Flavone and 3-hydroxyflavone supplementation in cryopreservation medium protects canine sperm against apoptosis and lipid peroxidation.

Cryopreservation is a pivotal technique in safeguarding genetic material across diverse species, despite its inherent challenges linked to induced spermatozoa damage, notably apoptosis and lipid peroxidation (LPO). Given the insufficient antioxidant defense of spermatozoa against LPO, there is a rising interest in integrating additional additives into extenders to ameliorate mammalian semen quality. Among these additives, flavonoids have garnered considerable attention due to their potent antioxidative properties. Hence, our study aimed to assess the efficacy of flavone (FL) and 3-hydroxyflavone (3-OH = ) supplementation in the cryopreservation medium to protect canine sperm against the damaging impacts of freezing and ensure the preservation of their reproductive potential. Semen was collected from five Beagle stud dogs and then pooled. Then, the sample was divided into 7 groups, each treated with 1) 0 mM, 2) 0.1 mM FL, 3) 0.2 mM FL, 4) 0.4 mM FL, 5) 0.1 mM 3-OH = , 6) 0.2 mM 3-OH = , 7) 0.4 mM 3-OH = . Semen samples were subjected to cryopreservation in French straws and glycerol as a cryoprotectant. In the frozen thawed semen, sperm motility parameters by CASA system and sperm membrane integrity, acrosome status, mitochondrial activity, DNA fragmentation, early apoptosis with capacitation, and LPO were assessed using flow cytometry just after thawing (0 h) and 4 h post thaw. Results reveal significant increase in the proportion of live spermatozoa with undamaged acrosomes in the FL 0.1 and 3-OH = 0.2 groups at 0 h post thaw. At this time point, 3-OH = 0.1 significantly reduced the DNA fragmentation index (DFI) compared to the FL 0.1 and 0.2 groups. However, after the next 4 h, 3-OH = 0.4 exhibited the lowest (P < 0.05) DFI compared to FL 0.2 and 3-OH = 0.1. Additionally, 3-OH = 0.4 showed the highest (P < 0.05) proportion of non apoptotic and non capacitated spermatozoa compared to FL 0.1 0 h post-thaw. Simultaneously, the same group demonstrated significant reduction in apoptotic and capacitated sperm cells, at 0 h and 4 h post-thaw. Moreover, 3-OH = at 0.1 (0 h and 4 h) and 0.2 mM (4 h) significantly enhances the proportion of live sperm without LPO post thaw. Whitin the FL groups, only 0.4 FL significantly increased the percentage of live sperm without LPO. No significant effect of the tested substances was observed on sperm motility, cell membrane integrity, or mitochondrial activity. These findings highlight the promising role of flavone and 3-hydroxyflavone in enhancing sperm resilience during cryopreservation, suggesting their protective function against acrosome damages, capacitation, apoptosis and lipid peroxidation.

In search of freezability predictors for feline spermatozoa - osmotic challenge tests and markers of sperm membrane structure.

Semen cryopreservation is a crucial part of assisted reproductive techniques (ART) in animals, and recently it is gaining more and more attention among cat breeders. Even if fresh semen quality is good, sometimes spermatozoa do not survive freezing. The freezability prediction was widely studied in many species, but not in the domestic cat. The aim of this study was to verify the usefulness of osmotic challenge tests and membrane structure markers (Yo-Pro 1 and Merocyanine 540) for the prediction of the quality of post-thawed feline semen. Semen was collected by urethral catheterization from 22 male cats. After a basic evaluation of semen, 20×106 spermatozoa were cryopreserved; the rest were evaluated by flow cytometry for membrane integrity (SYBR-14/PI), acrosome status (lectin PNA/PI), mitochondrial potential (JC-1) and membrane stability (Yo-Pro 1/M540 staining). Hypo- and hyperosmotic challenge tests were also performed. The thawed samples were evaluated as fresh ones. The Pearson correlation between all parameters in fresh semen and all parameters in cryopreserved spermatozoa was assessed. Although some moderate correlations were found between the results of the osmotic tests and markers of sperm membrane stability (Yo-Pro 1 and Merocyanine 540) and post-thaw semen quality parameters, the predictive value of studied markers was rather weak - no cut-off values could be established and, based on regression models, they explained less than 40 % of variability in post-thaw quality. Our results confirm that cryodamage is a complex matter, in which many different factors play a role, affecting sperm motility and membrane integrity differently.

