Domestic Cat Model Research Articles

Extracellular vesicles are involved in the paracrine communication between epithelial cells in different regions of the domestic cat epididymis†.

Sperm maturation depends on exposure to microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesized that epididymal extracellular vesicles could play a role in the process of maintaining microenvironments in different regions of the epididymis. Specifically, we tested whether the extracellular vesicles from different regions of the epididymis can ensure paracrine communication between cells in different segments. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to extracellular vesicles collected from upstream (i.e., caput) segments. Impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that extracellular vesicles from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1233 differentially expressed genes due to extracellular vesicle supplementation). Of note, expressions of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were influenced by the presence of extracellular vesicles. Together, our findings comprise the first report in any species of paracrine control of segmental gene regulation by epididymal extracellular vesicles. These results contribute to a better understanding of epididymis biology and could lead to strategies to enhance or suppress male fertility.

The study of the course of the infectious process in lungs of cats during experimental infection with SARS-CoV-2

Aim. To study and characterise the features of the course of coronavirus infection in a domestic cat model, as well as to assess the danger of SARSCoV-2 isolated in Russia for these animals based on the data obtained, with the possibility of extrapolating to wild felines.Material and Methods. For the study, model animals – Felis silvestris catus – were divided into 3 groups. The viral strain of SARS-Cov-2/human/RUS/Nsk-FRCFTM-1/2020 was cultivated on Vero E6 cell culture and then administered intranasally in a volume of 200 μl, 105TCID50/ml, to animals from the first and second groups. Model animals from the first group were euthanized and lung fragments were taken on 6–day post infection (d.p.i.), animals from the second group – on 14 d.p.i. The third group – the control group, was injected with saline in an equivalent volume. Histological sections colored with hematoxylin and eosin were investigated for pathomorphological changes in lung tissue using light microscopy.Results. According to the obtained results the pathomorphological picture in the lungs of experimental animals indicates the course of interstitial pneumonia for both 6 and 14 d.p.i. In addition, it has been determined that by 14 d.p.i. the growth of fibrous tissue in the lungs begins to be visualised, indicating the beginning of light regenerative processes.Conclusion. The study showed that cats are susceptible to the SARS-CoV-2 virus, and these model animals are characterized by the emergence of clinical manifestations and morphological patterns in the lungs, which correspond to interstitial pneumonia.

Transfer of Galectin-3-Binding Protein via Epididymal Extracellular Vesicles Promotes Sperm Fertilizing Ability and Developmental Potential in the Domestic Cat Model.

Key proteins transferred by epididymal extracellular vesicles (EVs) to the transiting sperm cells contribute to their centrosomal maturation and developmental potential. Although not reported in sperm cells yet, galectin-3-binding protein (LGALS3BP) is known to regulate centrosomal functions in somatic cells. Using the domestic cat model, the objectives of this study were to (1) detect the presence and characterize the transfer of LGALS3BP via EVs between the epididymis and the maturing sperm cells and (2) demonstrate the impact of LGALS3BP transfer on sperm fertilizing ability and developmental potential. Testicular tissues, epididymides, EVs, and spermatozoa were isolated from adult individuals. For the first time, this protein was detected in EVs secreted by the epididymal epithelium. The percentage of spermatozoa with LGALS3BP in the centrosome region increased as cells progressively incorporated EVs during the epididymal transit. When LGALS3BP was inhibited during in vitro fertilization with mature sperm cells, less fertilized oocytes and slower first cell cycles were observed. When the protein was inhibited in epididymal EVs prior to incubation with sperm cells, poor fertilization success further demonstrated the role of EVs in the transfer of LGALS3BP to the spermatozoa. The key roles of this protein could lead to new approaches to enhance or control fertility in clinical settings.

First Report of Successful Laser Warming for Frozen Gonadal Tissues and Oocytes in the Domestic Cat Model.

