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

The goal of the current study was to evaluate the action of the green tea plant (Camellia sinensis, L) on male rabbit reproduction and some non-reproductive indexes. Male rabbits were fed either a standard diet (control group) or a diet enriched with green tea powder (experimental groups; E): 5 g (E1) or 20 g (E2) per 100 kg of the milled complete feed mixture. Weight gain, sperm concentration, total and progressive motility, as well as haematological, and biochemical parameters and changes in testicular tissue histomorphology were evaluated. Feeding with green tea, at both tested concentrations, decreased weight gain per week and the total average weight gain compared to the control group (p < 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.

Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers.

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

The Cryopreserved Sperm Traits of Various Ram Breeds: Towards Biodiversity Conservation.

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.

Rabbit Endothelial Progenitor Cells Derived From Peripheral Blood and Bone Marrow: An Ultrastructural Comparative Study.

The present study was designed to compare the ultrastructure of early endothelial progenitor cells (EPCs) derived from rabbit peripheral blood (PB-EPCs) and bone marrow (BM-EPCs). After the cells had been isolated and cultivated up to passage 3, microphotographs obtained from transmission electron microscope were evaluated from qualitative and quantitative (unbiased stereological approaches) points of view. Our results revealed that both cell populations displayed almost identical ultrastructural characteristics represented by abundant cellular organelles dispersed in the cytoplasm. Moreover, the presence of very occasionally occurring mature endothelial-specific Weibel­Palade bodies (WPBs) confirmed their endothelial lineage origin. The more advanced stage of their differentiation was also demonstrated by the relatively low nucleus/cytoplasm (N/C) ratios (0.41 ± 0.19 in PB-EPCs; 0.37 ± 0.25 in BM-EPCs). Between PB-EPCs and BM-EPCs, no differences in proportions of cells occupied by nucleus (28.13 ± 8.97 versus 25.10 ± 11.48%), mitochondria (3.71 ± 1.33 versus 4.23 ± 1.00%), and lipid droplets (0.65 ± 1.01 versus 0.36 ± 0.40%), as well as in estimations of the organelles surface densities were found. The data provide the first quantitative evaluation of the organelles of interest in PB-EPCs and BM-EPCs, and they can serve as a research framework for understanding cellular function.

Factors affecting rabbit sperm cryopreservation: a mini-review.

Rabbits are an important animal species for meeting the nutritional requirements of the world's growing population due to the high conversion rate of feed. In most countries, the rabbit industry currently relies on artificial insemination with fresh or chilled and frozen-thawed spermatozoa. Various factors during the freezing process, including diluents, sperm preparation and freezing techniques, antioxidants, sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for the poor sperm quality post thaw. Despite the extensive progress reached in the field of rabbit sperm cryopreservation, new methodological approaches that could overcome problems in sperm cryopreservation are necessary. The aim of this review was to describe the factors that affect the cryopreservation of rabbit sperm.

Effect of selected natural and synthetic substances on rabbit reproduction-A mini review.

Numerous natural and synthetic substances have effects on reproduction through several mechanisms. This review aims to summarize the impact of green tea (GT), yucca schidigera (YS) extract, curcuma longa (CL), adenosine 3',5'-cyclic monophosphate (cAMP) and isobutyl-1-methyl-xanthine (IBMX) stimulators on rabbit reproduction performance. To obtain a comprehensive overview of this topic, the keywords "reproduction," "substances," "spermatogenesis," "embryogenesis,"hormonal profil", "green tea", "yucca schidigera" were searched in such databases as WOS and PubMed to obtain relevant information. Spermatozoa profile was positively effected by the GT and YS, however, cAMP inhibitors stimulated spermatozoa motility resulted in positive or negative effects depending on the doses. Similarly, embryogenesis and hormonal profile were positively influenced by the GT, YS, cAMP and IBMX in a proper administration dose. Further research is needed to improve current knowledge about these substances to identify potential effects on the other reproduction parameters. Furthermore, future studies should combine GT, YS and CL with different plant extracts to determine their effects on spermatozoa status, embryogenesis as well as hormonal profile as key outcomes. This review summarizes current knowledge about effect of natural and synthetic substances on rabbit reproduction.

