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.

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.

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.

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.

Comparison of 2 anesthetic protocols and surgical timing during cesarean section on neonatal vitality and umbilical cord blood parameters.

BACKGROUND: The objective of this study was to evaluate the relationship between the mode of anesthesia, the time form the induction to the extraction of a puppy and the immediate postnatal vitality and umbilical cord blood gases parameters in cesarean section derived-puppies. Two different anesthetic protocols were used: inhalation using isoflurane (ISO) and combined-inhalation and epidural (EPI) with propofol being the induction agent. RESULTS: Significant differences were found in ISO group in pH values, pCO2 levels and Apgar scores between puppies at different extraction times (< 30 vs. ≥ 30 min). In ISO group puppies extracted later were more acidic (7.16 vs. 7.22), had higher levels of pCO2 (69 vs. 57 mmHg) and lower Apgar scores at birth (1.2 vs. 2.5). On the contrary, in EPI group no differences were observed between the delivery time, umbilical blood gas parameters and puppies' vitality. Furthermore, the dams from the EPI group required lower concentrations of isoflurane (MAC 1.11 ± 0.19 vs.1.37 ± 0.16, p < 0.001). CONCLUSIONS: Multiple pregnancies frequent in dogs lead to significant differences in extraction times between the first and the last puppy during cesarean section. Obtained results showed that the mode of anesthesia and the surgical time would influence the neonatal outcome during cesarean section in dogs. The higher concentration of isoflurane with the longer time of exposure had a negative effect on the initial newborn vitality as well as the umbilical cord blood gas parameters. Therefore, when performing CS in giant dog breeds or expecting many puppies in the litter, it is worth considering epidural component that allow for lower concentrations of inhalant agents, which may contribute to a better clinical condition of newborns.

Whole genome sequencing identifies a missense polymorphism in PADI6 associated with testicular/ovotesticular XX disorder of sex development in dogs.

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa.

BACKGROUND: The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. RESULTS: Percoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. CONCLUSIONS: While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

Long-term stiripentol administration, an anticonvulsant drug, does not impair sperm parameters in rats.

In this study, we investigated the influence of long-term administration of stiripentol on sex hormones and semen quality in young Wistar rats. Investigated animals received for 6 months either stiripentol or saline solution. After one month, stiripentol increased temporarily serum level of testosterone (p < 0.05) and FSH (p < 0.01). However, after 6 months levels of testosterone, FSH, LH, prolactin and SHBG were comparable in both groups. After 6 months, semen analysis did not reveal differences in sperm concentration, total sperm count and sperm motility between groups. However, stiripentol increased the rate of head defect (p < 0.001) and midpiece abnormalities (p < 0.05). Flow cytometry revealed higher percentage of live cells without lipid peroxidation (p < 0.00001) and higher percentage of live spermatozoa with intact acrosomes (p < 0.000001) in rats receiving stiripentol. There was no significant difference between groups in sperm mitochondrial activity and DNA fragmentation index. However, percentage of high DNA stainability cells was increased in stiripentol group (p < 0.001). The data showed that stiripentol does not cause obvious disturbances in young rat's semen. Detected changes in semen morphology and chromatin structure need further explanation, and their influence on rat's fertility should be evaluated.

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification.

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.

The use of human and bovine commercial media for oocyte maturation and embryo development in the domestic cat (Felis catus).