First Report of Successful Laser Warming for Frozen Gonadal Tissues and Oocytes in the Domestic Cat Model.

A domestic cat (Felis silvestris catus) model of triarchic psychopathy factors: Development and initial validation of the CAT-Tri+ questionnaire

We operationalised the triarchic model of psychopathy (boldness, meanness, and disinhibition) in domestic cats using a cat triarchic (CAT-Tri) questionnaire. In study 1 (n = 549), we identified candidate items for CAT-Tri scales using thematically analysed cat owner questionnaire responses. In study 2 (n = 1463), owners completed a questionnaire battery; the preliminary CAT-Tri questionnaire, Feline Five, and Cat-Owner Relationship Subscales. In study 3 (n = 30), associations between feline daily activity and Cat-Tri scales were investigated. A five-factor cat triarchic plus (CAT-Tri+) solution emerged: boldness, disinhibition, meanness, pet-unfriendliness, and human-unfriendliness. Disinhibition and pet-unfriendliness predicted a higher quality cat-owner relationship; meanness and boldness predicted a lower quality relationship. Findings provide insight into the structure of triarchic psychopathy in cats.

A large animal model of RDH5-associated retinopathy recapitulates important features of the human phenotype.

Pathogenic variants in retinol dehydrogenase 5 (RDH5) attenuate supply of 11-cis-retinal to photoreceptors leading to a range of clinical phenotypes including night blindness because of markedly slowed rod dark adaptation and in some patients, macular atrophy. Current animal models (such as Rdh5-/- mice) fail to recapitulate the functional or degenerative phenotype. Addressing this need for a relevant animal model we present a new domestic cat model with a loss-of-function missense mutation in RDH5 (c.542G > T; p.Gly181Val). As with patients, affected cats have a marked delay in recovery of dark adaptation. In addition, the cats develop a degeneration of the area centralis (equivalent to the human macula). This recapitulates the development of macular atrophy that is reported in a subset of patients with RDH5 mutations and is shown in this paper in seven patients with biallelic RDH5 mutations. There is notable variability in the age at onset of the area centralis changes in the cat, with most developing changes as juveniles but some not showing changes over the first few years of age. There is similar variability in development of macular atrophy in patients and while age is a risk factor, it is hypothesized that genetic modifying loci influence disease severity, and we suspect the same is true in the cat model. This novel cat model provides opportunities to improve molecular understanding of macular atrophy and test therapeutic interventions for RDH5-associated retinopathies.

Age estimation using methylation-sensitive high-resolution melting (MS-HRM) in both healthy felines and those with chronic kidney disease

Age is an important ecological tool in wildlife conservation. However, it is difficult to estimate in most animals, including felines—most of whom are endangered. Here, we developed the first DNA methylation-based age-estimation technique—as an alternative to current age-estimation methods—for two feline species that share a relatively long genetic distance with each other: domestic cat (Felis catus; 79 blood samples) and an endangered Panthera, the snow leopard (Panthera uncia; 11 blood samples). We measured the methylation rates of two gene regions using methylation-sensitive high-resolution melting (MS-HRM). Domestic cat age was estimated with a mean absolute deviation (MAD) of 3.83 years. Health conditions influenced accuracy of the model. Specifically, the models built on cats with chronic kidney disease (CKD) had lower accuracy than those built on healthy cats. The snow leopard-specific model (i.e. the model that resets the model settings for snow leopards) had a better accuracy (MAD = 2.10 years) than that obtained on using the domestic cat model directly. This implies that our markers could be utilised across species, although changing the model settings when targeting different species could lead to better estimation accuracy. The snow leopard-specific model also successfully distinguished between sexually immature and mature individuals.

Exposure to epididymal extracellular vesicles enhances immature sperm function and sustains vitality of cryopreserved spermatozoa in the domestic cat model.

