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The evaluation of oocytes and zygotes, based on their size, shape and morphology, is a valuable tool for predicting subsequent embryo development. While this assessment is non-invasive and made possible with time-lapse monitoring systems, not all the assessment criteria used for zygotes with pale cytoplasm can be used for domestic cat zygotes, which have dark cytoplasm. In this study, feline presumptive zygotes were evaluated for shape, size, and morphology. Measurements were also made of the diameter of the entire zygote, its cytoplasm, and the zona pellucida. Differences in the dataset were assessed using the generalized linear model (GLM) procedure. While there was no relationship between a combination of the tested parameters with the potential for cleavage, blastocyst development, and hatching, the parameters of the shape and size of the entire oocyte, and of the zona pellucida, were related to the development potential. The results presented in this study indicate that the assessment procedure for human zygotes has to be adjusted to be used in the cat model, however the relationship between measurements of the diameter of presumptive feline zygotes and the thickness of zona pellucida with their developmental potential deserves further investigation to optimize assessment of cat presumptive zygotes. [ABSTRACT FROM AUTHOR]

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24–38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART. • We observed the internalization of ffEVs by both equine cumulus cells and oocytes. • ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted oocytes compared to the control group. • Supplementation of culture media with ffEVs from small follicles can improve the IVM rate of mare oocytes. [ABSTRACT FROM AUTHOR]

Despite extensive research on the cryopreservation of stallion semen, the process still requires improvement. Several diluent additives have been developed to overcome cryopreservation issues, however, semen from valuable individuals often shows poor quality after thawing. Mesenchymal stem cells (MSCs) are regarded as factors that may play an important role in the regenerative process of sperm by secreting extracellular vesicles (EVs) that are actively involved in intercellular communication, tissue repair and cell membrane maintenance. The study aimed to determine the effect of the addition of EV-MSCs obtained from equine adipose tissue on various parameters of cryopreserved stallion semen. All cells met the minimum criteria established by the ISCT, being a characteristic surface profile of the presence of CD90 and lack of MHC II, differentiation into three mesodermal lineages, and adherence. EVs were collected from the expanded cells by ultracentrifugation, and their characteristics were confirmed using nanoparticle tracking analysis (NTA) and flow cytometry to detect CD63 and CD81 antigens. Semen was collected from 11 stallions of the Hucul breed, and each parameter of sperm quality with (25 µg/ml, 50 µg/ml) and without EV addition (motility, morphology and viability) was analyzed. The results showed statistically significant differences in sperm motility, progressive movement and viability following EV treatment. While the amplitude of lateral head displacement (ALH) and morphology parameters did not change, the addition of EVs does not affect these sperm features. The results provide an initial insight into the role of EVs in the maintenance and regeneration of horse sperm after cryopreservation-induced shock. [ABSTRACT FROM AUTHOR]

The objective of this research was to assess the viability and developmental potential of feline oocytes following in vitro maturation (IVM), vitrification, and post-warming incubation with resveratrol. In the first experiment, warmed oocytes were incubated with 0.2 μM, 2 μM, or 20 μM resveratrol for 2 h. Oocytes treated with 0.2 μM resveratrol had the highest viability (68.89 %), as assessed by fluorescein diacetate and ethidium bromide staining, while higher concentrations were associated with diminished oocyte viability. In the second experiment, the warmed oocytes were inseminated following the 2-h incubation with the three concentrations of resveratrol. The presumptive zygotes were then maintained in culture and their development evaluated. The highest cleavage rate was observed when the oocytes had been incubated with 0.2 μM resveratrol (88.34 %), which was higher than for the control group (without resveratrol (75 %)). Moreover, this concentration of resveratrol also augmented the blastocyst formation rate. While the vitrification of oocytes often results in diminished developmental potential in the ensuing embryos, attributed to cryopreservation-induced injury, the utilization of low concentrations of resveratrol enhances the procedure's efficacy. • Resveratrol treatment is a possible countermeasure against cryopreservation‐induced mitochondrial damage. • Resveratrol improved the viability of oocytes after vitrification. • Oocytes vitrified and incubated with resveratrol have good developmental competence after IVF. • Resveratrol improves the quality of oocytes vitrified in minimal volumes. [ABSTRACT FROM AUTHOR]