Cryopreservation of ram semen: Manual versus programmable freezing and different lengths of equilibration.

The aim of our study was to examine effects of the length of semen equilibration as well as two freezing techniques on ram sperm post-thaw quality. The ejaculates of Wallachian sheep rams (n = 12) were collected by an electro-ejaculation, equilibrated in a Triladyl® (0, 2, 4, 6, and 8 h) containing glycerol and egg yolk and frozen by programmable freezing (PF) or manual freezing (MF). After thawing, sperm samples were subjected to the motility (computer-assisted sperm analysis [CASA]), viability (SYBR-14/PI), and fertilizing ability (FA) (in vitro penetration/fertilization test on bovine oocytes) assays. It was found that the equilibration of 6 h (E-6) ensured higher post-thaw sperm motility and progressive movement compared with other lengths tested, irrespective of a freezing technique. The E-6 sperm viability did not differ between PF and MF but was lower (P < 0.05) than control. Sperm FA (E-6) was similar in PF (60.44%) and MF (62%) but slightly lower than in fresh (72.8%). Our data demonstrate that the use of MF was comparable with PF, which can be applied in the field conditions without need in a piece of cost-expensive equipment, which can greatly benefit the gene bank of animal genetic resources.

Secretome Analysis of Rabbit and Human Mesenchymal Stem and Endothelial Progenitor Cells: A Comparative Study.

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) have been studied several years for their immunomodulatory effect through the paracrine mechanism and cytokine secretion. In combination with endothelial progenitor cells (EPCs), MSCs have great therapeutical potential for the repair of endothelium and wound healing. However, little is known about the cytokine profile of rabbit AT-MSCs or even EPCs. The aim of this study was to analyze the secretomes of these rabbit stem/progenitor cells. A large-scale human cytokine array (up to 80 cytokines) was used to identify and compare cytokines secreted into conditioned media of human and rabbit AT-MSCs as well as HUVECs and rabbit EPCs. Few cytokines were highly expressed by human AT-MSCs (TIMP-2, TIMP-1), HUVECs (MCP-1, TIMP-2, GRO, Angiogenin, IL-8, TIMP-1), or by rabbit EPCs (TIMP-2). Several cytokines have moderate expression by human (MCP-1, GRO, Angiogenin, TGF-ß 2, IL-8, LIF, IL-6, Osteopontin, Osteoprotegerin) and rabbit AT-MSCs (TIMP-2, TGF-ß 2, LIF, Osteopontin, IL-8, IL-5, IL-3) or by HUVECs (IL-6, MIF, TGF-ß 2, GCP-2, IGFBP-2, Osteoprotegerin, EGF, LIF, PDGF-BB, MCP-3, Osteopontin, Leptin, IL-5, ENA-78, TNF-ß) and rabbit EPCs (TGF-ß 2, Osteopontin, GRO, LIF, IL-8, IL-5, IL-3). In conclusion, the proposed method seems to be useful for the secretome analysis of rabbit stem/progenitor cells.

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

Although the rabbit is a frequently used biological model, the phenotype of rabbit adipose-derived mesenchymal stem cells (rAT-MSCs) is not well characterized. One of the reasons is the absence of specific anti-rabbit antibodies. The study aimed to characterize rAT-MSCs using flow cytometry and PCR methods, especially digital droplet PCR, which confirmed the expression of selected markers at the mRNA level. A combination of these methods validated the expression of MSCs markers (CD29, CD44, CD73, CD90 and CD105). In addition, cells were also positive for CD49f, vimentin, desmin, α-SMA, ALDH and also for the pluripotent markers: NANOG, OCT4 and SOX2. Moreover, the present study proved the ability of rAT-MSCs to differentiate into a neurogenic lineage based on the confirmed expression of neuronal markers ENO2 and MAP2. Obtained results suggest that rAT-MSCs have, despite the slight differences in marker expression, the similar phenotype as human AT-MSCs and possess the neurodifferentiation ability. Accordingly, rAT-MSCs should be subjected to further studies with potential application in veterinary medicine but also, in case of their cryopreservation, as a source of genetic information of endangered species stored in the gene bank.