The neonicotinoid, imidacloprid, disrupt the chicken sperm quality through calcium efflux.

Imidacloprid (IMI), an insecticide from the neonicotinoid group widely used in agriculture, has drawn attention due to its potential harmful effects on non-target species, including bird populations. In the present work, we investigated the effect of IMI on avian semen by in vitro exposure of rooster spermatozoa to this pesticide. The semen was collected twice a week. Samples collected on one day were pooled and incubated with the following IMI concentrations: 0 mM, 0.5 mM, 5 mM, 10 mM, and 50 mM at 36°C for 3 h. Comprehensive semen analysis was carried out after 1 h and 3 h of incubation, evaluating sperm motility parameters with the CASA system and using flow cytometry to assess membrane integrity, mitochondrial activity, acrosome integrity, chromatin structure, intracellular calcium level and apoptosis markers such as: early apoptosis and caspase activation and lipid peroxidation. The results of the first experiment suggest that low concentrations of IMI have a different effect on sperm motility compared to higher concentrations. In IMI samples, we also observed a lower percentage of cells with a high level of calcium ions compared to the control, and a lower level of lipid peroxidation. We concluded that IMI may act as a blocker of calcium channels, preventing the influx of these ions into the cell. To confirm this mechanism, we conducted a second experiment with calcium channel blockers: SNX 325, MRS-1845, and Nifedipine. The results of this experiment confirmed that the mechanism of action of IMI largely relies on the blockade of calcium channels in rooster sperm. Blocking the influx of calcium ions into the cell prevents the formation of Ca²âº-dependent pores, thereby preventing an increase in cell membrane permeability, ultimately blocking early apoptosis and lipid peroxidation in chicken spermatozoa.

Fatty acid composition and biophysical characteristics of the cell membrane of feline spermatozoa.

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

Vitrification of feline ovarian tissue: Comparison of protocols based on equilibration time and temperature.

Global contraction of biodiversity pushed most members of Felidae into threatened or endangered list except the domestic cat (Felis catus) thence preferred as the best model for conservation studies. One of the emerging conservation strategies is vitrification of ovarian tissue which is field-friendly but not yet standardized. Thus, our main goal was to establish a suitable vitrification protocol for feline ovarian tissue in field condition. Feline ovarian tissue fragments were punched with biopsy punch (1.5 mm diameter) and divided into 4 groups. Group 1 was fresh control (Fr), while the other three were exposed to 3 vitrification protocols (VIT_CT, VIT_RT1 and VIT_RT2). VIT_CT involved two step equilibrations in solutions containing dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 10 min each at 4 °C. VIT_RT1 involved three step equilibration in solutions containing DMSO, EG, polyvinylpyrrolidone and sucrose for 14 min in total at room temperature, while in VIT_RT2 all conditions remained the same as in VIT_RT1 except equilibration timing which was reduced by half. After vitrification and warming, fragments were morphologically evaluated and then cultured for six days. Subsequently, follicular morphology, cellular proliferation (expression of Ki-67, MCM-7) and apoptosis (expression of caspase-3) were evaluated, and data obtained were analysed using generalised linear mixed model and chi square tests. Proportions of intact follicles were higher in Fr (P = 0.0001) and VIT_RT2 (P = 0.0383) in comparison to the other protocols both post warming and after the six-day culture. Generally, most follicles remained at primordial state which was confirmed by the low expression of Ki-67, MCM-7 markers. In conclusion, VIT_RT2 protocol, which has lower equilibration time at room temperature has proven superior thus recommended for vitrification of feline ovarian tissue.