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer key factors to maturing spermatozoa. Using an in vitro system previously developed in our laboratory, the objective was to (1) characterize the impact of EV exposure on the fertilizing ability and developmental potential of immature sperm cells from the caput epididymidis and (2) examine the benefit of EV exposure to restore vitality of mature spermatozoa from the cauda epididymidis after freezing-thawing. EVs were isolated from entire epididymides and collected into pellets via ultracentrifugation. Immature spermatozoa from adult cats were isolated from the caput epididymis and incubated with EVs prior to in vitro fertilization. Similarly, mature spermatozoa were isolated from the cauda segment and cryopreserved prior to EV exposure and subsequent analysis of motility and developmental potential after fertilization. EV exposure did not affect the percentage of caput sperm penetration; however, it improved the fertilizing ability (faster pronuclear apposition) and the developmental potential (higher proportions of morula-blastocysts) of those immature sperm cells. While EV exposure was beneficial to the frozen-thawed sperm motility, it did not significantly improve the fertilizing ability and the developmental potential. Epididymal EVs contain multiple factors contributing to immature sperm function, specifically enhancing the ability to complete a faster pronuclear apposition with subsequently improved early embryonic development. Supplementation was also beneficial to the motility of spermatozoa that had undergone cryopreservation. Those new findings could lead to new options for male fertility treatment in animal models and humans.

Oocyte Meiotic Competence in the Domestic Cat Model: Novel Roles for Nuclear Proteins BRD2 and NPM1.

To participate in fertilization and embryo development, oocytes stored within the mammalian female ovary must resume meiosis as they are arrested in meiotic prophase I. This ability to resume meiosis, known as meiotic competence, requires the tight regulation of cellular metabolism and chromatin configuration. Previously, we identified nuclear proteins associated with the transition from the pre-antral to the antral follicular stage, the time at which oocytes gain meiotic competence. In this study, the objective was to specifically investigate three candidate nuclear factors: bromodomain containing protein 2 (BRD2), nucleophosmin 1 (NPM1), and asparaginase-like 1 (ASRGL1). Although these three factors have been implicated with folliculogenesis or reproductive pathologies, their requirement during oocyte maturation is unproven in any system. Experiments were conducted using different stages of oocytes isolated from adult cat ovaries. The presence of candidate factors in developing oocytes was confirmed by immunostaining. While BRD2 and ASRGL1 protein increased between pre-antral and the antral stages, changes in NPM1 protein levels between stages were not observed. Using protein inhibition experiments, we found that most BRD2 or NPM1-inhibited oocytes were incapable of participating in fertilization or embryo development. Further exploration revealed that inhibition of BRD2 and NPM-1 in cumulus-oocyte-complexes prevented oocytes from maturing to the metaphase II stage. Rather, they remained at the germinal vesicle stage or arrested shortly after meiotic resumption. We therefore have identified novel factors playing critical roles in domestic cat oocyte meiotic competence. The identification of these factors will contribute to improvement of domestic cat assisted reproduction and could serve as biomarkers of meiotically competent oocytes in other species.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

Novel Proteomic Profiling of Epididymal Extracellular Vesicles in the Domestic Cat Reveals Proteins Related to Sequential Sperm Maturation with Differences Observed between Normospermic and Teratospermic Individuals

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals.

Initial response of ovarian tissue transcriptome to vitrification or microwave-assisted dehydration in the domestic cat model