Simple Summary: This study was conducted to compare the minimum volume vitrification method using material from female felids to protect them in wild felid conservation programs. In the experiment, the three most popular techniques for oocyte vitrification were compared in terms of their survival and developmental competence. The results show that using this method as an emergency protocol is essential to protecting the gametes of wild felids threatened with extinction in conservation programs based on assisted reproductive techniques (ART). This project aimed to compare the three most popular commercial oocyte vitrification techniques to determine their suitability for the vitrification of felid germlines in rescue and conservation programs. The present study aimed to determine the viability and developmental competence of feline oocytes after IVM and vitrification using a commercial vitrification method. In the first experiment, oocytes were vitrified after in vitro maturation (IVM) using the Kitazato, Cryotech, and Vitrolife methods. The oocytes were stained with fluorescein diacetate and ethidium bromide to evaluate their viability. The differences between Vitrolife and the control, Cryotech and Kitazato were statistically significant (p < 0.05), and between the control and Kitazato, were highly significant (p < 0.01). There were no significant differences between the control and Cryotech, Vitrolife and Cryotech, or Kitazato and Vitrolife. In the second part of the experiment, oocytes, after IVM and vitrification using three commercial methods, were subjected to fertilization. After vitrification, IVF was performed. We observed 35% of embryonic divisions in the group where Vitrolife and Kitazato media were used and 45% in the control group. In the presented experiment, vitrification with Vitrolife media gave slightly better results for survival and fertilization, while in the case of emergency protocol vitrification, all of the above methods may be useful to protect material derived from valuable wild felids. [ABSTRACT FROM AUTHOR]

Simple Summary: The study was conducted with the aim of visualizing sex chromosomes in domestic cat sperm. Our research was carried out using fluorescence in situ hybridization (FISH) technique and the microscopic analysis showed the presence of X and Y chromosomes in domestic cat spermatozoa. The procedure used for sperm decondensation and FISH is adequate to visualize chromosomes in domestic cat sperm. Fluorescence in situ hybridization is a molecular cytogenetics technique that enables the visualization of chromosomes in cells via fluorescently labeled molecular probes specific to selected chromosomes. Despite difficulties in carrying out the FISH technique on sperm, related to the need for proper nuclear chromatin decondensation, this technique has already been used to visualize chromosomes in human, mouse, cattle, swine, horse, and dog spermatozoa. Until now, FISH has not been performed on domestic cat sperm; therefore, the aim of this study was to visualize sex chromosomes in domestic cat sperm. The results showed the presence of X and Y chromosomes in feline spermatozoa. The procedure used for sperm decondensation and fluorescence in situ hybridization was adequate to visualize chromosomes in domestic cat spermatozoa and, in the future, it may be used to determine the degree of chromosomal abnormalities in these gametes. [ABSTRACT FROM AUTHOR]

The present experiment employed time-lapse (TL) imaging to assess the effects of vitrification on the development of ovine blastocysts and to see if the timing of blastocyst formation and expansion was correlated with the numbers of embryo- and trophoblasts determined through differential staining of the expanded blastocysts. Ovaries were obtained after slaughter from cycling (October–March) Polish Longwool ewes aged 1–3 years and cumulus-oocyte complexes were collected by ovarian scarification. In vitro maturation was performed in TCM 199 medium supplemented with Earle's Salt, 10% of FBS, and 5 μg/mL of LH/FSH at 38 °C for 24 h. After maturation, the oocytes were incubated with thawed ram semen (IVF) for 19 h and all presumptive zygotes were transferred to a 16-well dish containing Cult medium for monitoring with the Primo Vision TL system. A portion of ovine embryos were vitrified (Cryotop system) at the early cavitation stage and TL observations of warmed (n = 30) and non-vitrified (n = 32) blastocysts continued until the attainment of the expanded blastocyst stage, at which point they were differentially stained with bisbenzimide and propidium iodide for microscopic enumeration of embryoblasts (inner cell mass blastomeres) and trophoblast-cells. There were no statistically significant differences in the timing of blastulation (tB) and formation of expanded blastocysts (tEB) between vitrified and non-vitrified ovine embryos, but non-vitrified blastocysts exceeded (P < 0.05) their vitrified and warmed counterparts in the mean number of embryo- and trophoblasts. In addition, the number of trophoblasts was negatively and moderately correlated with tB and tEB, for both vitrified and non-vitrified embryos. It can be concluded that even though vitrification of ovine embryos is associated with a significant reduction in the number of blastomeres, the rate of blastocyst development remains closely linked to the numbers of trophoblastic cells. • Vitrification reduced the number of trophoblasts cells in warmed ovine embryos. • Vitrification of ovine embryos is associated with a significant reduction in the number of blastomere. • There is relationship between the timing of blastocyst formation and expansion and the number of trophoblasts. • Embryonic morphokinetics are determinants and predictors of in vitro fertilization outcomes. • The rate of trophoblast expansion is a significant potential marker of embryo quality. [ABSTRACT FROM AUTHOR]