Effect of Fixatives and Fixation Period on Morphology and Immunohistochemistry of Feline Ovarian Tissue.

Fixatives and fixation protocol have a profound effect on both the morphology and epitope sensitivity of ovarian tissue, which hampers accurate ovarian tissue evaluation. We aimed to establish the most suitable fixation protocol for feline (Felis catus) ovarian tissue. Fragments (1.5 mm diameter) were punched from 1 mm-thick feline ovarian tissue, divided into three groups then fixed with three different fixatives (Bouin, neutral buffered formalin [NBF] and form acetic acid [new compound fixative formulation for ovarian tissue composed of 5% acetic acid in NBF]) for five fixation periods. Subsequently, fragments were processed and evaluated for the morphology and intensity of immunohistochemical signals against three antigens (Ki-67, MCM-7 and activated caspase-3). Proportions of grade 1 or morphologically intact follicles were significantly lower in NBF when compared with Bouin and form acetic acid fixatives. However, Bouin fixative had the lowest mean DAB intensity (p < 0.05) in all three antigen targets, while NBF had the highest (p < 0.05) in Ki-67 and caspase-3, but in MCM-7, it was no different from form acetic acid. In conclusion, form acetic acid maintained ovarian tissue architecture with excellent follicular morphology in the same manner as Bouin fixative, and it also maintained reasonable DAB signals similar to NBF, thus providing a better alternative for feline ovarian tissue studies.

Evaluation of a commercial proAKAP4 kit for the assessment of fresh and frozen-thawed feline spermatozoa.

ProAKAP4 is gaining increasing attention as a potential marker of semen quality in many species, but while there is a commercial kit for assessing proAKAP4 in the domestic cat, there are no publications about its use. The aim of this study was to evaluate the commercial proAKAP4 kit - Cat 4MID® Kit (SPQI - 4BioDx, Lille, France) for the assessment of feline semen. Semen was collected from 54 male cats by urethral catheterization. After a basic semen evaluation (subjective motility, CASA, viability, morphology), proAKAP4 levels in each sample were assessed using the Cat 4MID® Kit according to the manufacturer's protocol or with some modifications related to incubation time, sample storage conditions and number of spermatozoa used. Finally, the Spearman correlation of proAKAP4 concentration and sperm motility parameters was calculated. The most reliable results (acceptable intraassay coefficient of variation) were obtained with an optimized protocol of overnight incubation and isolation of proAKAP4 protein from 1 × 106 spermatozoa stored at -80°C. For fresh semen, there were no significant correlations between proAKAP4 concentration and sperm motility parameters, despite a strong correlation between motility parameters and sperm viability and morphology. A predominant effect of other sperm parameters and highly variable performance of lysis buffer question the usefulness of Cat 4MID® Kit for the assessment of feline semen. For frozen-thawed semen, there was a moderate, negative correlation between proAKAP4 concentration and two CASA parameters, VAP and VSL. As there were no correlations between proAKAP4 concentration in fresh semen and motility parameters in cryopreserved samples, proAKAP4 cannot be used as freezability marker in cats. More studies are needed to establish potential correlation with long-lasting motility.

Prolonged cold-preservation of domestic cat ovarian tissue is improved by extracellular solution but impaired by the fragmentation of ovary.

