Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

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.

Morphokinetic changes and apoptotic cell death in vitrified and non-vitrified in vitro-produced ovine embryos.

This article addresses morphokinetic changes and the extent of apoptosis in vitrified and non-vitrified in vitro-derived ovine blastocysts. Cumulus-oocyte complexes were collected after ovarian scarification obtain after slaughter and 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 co-incubated with thawed ram semen (IVF) for 19 h.Embryo development was monitored with the aid of the Primo Vision Time-Lapse (TL) system. Twenty-five out of thirty-one ovine blastocysts that were vitrified using the Cryotop system at the early blastulation stage of development subsequently re-expanded. Both the vitrified (n = 25) and non-vitrified (control group: n = 28) blastocysts were examined for detection of apoptosis (TUNEL assay) and total blastomere counts at the time they attained the expanded blastocyst stage. Blastocyst formation occurred earlier in non-vitrified than in vitrified ovine embryos (147:49 ± 20:23 compared with 156:46 ± 19:24; hours:minutes post-insemination; mean ± SD; P < 0.05). The average number of blastocyst collapses was greater (2.45 ± 1.64 compared with 1.45 ± 1.64), but the number of weak contractions was less for vitrified than non-vitrified ovine blastocysts (P < 0.05). The mean number of blastomeres was greater (131.8 ± 38.6 compared with 91.5 ± 18.3; P < 0.05) while the number of TUNEL-positive cells (4.4 ± 1.6 compared with 6.3 ± 2.3) and apoptotic index (3.4 ± 1.2 % compared with 6.9 ± 2.6 %) were less (P < 0.05) in non-vitrified compared with vitrified blastocysts. Vitrification of ovine embryos was associated with a delayed blastocyst formation, greater numbers of apoptotic cells, significant reduction in the number of blastomeres, and higher/lower incidence of blastocyst collapse/weak contractions.

Prevalence of Sex-Related Chromosomal Abnormalities in a Large Cohort of Spanish Purebred Horses.

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.

Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes.

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.

Morphokinetic changes in vitrified and non-vitrified in vitro-derived ovine embryos.

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.

Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence in vitro: a preliminary assessment using time-lapse imaging.

The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm2, respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.

The Use of Commercial Microvolume Techniques for Feline Oocyte Vitrification.

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.

Characteristics of diluted-stored and post-thawed semen of Hutsul stallions.

The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, using a CASA system. There were no differences in sperm motility (P = 0.3372) or morphology between the groups, although the progressive motility was higher in thawed semen (P = 0.0151), while the sperm vitality was higher in diluted-stored semen (P = 0.00517). This study demonstrates that semen from Hutsul horses is suitable for cryopreservation, thus supporting the creation of a sperm bank as a genetic reserve for representatives of this breed.

Application of the FISH Technique to Visualize Sex Chromosomes in Domestic Cat Spermatozoa.

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.