BackgroundLong term preservation of living ovarian tissues is a critical approach in human reproductive medicine as well as in the conservation of rare animal genotypes. Compared to single cell preservation, optimization of protocols for tissues is highly complex because of the diversity of cells responding differently to non-physiological conditions. Using the prepubertal domestic cat as a model, the objective was to study immediate effects of vitrification or microwave-assisted dehydration on the global transcriptome dynamics in the ovarian cortex. RNA sequencing was performed on ovarian tissues (n = 6 individuals) from different conditions: fresh tissue after dissection (F), vitrified/warmed tissue (V), tissue dehydrated for 5 min (D5) or 10 min (D10) followed by rehydration. Differential gene expression analysis was performed for comparison pairs V vs. F, D10 vs. F, D5 vs. F and D10 vs. D5, and networks were built based on results of functional enrichment and in silico protein-protein interactions.ResultsThe impact of the vitrification protocol was already measurable within 20 min after warming and involved upregulation of the expression of seven mitochondrial DNA genes related to mitochondrial respiration. The analysis of D10 vs. F revealed, 30 min after rehydration, major downregulation of gene expression with enrichment of in silico interacting genes in Ras, Rap1, PI3K-Akt and MAPK signaling pathways. However, comparison of D5 vs. F showed negligible effects of the shorter dehydration protocol with two genes enriched in Ras signaling. Comparison of D10 vs. D5 showed downregulation of only seven genes. Vitrification and dehydration protocols mainly changed the expression of different genes and functional terms, but some of the differentially expressed genes formed a major in silico protein-protein interaction cluster enriched for mitochondrial respiration and Ras/MAPK signaling pathways.ConclusionsOur results showed, for the first time, different effects of vitrification and microwave-assisted dehydration protocols on the global transcriptome of the ovarian cortex (using the domestic cat as a biomedical model). Acquired data and networks built on the basis of differentially expressed genes (1) can help to better understand stress responses to non-physiological stresses and (2) can be used as directions for future preservation protocol optimizations.

Influence of Microwave-Assisted Drying on Structural Integrity and Viability of Testicular Tissues from Adult and Prepubertal Domestic Cats.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.

Resveratrol treatment during maturation enhances developmental competence of oocytes after prolonged ovary storage at 4 °C in the domestic cat model

Resveratrol (Resv; 3,4,5-trihydroxy-trans-stilbene) is a phytoalexin with antioxidant activity that modulates redox homeostasis in oocytes and improves in vitro embryo production. Cold storage of cat ovaries for a period longer than 24 h alters oxidative status of oocytes after in vitro maturation and reduces their developmental competence. The aim of this study was to evaluate the effect of resveratrol supplementation to the maturation medium on embryo development of oocytes after storage of domestic cat ovaries at 4 °C for 24 h or 48 h. Cumulus–oocyte complexes (COCs) were recovered from ovaries of domestic queens and cultured in maturation medium supplemented with (+) or without (−) 5 μM resveratrol for 24 h. COCs collected from fresh ovaries were matured in vitro (IVM) in standard conditions as control. After IVM, oocytes were in vitro fertilized (IVF) and presumptive zygotes cultured for 7 days. Oocyte nuclear maturation, reactive oxygen species (ROS) and glutathione (GSH) levels as well as cleavage, blastocyst formation and blastocyst cell number were determined. There were no differences in the maturation rates of oocytes between the control and stored groups, irrespective of resveratrol supplementation. Resveratrol treatment during IVM significantly increased the level of GSH and reduced the level of ROS of oocytes recovered from ovaries stored for 48 h as compared to the non-treated group (48 h-). The rate of blastocyst formation from oocytes recovered from ovaries after 48 h storage that underwent IVM with resveratrol was higher (P < 0.05) than that of oocytes matured without resveratrol and similar to that of control oocytes. Resveratrol treatment increased (P < 0.05) cell number in blastocysts from 24 h + and 48 h + groups as compared to their respective counterparts. In conclusion, our results demonstrated that resveratrol supplementation during IVM can reverse the adverse effect of oxidative stress on oocytes, and enhances embryo development after ovary storage at 4 °C for 48 h. These results may provide a basis for improving culture conditions and extend the possibility of storage of cat ovaries for more than 24 h thus ensuring successful in vitro embryo production.