The aim of this study was to examine the suitability of commercial media designed for humans and cattle for oocyte maturation and embryo culture in the domestic cat. In Exp. I, feline oocytes collected ex vivo were subjected to in vitro maturation in a laboratory-made culture medium (based on M199) or a commercial medium designed for cattle cells (BO-IVM® ). In Exp. II, ICSI-derived feline embryos were cultured for 7 days in a commercial human (Continuous Single Culture® ) or bovine (BO-EC® ) cell medium. The rates of cleavage, morula and blastocyst formation were evaluated at 24 hr, 6 days and 7 days after ICSI, respectively, and compared between experimental groups. At the end of culture, embryos were assessed for viability and apoptotic changes. In Exp. I, no statistically significant difference in oocyte maturation outcome between laboratory-made (52.7%) and commercial media (58.9%) was observed. However, the use of a commercial medium prepared for use with bovine cells resulted in a significantly lower variance of the maturation rate. In Exp. II, no statistically significant differences between two commercial media were observed for cleavage (67.5% and 64.5%), morula (39.3% and 47.1%) and blastocyst rates (25.0% and 19.6%), as well as for the percentage of late apoptotic blastomeres. Morulae cultured in medium marketed for humans exhibited significantly more early apoptotic (43.2 ± 31.2% vs. 23.4 ± 23.2%) and necrotic (60.6 ± 47.6% vs. 29.4 ± 22.6%) blastomeres. In conclusion, both commercial media tested are suitable for in vitro oocyte maturation and embryo culture procedures in cats. It is remarkable that a culture medium designed for use in cattle for in vitro maturation of cat oocytes provides more reproducible results.

Developmental competence of cat (Felis domesticus) oocytes and embryos after parthenogenetic stimulation using different methods.

The aim of this study was to compare the effects of various activating factors on feline oocytes. The study included activation within the ovary (natural), activation during in vitro maturation (spontaneous activation), chemical activation (ionomycin + 6-DMAP), activation by spermatozoa and injection (ICSI) and mechanical activation (sham ICSI). According to our results, parthenogenetic embryos could emerge at every step of in vitro embryo production (IVP) procedures. After oocyte collection, 6% of parthenogenetic embryos were observed, mainly at the 2-4-blastomere stages. After 24 h of in vitro maturation, parthenogenetic activation was observed in 7% of oocytes. Using ionomycin and 6-DMAP to artificially activate oocytes, 53% of cleaved embryos were obtained. The results after ICSI (54% cleaved embryos) were not significantly different from the results in Group III using chemical activation (53% cleaved embryos). But only after ICSI were blastocysts obtained (5/73.7%) as a result of in vitro culture. Moreover, embryos after ICSI were of the best morphological quality with minor levels of fragmentation evident in the embryos. After sham mechanical activation, 'sham ICSI', 8% of cleaved embryos were noted. Therefore, it is advised to maintain a negative control in parallel with each step of IVP techniques, to avoid misleading results. Chemical methods for artificial activation of feline oocytes are the most promising for application to the cloning and production of parthenogenetic embryos for experimental studies.

Comparison of the characteristics of chinchilla epidydimal semen after collection, storage at 5°C and cryopreservation.

The aim of the study was to compare the characteristics of chinchilla epididymal sperm: fresh, stored at liquid state and cryopreserved. Epididymal spermatozoa obtained from 11 males were assessed for subjective motility, concentration, motility parameters measured by CASA, viability, morphology, membrane integrity, acrosome integrity, mitochondrial potential, lipid peroxidation, chromatin structure, apoptotic changes and capacitation. Then half of the spermatozoa were stored at 5°C for 30 hr, and the second half was cryopreserved. After storage and thawing the same parameters as in fresh semen were assessed. Fresh semen showed good quality, with low levels of lipid peroxidation, chromatin fragmentation and capacitation. CASA evaluation showed significantly lower values for MOT, PMOT, RAPID, VCL, VAP and VSL after both storage at liquid state and cryopreservation (p < 0.05). Cold storage did not induce membrane and acrosome damage (p > 0.05), conversely to cryopreservation (p < 0.05). After storage, there was a drop in high mitochondrial potential in live cells (p < 0.05) and an increase in the percentage of non-apoptotic, capacitated cells (p < 0.05). These changes were not seen after cryopreservation (p > 0.05). Lipid peroxidation in live cells and chromatin structure remained unchanged both after storage and cryopreservation (p > 0.05). The study showed that examined methods of semen preservation exerted different patterns of changes in spermatozoa and that sperm quality after both of them allowed for further use of preserved spermatozoa in artificial reproductive techniques.