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus–oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups (p < 0.01), but decreased in Ex groups (p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation. [ABSTRACT FROM AUTHOR]

Simple Summary: Horses are well known for the increased number of individuals carrying chromosomal abnormalities related to the sex pair, which have been identified as a major cause of idiopathic infertility. However, large-scale populational studies evaluating the occurrence of these chromosomal aberrations in commercial or wild populations are extremely scarce. We, therefore, performed a cytogenetic analysis on a large dataset of 25,237 individuals, gathered over a period of 24 months, using a two-step genomic-based diagnostic methodology. We first screened the entire population, analyzing the results of short tandem repeats parentage testing to determine individuals showing abnormal results. Thereafter, the positive samples, together with the individuals showing morphological abnormalities in the reproductive tract, were reanalyzed using a single nucleotide polymorphism (SNP)-based procedure to determine the occurrence of chromosomal abnormalities. Our results showed that the overall prevalence of individuals carrying chromosomal alterations was close to 0.05%, with blood chimerism and 64,XY sex-reversed mares the most common type of aberrations detected. In addition, one case of Turner and one of Klinefelter syndrome, as well as a small number of individuals carrying complex karyotypes, were also detected. However, these results should be taken with caution since the occurrence of X chromosome monosomy, a sex-related chromosomal aberration commonly reported in mares, cannot be screened using the methodology employed in this study. To our knowledge, this is the largest study performed aimed at determining the prevalence of the most important chromosomal abnormalities in the domestic horse. Chromosomal abnormalities are largely associated with fertility impairments in the domestic horse. To date, over 600 cases of individuals carrying abnormal chromosome complements have been reported, making the domestic horse the species with the highest prevalence. However, studies analyzing the prevalence of chromosomal diseases in whole populations are scarce. We, therefore, employed a two-step molecular tool to screen and diagnose chromosomal abnormalities in a large population of 25,237 Pura Raza Español horses. Individuals were first screened using short tandem repeats parentage testing results and phenotypic evaluations. Those animals showing results suggesting chromosomal abnormalities were re-tested using a single nucleotide polymorphism (SNP)-based diagnostic methodology to accurately determine the chromosomal complements. Thirteen individuals showed a positive screening, all of which were diagnosed as chromosomally abnormal, including five 64,XY mares with sex development disorders (DSD) and four cases of blood chimerism (two male/female and two female/female cases). In addition, we detected one Turner and one Klinefelter syndrome and two individuals carrying complex karyotypes. The overall prevalence in the entire population was ~0.05%, with the prevalence of 64,XY DSD and blood chimerism ~0.02% and ~0.016%, respectively. However, the overall results should be taken with caution since the individuals carrying Turner syndrome (in full (63,X) or mosaic (mos 63,X/64,XX) forms) cannot be detected due to limitations in the methodology employed. Finally, the lack of agreement between populational studies performed using karyotyping or molecular methods is discussed. To our knowledge, this is the largest populational study performed evaluating the prevalence of the most common chromosomal abnormalities in the domestic horse. [ABSTRACT FROM AUTHOR]