78 Radio electric asymmetric conveyer treatment during prolonged cold storage of ovaries enhances developmental competence of oocytes in the domestic cat model

Hypothermic storage (4°C) of ovaries for long-distance transport holds great potential to expand access to fertility preservation in animals and humans (Duncan et al. 2016 Reproduction 152, 201-210). However, storage for prolonged periods (over 24h) leads to structural/functional changes in the ovarian tissue with a critical reduction of follicular viability and oocyte quality (Piras et al. 2018 Reprod. Biol. Endocrinol. 10, 16-76; Isachenko et al. 2009 Fertil. Steril. 91, 1556-1559). The radio electric asymmetric conveyer (REAC) is a novel technology platform for neuro and bio-modulation that optimize the ion fluxes and the mechanisms driving cellular asymmetry and polarization in biological structures (Maioli et al. 2016 Sci. Rep. 6, 28682). The REAC has already proved to be effective in promoting cell differentiation and reprogramming and to counteract the biological mechanisms linked to aging and the degenerative process (Berlinguer et al. 2017 Reprod. Biol. Endocrinol. 15, 11; Maioli et al. 2014 Age 36, 9-20). In the present study, the domestic cat was used as an experimental model to evaluate the effect of REAC treatment during ovary storage at 4°C for 48h on invitro developmental ability of oocytes retrieved from antral follicles. Ovaries harvested from healthy domestic queens during ovariectomy were randomly assigned to the REAC-treated (R: n=13) and untreated (C: n=13) groups. In detail, ovaries were maintained in 4mL of phosphate-buffered saline at 4°C for 48h. The REAC device was set at 2.4 GHz, and its conveyer electrodes were immersed into the phosphate-buffered saline. After 48h, ovaries were sliced to release cumulus-oocyte complexes, which were selected according to their morphological characteristics (Johnston et al. 1991 Biol. Reprod. 45, 898-906) for IVM (R: n=130; C: n=133). Matured oocytes were fertilised (IVF) with frozen-thawed epididymal spermatozoa and presumptive zygote were invitro cultured (IVC) for 7 days. On Day 2 and Day 7 of IVC, respectively, the number of embryos cleaved and developing to the blastocyst stage was determined. The IVM, IVF, and IVC were performed according to the procedure of Piras et al. (2018 Reprod. Biol. End. 16, 76). Data were analysed by chi-square test with STATA\IC 11.0. Maturation rate of oocytes did not differ between groups (R: n=59/130, 45.4%; C: n=66/133, 49.6%). Cleavage rate was higher (P&amp;lt;0.05) in the R group (n=34/59, 57.6%) compared with the C group (n=25/66, 37.9%). The percentages of blastocyst formation relative to the number of cleaved embryos (R: n=12/34, 35.3%; C: n=3/25 12.0%) and to the total number of MII oocytes (R: n=12/59, 20.4%; C: n=3/66, 4.5%) increased (P&amp;lt;0.05) after REAC treatment compared with the untreated counterpart. In conclusion, REAC treatment during cold storage of cat ovaries for 48h positively affected the quality of oocytes as assessed by invitro embryo production outcome. The REAC technology could provide a useful approach for the optimization of ovarian tissue transport conditions for fertility preservation especially for endangered species and patients with fertility-threatening conditions.

Activation of adenosine monophosphate-activated protein kinase (AMPK) enhances energy metabolism, motility, and fertilizing ability of cryopreserved spermatozoa in domestic cat model.