Accuracy of ultrasonography and fine-needle aspiration cytology in the diagnosis of prostate diseases in dogs.

Clinical signs of prostatic diseases in dogs are often non-specific. Appropriate treatment should be based on a detailed investigation using reliable diagnostic tools. The aim of our study was to evaluate the diagnostic value of ultrasonography (US) and fine-needle aspiration (FNA) cytology in dogs' prostate diseases. The mean accuracy of FNA cytology and US were 0.72 and 0.88 (n = 13), respectively. US gland size measurements and actual gland dimensions were highly concordant. Obtained results confirm the high diagnostic value of US and FNA biopsy and in prostatic diseases. Diagnosis based on US is highly reliable; however, it should be combined with clinical signs. Therefore, cytological evaluation of prostate gland material may be performed to differentiate or confirm presumptive diagnosis.

Disorders of sex development in cats with different complements of sex chromosomes.

The genetic background of disorders of sex development (DSDs) in cats is poorly understood, due to a relatively low number of such studies in this species. Here we present three new DSD cases with different complements of sex chromosomes. The first, an Oriental Shorthair cat with a rudimentary penis, abdominal atrophic testicles and lack of uterus appeared to be a freemartin, since leucocyte chimerism XX/XY and a lack of Y-linked genes (SRY and ZFY) were observed in DNA isolated from hair follicles. XXY trisomy was identified in the second case, a tortoiseshell Devon Rex male cat with atrophic scrotal testicles and a normal penis. Finally, a European Shorthair cat with atrophic testicles in a bifid scrotum, rudimentary penis and a lack of uterus had XY complement, including Y chromosome of normal size and morphology. Also presence of eight Y-linked genes, detected by PCR, was confirmed. Due to the low testosterone level in this last patient, we searched for a causative mutation in two candidate genes (HSD3B2 and HSD17B3) involved in the metabolism of this steroid hormone. Altogether, five polymorphic sites in HSD3B2 and two in HSD17B3 were found, but none of them showed associations with DSD phenotype. We thus excluded a possibility that the causative mutation is present in these genes. In conclusion, we confirmed that analysis of the sex chromosome complement is a crucial step in diagnosis of DSDs. However, extensive molecular studies of the genes involved in sex development are needed to elucidate the causes of DSDs in cats with normal complements of sex chromosomes.

Modification of membrane cholesterol and its impact on frozen-thawed chicken sperm characteristics.

This study was conducted to determine the changes in chicken sperm plasma membranes fluidity and polarity as lipid packing arrangement induced by cholesterol-loaded cyclodextrin (CLC) and 2-hydroxypropyl-ß-cyclodextrin (HBCD) and how sperm cryopreservation outcomes are improved by these changes. Treatment with 2 mg HBCD supported the highest (P < 0.01) percentage of viable spermatozoa compared with the control and CLCs groups after cryopreservation. The percentage of post-thaw progressive and rapid sperm motility was highest in 2 mg HBCD (P < 0.01). After thawing, sperm treated with 1 or 2 mg CLC showed the highest anisotropy at 5, 21, 25 and 40°C (P < 0.01). At 25°C, the lowest anisotropy was observed in the thawed semen from the control group. The highest value (P < 0.01) of generalized polarization (GP) (0.5) at 5°C was observed in the 1 mg CLC treated sample. After 2 h of incubation, the highest percentage of viable spermatozoa was observed in the HBCD group in relation to the other treatments (P < 0.01). Exposure to 1 mg or 2 mg of CLC significantly decreased the percentage of live spermatozoa after thawing (P < 0.01). In conclusion, HBCD appears to play a role in the modification of sperm membranes, increasing their fluidity and preventing them against membrane phase transition to gel, thus minimizing freezing-thaw sperm damage. HBCD treatment enhances chicken sperm viability and motility after cryopreservation and subsequent storage. This novel procedure may be useful for improving the technology for cryopreservation of fowl 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.

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.

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.

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.

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.