For domestic cats ovaries, recommended cold-storage limit is 24 h in Phosphate Buffered Saline (PBS) or Dulbecco`s PBS (DPBS). Here, we attempted to verify wheatear cat ovaries may benefit from more complex solutions during prolonged cold-storage (>24 h). First, the preservation capabilities of extracellular (SP+), intracellular (UW) solutions and DPBS supplemented with glutathione (DPBS+GSH) were compared using ovary fragments from the same ovary (n=10). Intact ovary stored in DPBS served as a control. Ovaries were kept at 4 °C for 48 h, and 72 h. In the second experiment, first ovary was stored in DPBS, second in SP+ or UW solution for 48 h (n = 12). Ovaries pairs stored in DPBS for 24 h served as a control (n=8). Tissue samples were evaluated directly after cold-storage and after following 24 h in vitro culture. Ovarian follicle morphology, apoptosis rates (cleaved caspase-3, TUNEL), and follicular growth activation (Ki-67) were assessed. Ovary fragmentation impaired follicular morphology preservation upon cold-storage comparing to intact ovary. However, ovarian fragments stored in UW for 48 h and in SP+ for 72 h presented better morphology than DPBS+GSH group. Comparison of intact ovaries cold-storage for 48 h showed that SP+ provided superior follicular morphology over DPBS, and it was comparable to the outcome of 24-hour storage. No follicular activation after in vitro culture was observed. Nevertheless, tissue culture increased considerably caspase-3 cleavage and TUNEL detection. The ovary fragmentation prior to cold-storage is not recommended in domestic cats. Replacement of DPBS with SP+ solution for whole ovary and UW solution for ovarian tissue fragments improves follicular structure preservation during 48-hour cold-storage.

Addition of low concentration of cholesterol-loaded cyclodextrin (CLC) has a positive effect on cryopreserved canine spermatozoa evaluated by andrological and biophysical methods.

BACKGROUND: This study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP). RESULTS: An addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05). CONCLUSIONS: In conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters.

Feline Wharton's jelly-derived mesenchymal stem cells as a feeder layer for oocytes maturation and embryos culture in vitro.

Introduction: Due to their capacity to release growth factors and cytokines, co-culture using mesenchymal stem cells has been considered a good alternative to promoting the maturation of the oocytes and the embryo's development quality in vitro in different mammalian species. In this regard, we investigated the effect of feline Wharton's jelly MSCs as feeders layer in oocyte maturation-consequently, the development of resulting embryos in co-culture. Methods: Oocytes with dark cytoplasm and a few layers of cumulus cells were collected and subjected to in vitro maturation and embryo culture using commercial media with and without MSCs addition. The oocytes' nuclear maturation and the degree of cumulus expansion in different groups were assessed after 24 h; the development of the embryo was evaluated every 12 h until day eight. Results: Although MSCs increased the proportion of cumulus cells oocytes exhibiting cumulus expansion, there were no significant differences in the percentage of matured oocytes (metaphase II) among the groups (p > 0.05). However, the embryo development differs significantly, with a higher cleavage, morula, and blastocyst percentage in oocytes matured with MSC co-culture conditions than in commercial media alone (p < 0.05). Also, we observed higher morula and blastocyst rates in the embryos co-cultured with MSCs during the in vitro culture (p > 0.05). Conclusion: Based on our results, the co-culture with MSCs during the oocyte maturation resulted in better embryo development, as well as the MSCs addition during embryo culture returned an increased number of morula and blastocysts. Further research is needed to fully understand and optimize the use of MSCs in oocyte maturation and embryo development.

Culture of vitrified bovine ovarian tissue on agarose gel inserts maintains follicle integrity.

In brief: Ovarian tissue cryopreservation and culture provide an option for fertility preservation without tissue grafting, but need optimization. This study reveals that vitrified bovine ovarian tissue can be cultured on agarose gel and maintain follicle morphology, low activation, and low apoptosis. Abstract: Ovarian tissue preservation is hitherto a promising fertility insurance option for precious animals. Ovarian tissue vitrification and culture combined approach would eliminate the need of transplanting ovarian tissue to obtain mature oocytes. We aimed at optimizing vitrification and in vitro culture conditions for improved bovine ovarian tissue viability. Ovaries obtained from the slaughterhouse were punched into fragments and divided into three groups. Group 1 (fresh) was divided into two and immediately placed in two-culture systems (culture inserts and agarose inserts). Group 2 was vitrified, warmed, and placed in the two-culture systems, while group 3 was only equilibrated and then placed in the two-culture systems. All cultures were maintained for 6 days and spent media were collected on alternate days for cytokine (interleukin 1ß and interleukin 6) evaluation. Fragments were fixed for morphology assessment and immunohistochemistry. Higher percentages (P < 0.05) of grade 1 (morphologically intact) follicles were observed in fragments on agarose compared to those on culture inserts on days 2 and 4 of the culture. Conversely, we found higher (P < 0.05) shifts of primordial follicles to transitional follicles in fragments on culture inserts vis-à-vis agarose inserts which was consistent with a higher proportion of Ki-67 and MCM-7 and activated caspase-3-positive follicles. In conclusion, in vitro culture of bovine ovarian tissue on agarose inserts maintained follicle morphology, low follicle activation, and low apoptosis compared to culture inserts.