Increasing intracellular energy storage by chemically activating adenosine monophosphate-activated protein kinase (AMPKα) prior to sperm cryopreservation may improve post-thawed sperm function. Using the domestic cat as a biomedical model, the objectives were to (1) confirm the expression of AMPKα and its regulatory kinases in epididymal spermatozoa and (2) assess the influence of AMPK activator, 5'-aminoimidasole-4-carboxamide-1-β-d-ribofuranoside (AICAR) on epididymal sperm function before and after cryopreservation. In study I, sperm samples of different qualities were obtained from cauda epididymides of domestic cats and evaluated for AMPKα expression. In study II, epididymal spermatozoa were equilibrated for either 30 or 60min in the presence of 0 (control), 0.5, 2.0, and 5.0mM AICAR and sperm functions were assessed before and after cryopreservation. In study III, epididymal spermatozoa were treated as in study II and evaluated for AMPKα signaling protein expressions (phospho-AMPKα Thr172 and GLUT1) as well as ATP levels. AMPKα protein expression was higher in high-motility vs poor-motility samples. Thirty-minute equilibration with 0.5mM AICAR improved motion characteristics and fertilizing ability of cryopreserved sperm to the control. Increased expressions of phospho-AMPKα Thr172 and GLUT1 as well as intracellular ATP level were confirmed in sperm samples equilibrated with 0.5 or 2.0mM AICAR for 30min. Presence and role of AMPKα protein in cat regulating sperm function were demonstrated before and after cryopreservation. Findings could be used to potentially enhance cryopreserved sperm function in sub-fertile men.

Oocytes vitribanking in the domestic cat model: Developmental competence in 3d culture

Oocytes vitribanking in the domestic cat model: Developmental competence in 3d culture

Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model.

Understanding critical roles of warming and reanimation is critical to improve the survival of vitrified testicular tissue in domestic cats. The objective was to study structural and functional properties of testicular tissues from prepubertal domestic cats after standard vitrification followed by two warming protocols (directly at 37°C or with a 5-second pre-exposure to 50°C) and three reanimation time points (immediately, 24 h and 5 days post-warming). In Experiment 1, tissues were evaluated for histo-morphology and mitochondrial activity immediately or 24 h after warming protocols. In Experiment 2, cell viability, DNA fragmentation, and germ cell composition were assessed immediately, 24 h, or 5 days after optimal warming. Preservation of seminiferous tubule structure was better using warming at 50°C for five seconds, and survival of somatic as well as germinal cells was higher compared to direct warming at 37°C for one minute. Short term in vitro culture (for reanimation) also proved that cellular composition and functionality were better preserved when warmed for a short time at 50°C. Collective data showed that short warming at 50°C led to better quality of seminiferous tubule structure and cell composition after vitrification and short-term culture. In addition, data suggest clear directions to further understand and optimize testicular tissue survival after fertility preservation procedures.

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4\xa0\xb0C in the domestic cat model

BackgroundStorage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model.MethodsOvaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/β tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes.ResultsThe results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage.ConclusionsStorage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers.

Key factors enhancing sperm fertilizing ability are transferred from the epididymis to the spermatozoa via epididymosomes in the domestic cat model.

Spermatozoa undergo critical changes in structure and function during the epididymal transit. Our previous studies in the domestic cat demonstrated that incidence of cenexin-a key protein involved in the centrosomal maturation-progressively increases in sperm cells from caput to cauda epididymidis. The objectives of the study were to (1) characterize mechanisms involved in transferring key factors-using the cenexin as a marker-between the epididymis and maturing sperm cells and (2) demonstrate the impact of such mechanisms on the acquisition of functional properties by spermatozoa. Epididymides were dissected from adult cat testes to assess the presence and localization of cenexin in testicular tissues and each epididymal segment (caput, corpus, and cauda) via immunofluorescence, Western blot, and mass spectrometry. Results showed that tissues, luminal fluid, and isolated epididymosomes from each segment contained cenexin. Co-incubation of immature sperm cells for 3h with luminal fluid or epididymosomes followed by immunostaining revealed that percentages of sperm cells containing cenexin significantly increased in samples co-incubated with epididymosome suspensions. Additionally, epididymosome co-incubation with immature spermatozoa resulted in sustained motility compared to untreated spermatozoa while there was no significant effect on acrosome integrity. Taken together, these results suggest that epididymosomes play a critical role in epididymal sperm maturation and could be ideal vehicles to assist in the enhancement or suppression of male fertility.