digital CCD camera with linear image sensor CCD L133C, 8-bit interface and a personal computer. The scanning digital CCD camera generated a video signals, which are processed in the analog signal processor. The output signal is continually converted to 8-bit data words in A/D converter. This data words maybe transfer over a bus driver to the operation memory of personal computer, by setting one of the three work regimes of digital CCD camera. Some application possibilities and basic technical parameters of this system are given.

Abstract Background Nickel (Ni) and zinc (Zn) are trace elements present at low concentrations in agroecosystems. Nickel, however, may have toxic effects on living organisms and is often considered as a contaminant. This study reports the effect of peroral administrated Ni or a combination of Ni and Zn on femoral bone structure in rabbits. Methods One month-old female rabbits were divided into three groups of five animals each. Group 1 rabbits were fed a granular feed mixture with addition of 35 g NiCl2 per 100 kg of mixture for 90 days. In group 2, animals were fed a mixture containing 35 g NiCl2 and 30 g ZnCl2 per 100 kg of mixture. Group 3 without administration of additional Ni or Zn served as control. After the 90-day experimental period, femoral length, femoral weight and histological structure of the femur were analyzed and compared. Results The results did not indicate a statistically significant difference in either femoral length or weight between the two experimental groups and the control group. Also, differences in qualitative histological characteristics of the femora among rabbits from the three groups were absent, except for a fewer number of secondary osteons found in the animals of groups 1 and 2. However, values for vascular canal parameters of primary osteons were significantly lower in group 1 than in the control one. Peroral administration of a combination of Ni and Zn (group 2) led to a significant decreased size of the secondary osteons. Conclusions The study indicates that dietary supplementation of Ni (35 g NiCl2 per 100 kg of feed mixture) and Ni-Zn combination (35 g NiCl2 and 30 g ZnCl2 per 100 kg of the mixture) affects the microstructure of compact bone tissue in young rabbits.

Abstract Background While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits. Methods Seventeen clinically healthy 2–3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70–80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed. Results Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter. Conclusion The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.