Mito-Tempo improves acrosome integrity of frozen-thawed epididymal spermatozoa in tomcats.

Introduction: In tomcats, epididymal spermatozoa provide an additional source of male gametes available for cryopreservation. While this procedure is feasible, the survival rate and motility of epididymal cat spermatozoa are both low after thawing. Cryopreservation is known to induce oxidative stress in spermatozoa, with mitochondria and the plasma membrane being the two major generation sites, and an imbalanced presence of free radicals is a possible cause for this low survival rate. Different antioxidants have been tested before for their effect on cryopreserved cat spermatozoa quality, with varying results. Here, we used Mito-Tempo, which is a synthetic mitochondria-targeted antioxidant and a specific scavenger of the mitochondrial superoxide system. By supplementing Mito-Tempo with the freezing extender, we aimed to improve the sperm quality of frozen-thawed cat epididymal spermatozoa. Methods: Epididymal spermatozoa obtained from twelve tomcats were assessed for motility and concentration. Prior to freezing, samples were diluted in TRIS buffered extender with egg yolk and glycerol and divided into five aliquots supplemented with 0 (control), 0.5, 5, 50, and 1005M of Mito-Tempo. After thawing, sperm motility, concentration, morphology, plasma membrane integrity, acrosome integrity, and mitochondrial membrane potential were evaluated. A Friedman rank sum test with a Bonferroni post-hoc test was used to determine statistical in-between group differences in post-thaw semen parameters. Results and discussion: The results indicated a slight improvement in acrosome integrity across all groups that were supplemented with Mito-Tempo, with the group that received 55M of Mito-Tempo showing the greatest improvement [(median of 67.99%, IQR of 5.55) compared to the control group (median of 65.33%, IQR of 7.75; P = 0.05)]. For all other sperm parameters, no significant differences (P > 0.05) were detected between different Mito-Tempo concentrations. These findings highlight the protective effect of Mito-Tempo on acrosome integrity and suggest that 55M is the most effective concentration for maintaining acrosome integrity. Since Mito-Tempo has shown a positive effect on multiple sperm parameters in other species, such as men, boars, roosters, rams, and bulls, we need to conclude that species-specificity may play a role here.

The morphology, morphometry and functionality of fresh and cryopreserved wisent (Bison bonasus) epididymal spermatozoa.