Molecular Profiling and Gene Banking of Rabbit EPCs Derived from Two Biological Sources.

Endothelial progenitor cells (EPCs) have been broadly studied for several years due to their outstanding regenerative potential. Moreover, these cells might be a valuable source of genetic information for the preservation of endangered animal species. However, a controversy regarding their characterization still exists. The aim of this study was to isolate and compare the rabbit peripheral blood- and bone marrow-derived EPCs with human umbilical vein endothelial cells (HUVECs) in terms of their phenotype and morphology that could be affected by the passage number or cryopreservation as well as to assess their possible neuro-differentiation potential. Briefly, cells were isolated and cultured under standard endothelial conditions until passage 3. The morphological changes during the culture were monitored and each passage was analyzed for the typical phenotype using flow cytometry, quantitative real-time polymerase chain reaction (qPCR) and novel digital droplet PCR (ddPCR), and compared to HUVECs. The neurogenic differentiation was induced using a commercial kit. Rabbit cells were also cryopreserved for at least 3 months and then analyzed after thawing. According to the obtained results, both rabbit EPCs exhibit a spindle-shaped morphology and high proliferation rate. The both cell lines possess same stable phenotype: CD14-CD29+CD31-CD34-CD44+CD45-CD49f+CD73+CD90+CD105+CD133-CD146-CD166+VE-cadherin+VEGFR-2+SSEA-4+MSCA-1-vWF+eNOS+AcLDL+ALDH+vimentin+desmin+α-SMA+, slightly different from HUVECs. Moreover, both induced rabbit EPCs exhibit neuron-like morphological changes and expression of neuronal markers ENO2 and MAP2. In addition, cryopreserved rabbit cells maintained high viability (>85%) and endothelial phenotype after thawing. In conclusion, our findings suggest that cells expanded from the rabbit peripheral blood and bone marrow are of the endothelial origin with a stable marker expression and interesting proliferation and differentiation capacity.

The Impact of Bacteriocenoses on Sperm Vitality, Immunological and Oxidative Characteristics of Ram Ejaculates: Does the Breed Play a Role?

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.

Ram semen quality can be assessed by flow cytometry several hours after post-fixation.

Ram spermatozoa are very sensitive to any cold shock or oxidative damage, therefore making them unsuitable for prolonged storage or distant transport to specialized laboratories for flow-cytometric analysis. The aim of this study was to stain ram semen samples with several fluorescent markers and analyse their stability during formaldehyde fixation. Briefly, freshly collected semen samples were stained for apoptosis (annexin V-FITC, YO-PRO™-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX™ Green] and cell viability (live/dead fixable viability dyes). Next, samples were fixed in buffer containing formaldehyde and then washed. Stained sample were analyzed using flow cytometer before fixation, immediately after fixation, and at 5 h and 20 h post-fixation. Fluorescent signals and the proportion of positively stained spermatozoa were compared statistically in fresh and post-fixed samples. All examined markers, except YO-PRO-1 (decreased significantly, P < 0.05), retained their fluorescence intensities after fixation. In conclusion, several tested markers were able to withstand formaldehyde fixation of ram semen samples as follows: annexin V and FLICA for apoptosis; PNA for acrosomal status; MitoTracker Red CMXRos for mitochondrial activity; and CellROX Green for oxidative status in combination with a suitable live/dead fixable viability dye. This optimized methodology could help to comprehensively analyse the quality of ram semen from local farms countrywide.

Combined approach for characterization and quality assessment of rabbit bone marrow-derived mesenchymal stem cells intended for gene banking.