Epididymal spermatozoa obtained post mortem are considered a valuable source of genetic material which is often irrevocably lost. This makes these gametes constitute a key element in protection and restitution programs. The wisent (Bison bonasus, Linnaeus 1758) is a species that survived in zoos after extinction from its natural habitat. This resulted in a narrowing of the genetic pool of the whole population, which is at present derived from only 12 ancestors. Currently, wisent protection programs are aimed at preserving the genetic diversity by establishing a germplasm bank. The objective of this study was to comprehensively characterize the morphology, morphometry and functionality of wisent epididymal spermatozoa and evaluate the effectiveness of their cryopreservation in extender based on Tris buffer and chicken egg yolk. The median total number of spermatozoa obtained from one individual was 1985.0 × 106 (62.5 × 106-7452.0 × 106). These gametes were characterized by median: 40.0% (0.5-70.0%) subjective motility, 69.8% (32.5-90.0%) viability and 54.3% (10.5-83.3%) normal morphology. The sperm head had a median size of 5.0 µm (3.5-6.7 µm) width, 8.5 µm (6.4-11.3 µm) length and 36.9 µm2 (23.7-48.6 µm2) surface area. The viable population of the obtained gametes was characterized by median values 53.2% (4.5-80.3%) of intact sperm membrane, 50.8 (26.0-76.6%) of intact acrosome, 0.4% (0-98.7%) of fragmented chromatin, 5.9% (0.0-88.8%) of cells with high mitochondrial potential and 42.1% (8.3-63.7%) without lipid peroxidation. The viable population of the frozen/thawed gametes was characterized by median values: 18.4% (2.4-57.9%) of intact sperm membrane, 35.1 (11.9-56.7%) of intact acrosome, 0.07% (0-89.2%) of fragmented chromatin, 12.8% (0.0-49.7%) of cells with high mitochondrial potential and 16.3% (2.2-53.6%) without lipid peroxidation. Due to the material originating from a relatively large number of wild individuals, the research presented here contributed to the description of certain species standards for the assessment of wisent epididymal spermatozoa. The presented effect of cryopreservation on these gametes justifies the use of an extender based on Tris buffer with the addition of chicken egg yolk. The obtained effects are satisfactory from the point of view of preserving valuable genetic material and their use in ART.

Effect of male age on semen quality in domestic animals: potential for advanced functional and translational research?

Age and other factors like season and breed are often associated with sperm quality and fertility in domestic animals. Even though many studies assessed the relationship between the age of the male and sperm parameters, the effects have not been comprehensively evaluated. Changes in semen quality from pubertal (young) to adult and old age were identified in the bull, ram, buck, boar, dog, and stallion, respectively. The review discusses the association between male age and semen volume, the total number of spermatozoa per ejaculate, sperm concentration, motility, morphology, sperm cell function, sperm DNA integrity, oxidative stress, and antioxidant activity in these species of animals. Generally, semen characteristics improve to a certain age, which declines as the animal ages. Only a few studies evaluated the impact of advanced age or employed advanced functional sperm assessment methods to assess age-related changes in sperm quality and male fertility. Such studies in the dog or stallion, for instance, may contribute to advancing knowledge in human-assisted reproductive techniques used in patients of advanced paternal and maternal age.

Feline herpesvirus 1 (FHV-1) enters the cell by receptor-mediated endocytosis.

Feline herpesvirus type 1 (FHV-1) is an enveloped dsDNA virus belonging to the Herpesviridae family and is considered one of the two primary viral etiological factors of feline upper respiratory tract disease. In this study, we investigated the entry of FHV-1 into host cells using two models: the AK-D cell line and primary feline skin fibroblasts (FSFs). We employed confocal microscopy, siRNA silencing, and selective inhibitors of various entry pathways. Our observations revealed that the virus enters cells via pH and dynamin-dependent endocytosis, as the infection was significantly inhibited by NH4Cl, bafilomycin A1, dynasore, and mitmab. Additionally, genistein, nystatin, and filipin treatments, siRNA knock-down of caveolin-1, as well as FHV-1 and caveolin-1 colocalization suggest the involvement of caveolin-mediated endocytosis during the entry process. siRNA knock-down of clathrin heavy chain and analysis of virus particle colocalization with clathrin indicated that clathrin-mediated endocytosis also takes part in the primary cells. This is the first study to systematically examine FHV-1 entry into host cells, and for the first time, we describe FHV-1 replication in AK-D and FSFs. IMPORTANCE Feline herpesvirus 1 (FHV-1) is one of the most prevalent viruses in cats, causing feline viral rhinotracheitis, which is responsible for over half of viral upper respiratory diseases in cats and can lead to ocular lesions resulting in loss of sight. Although the available vaccine reduces the severity of the disease, it does not prevent infection or limit virus shedding. Despite the clinical relevance, the entry mechanisms of FHV-1 have not been thoroughly studied. Considering the limitations of commonly used models based on immortalized cells, we sought to verify our findings using primary feline skin fibroblasts, the natural target for infection in cats.

Mesenchymal Stem Cells: Generalities and Clinical Significance in Feline and Canine Medicine.

Mesenchymal stem cells (MSCs) are multipotent cells: they can proliferate like undifferentiated cells and have the ability to differentiate into different types of cells. A considerable amount of research focuses on the potential therapeutic benefits of MSCs, such as cell therapy or tissue regeneration, and MSCs are considered powerful tools in veterinary regenerative medicine. They are the leading type of adult stem cells in clinical trials owing to their immunosuppressive, immunomodulatory, and anti-inflammatory properties, as well as their low teratogenic risk compared with pluripotent stem cells. The present review details the current understanding of the fundamental biology of MSCs. We focus on MSCs' properties and their characteristics with the goal of providing an overview of therapeutic innovations based on MSCs in canines and felines.

Feline sperm head morphometry in relation to male pedigree and fertility.

Computer-assisted sperm morphometry analysis is an advanced tool which allows to precise measure sperm head parameters like length, width, area, and perimeter. On the basis of these and calculated parameters, morphometric subpopulations of spermatozoa can be distinguished. In many species, the distribution of subpopulation within the ejaculate is related to male fertility. There is no information about such a relation for domestic cats; therefore, the aim of this study was to evaluate whether spermatozoa from non-pedigree and purebred domestic cats differ in morphometric parameters. The second aim was to check if there is a relationship between sperm morphometry and fertility. Urethral semen was collected from 27 tomcats, divided into three study groups: non-pedigree cats of unknown fertility, purebred infertile cats and purebred fertile cats. The morphometric assessment was performed by CASMA, followed by principal component analysis and clustering. The results revealed huge intra- and inter-individual variation in sperm head morphometric parameters and three sperm-head morphometric subpopulations were identified in feline semen. Neither mean values of morphometric parameters nor the distribution of spermatozoa between morphometric subpopulations differ between non-pedigree cats of unknown fertility and purebred infertile and fertile cats. We hypothesize that other factors, especially abnormalities of the midpiece and tail, and overall worse quality of the semen of infertile males could have masked the effect of subtle changes in the sperm head morphometry.

Does Better Post-Thaw Motility of Dog Sperm Frozen with CLC Mean Better Zona Pellucida Binding Ability?

Even though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen-thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa.

Preliminary study on fluid bolus administration for prevention of spinal hypotension in dogs undergoing elective cesarean section.

Introduction: This study aimed to investigate the effect of fluid bolus administration during epidural anesthesia (coload) in female dogs scheduled for elective cesarean section (CS). Hypotension is one of the most common complications of epidural (EA) and spinal (SA) analgesia, and in the case of cesarean section, it may pose a significant risk for placental perfusion and subsequent fetal vitality and puppy survival. Methods: Pregnant bitches scheduled for elective CS underwent EA with (treatment group) or without (control group) intravenous fluid bolus administration. The following parameters were measured and compared between both groups: HR, RR, etCO2, SpO2, systolic, diastolic and mean arterial blood pressure were measured at three time points (T1: before surgery, T2: after the last puppy removal, and T3: end of surgery) in dams; vitality (Apgar score at 0, 5, and 20 min) and umbilical cord blood parameters (pH, pCO2, HCO3, base excess, lactate and glucose) in newborns. Results: The results indicated that crystalloid coloading increased maternal systolic, diastolic, and mean blood pressure (treatment vs. control group, 101.46 ± 9.18, 48.01 ± 13.47, and 67.07 ± 13.15 mmHg vs. 80.68 ± 7.29, 36.52 ± 8.75, and 180 52.30 ± 7.77, p < 0.05) with significantly fewer episodes of hypotension. Additionally, puppies in the treatment group received higher scores in the 5-min (7.91 ± 1.67 vs. 6.74 ± 2.20) and 20-min (9.38 ± 0.87 vs. 8.39 ± 2.50) assessments without the favorable effect on umbilical blood gas parameters. Discussion: Based on the obtained results, it can be stated that crystalloid coload offers an effective option in cases of hypotension during cesarean section, with clear benefits for both mothers and newborns.