Rabbit mesenchymal stem cells (rMSCs) are promising agents for the preservation of genetic biodiversity in domestic rabbit breeds. However, rMSCs must meet certain requirements to be used for cryopreservation in animal gene banks. Currently, there are numerous discrepancies in the published data regarding the rMSC phenotype, which may complicate efforts to evaluate their purity and suitability for reuse after cryopreservation in gene and tissue banks. We propose a combined approach (flow cytometry, PCR, differentiation and ultrastructure studies) for the characterization and recovery of rMSCs after cryopreservation. Flow cytometric analyses of rMSCs confirmed the expression of CD29, CD44, vimentin, desmin and α-SMA. RT-PCR revealed the expression of other markers at the mRNA level (SSEA-4, CD73, CD90, CD105, CD146 and CD166). rMSCs showed efficient multilineage differentiation into adipo-, chondro- and osteogenic lineages, SOX2 expression (pluripotency) and typical MSC morphology and ultrastructure. The confirmed rMSCs were subsequently used for cryopreservation. Efficient recovery of rMSCs after cryogenic freezing was demonstrated by high cell viability, normal ultrastructure of reseeded rMSCs, high expression of CD29 and CD44 and lineage differentiation capacity. The proposed combined approach could be used for characterization, cryopreservation and recovery of rMSCs as genetic resources for native rabbit breeds.

Mechanisms of the direct effects of oil-related contaminants on ovarian cells.

We studied the influence of oil-related environmental contaminants (OREC) on the viability, hormone secretion, and protein expression using cultured porcine ovarian granulosa cells. Addition of benzene and xylene promoted proliferation and apoptosis and reduced ovarian cell viability whereas toluene induced apoptosis only. The release of progesterone (P4) and oxytocin (OT) was promoted by benzene and xylene, and suppressed by toluene while prostaglandin F (PGF) output was stimulated by benzene and toluene, but not xylene. The addition of FSH to the culture medium increased ovarian cell proliferation and hormone release, but did not affect apoptosis. However, this FSH's proliferative effect has been prevented in presence of benzene. On the other hand and in the presence of FSH, toluene prevented P4 release and decreased PGF release, while xylene prevented PGF release. We concluded that OREC can affect reproductive processes by directly influencing ovarian cell proliferation, apoptosis, viability, hormone release, and response to gonadotropins.

Effects of benzene, quercetin, and their combination on porcine ovarian cell proliferation, apoptosis, and hormone release.

We hypothesized that the environmental contaminant benzene and the plant antioxidant quercetin may affect ovarian cell functions and that quercetin could offer protection against the adverse effects of benzene. This study aimed to examine the action of benzene, quercetin, and their combination on porcine ovarian granulosa cell functions. We elucidated the effects of benzene (20  µ g mL - 1 ), quercetin (at the doses 0, 1, 10, 100  µ g mL - 1 ), and their combination on ovarian granulosa cell functions (proliferation, apoptosis, and hormone release) in vitro using immunocytochemistry and enzyme immunoassay respectively. Benzene alone stimulated proliferation, apoptosis, and oxytocin release and inhibited progesterone and prostaglandin F release. Quercetin alone inhibited proliferation, apoptosis, and stimulated oxytocin release but did not affect progesterone and prostaglandin F release. When used in combination with benzene, quercetin promoted the inhibitory effect of benzene on progesterone release. Overall, these data suggest that benzene and quercetin have direct stimulatory and inhibitory effects, respectively, on basic ovarian functions. Moreover, no protective action of quercetin against the effects of benzene was found. Rather, it was found to enhance the effect of benzene on progesterone release. Therefore, quercetin cannot be considered for preventing or mitigating the effects of benzene on reproductive processes.

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification.

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48-72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.

Low dose exposure of patulin and protective effect of epicatechin on blood cells in vitro.

In the present study, we aimed to assess antioxidant status in erythrocytes in vitro after patulin (PAT) and epicatechin exposure by measuring antioxidant enzymes (superoxide-dismutase - SOD, glutathione peroxidase - GPx and catalase - CAT) and parameters associated with oxidative stress (malondialdehyde - MDA and ROS). We also investigated the effect of PAT on viability and count of lymphocytes and lymphocyte subpopulations in rabbit blood in vitro. Whole blood of rabbits was used for analysis of antioxidant changes in rabbit erythrocytes after epicatechin and PAT treatment (separately or in combination, at concentrations of 0.2; 2; 20; 200 µg mL-1 of epicatechin and 0.5; 5; 10 µg mL-1 of PAT). Whole blood of rabbits was also used for analysis of count and viability of lymphocytes after PAT treatment at concentrations of 10; 25 and 50 µg mL-1. Results from our experiment confirmed the ability of epicatechin to protect cells against oxidative stress and lipoperoxidation. Our findings indicate that mycotoxin PAT in low concentrations did not affect the activity of antioxidant enzymes in erythrocytes of rabbits significantly. Only slight non-significant changes in lymphocytes count after treatment with low doses of PAT in rabbit blood were observed.

Green tea can supress rabbit ovarian functions in vitro and in vivo.

The aim of present study was to evaluate the action of green tea and its constituents on rabbit ovarian functions and some non-reproductive indexes. In in vitro experiments, rabbit ovarian fragments were cultured with green tea constituents - epigallocatechin-3-gallate (EGCG), green tea polyphenols (GTPP) and resveratrol (RSV) (at 0, 1, 10 or 100 µg/mL medium). The accumulation of an apoptosis marker - caspase 3 and the release of progesterone (P4) and testosterone (T) were measured. In in vivo experiments, does were fed a standard diet or a diet enriched with green tea powder. The weight gain, mortality, ovarian length and weight, conception and kindling rate, number of liveborn, stillborn, and weaned pups, diameter of ovarian follicles and some blood haematological and biochemical parameters were analysed. Culture of ovarian fragments with EGCG increased accumulation of caspase 3, whilst both GTTP and RSV decreased it. EGCG inhibited both P4 and T output, GTPP stimulated P4 and inhibited T, whilst RSV promoted release of both P4 and T. Feeding with green tea increased ovarian length and diameter of ovarian non-ovulated peri-ovulatory haemorrhagic but not of primary and secondary growing follicles. Furthermore, green tea reduced conception and kindling rate, the number of liveborn and weaned pups, increased female mortality but not their weight gain. It reduced platelet distribution width, but it did not affect other haematological and biochemical indexes. These observations suggest that dietary green tea can reduce rabbit doe's viability, ovarian functions and fecundity, perhaps due to changes in ovarian cell apoptosis, steroid hormones release and blockade of the ovulation of large ovarian follicles. The anti-reproductive action of green tea could be due to its constituent - EGCG with pro-apoptotic and anti-steroid hormone properties.

Cryodamage of plasma membrane and acrosome region in chicken sperm.

Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen-thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil+ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non-permeating cryoprotectants: trehalose (KEG-TRE) or glycine (KEG-GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo-PRO-1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG-GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome-sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo-PRO-1) was noted in the KEG-GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.

In vitro effect of various cryoprotectants on the semen quality of endangered Oravka chicken.

We aimed to compare the effect of three different permeating cryoprotectants on the post-thaw spermatozoa quality. Pooled semen from Oravka cock line (n = 6) was diluted in Kobidil+ extender and frozen in cryoprotectant solutions containing 8% dimethylsulfoxide (DMSO), 8% ethylene glycol (EG) or 8% glycerol (GL) in liquid nitrogen vapours before being plunged into the liquid nitrogen. Spermatozoa motility parameters were assessed in vitro after freezing-thawing by a computer-assisted semen analysis (CASA) system and viability status was examined using fluorescent probes. The lower percentage (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing were obtained in all experimental groups (DMSO, EG, GL) compared with the control. Significant (P < 0.05) differences in total motility and progressive movement between GL and DMSO, EG groups were observed. However, the higher number (P < 0.05) of acrosome damaged spermatozoa was found in the DMSO and EG groups and no significant differences were observed in the GL group compared with the control. Differences (P < 0.05) between experimental groups and the control in the results of spermatozoa necrosis were observed. No significant differences in the percentage of apoptotic spermatozoa were found between control and experimental groups. However, significant differences (P < 0.05) in number of live and necrotic spermatozoa between GL and DMSO, EG groups were examined. The findings of the present study indicate that glycerol seems to be suitable for semen cryopreservation in the gene banks. In addition, fertility evaluation in vivo is needed in order to evaluate the possible contribution for the bank of animal genetic resources.