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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 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]

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]

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. [ABSTRACT FROM AUTHOR]

Routine semen preparation techniques are not always satisfactory for all stallions. Some stallions, despite poor semen parameters, are valuable for breeders who want to produce equally worthwhile offspring. In recent years, advanced sperm selection methods such as the microflow technique have been developed and successfully applied to humans. The aim of this study was to adapt the microflow technique for selection of stallion semen, focusing on their predicted benefits for sperm quality. The experiment was performed with semen of 6 stallions, which after combining in equal volumes created pooled research sample. Semen was left for 24 h in order to reduce the viability and motility of spermatozoa and divided into 3 parts, the control and the two research groups separated using ZyMōt Devices with two different incubation times (30 and 45 minutes). For each group, the basic sperm characteristics were assessed by SCA/CASA system, and fragmentation was evaluated using the TUNEL technique. The Z-score test was used for statistical analysis. In the sample incubated for 30 min in ZyMōt Devices, sperm motility increased from 28% to 52.15% (p=0.001), including sperm with progressive motility which increased from 8.47% to 41.91% (p<0.00001). After extending the incubation time for 45 minutes, these parameters were even higher: motility 62.43% (p<0.00001), progressivemovement 55.33% (p<0.00001). It should be noted, however, that in the case of both research samples, the concentration of sperm decreased from 804.68 M/ml in the control to 32.35 M/ml (p<0.00001) after 30 min incubation and 40.37 M/ml after 45 min incubation, respectively (p<0.0001). Sperm selection did not affect vitality. The TUNEL technique showed a very low percentage of spermatozoa with apoptosis in the semen fraction after selection at both incubation times. In summary, the microflow technique is an effective method for selection of stallion sperm with high motility, progressive movement and low DNA sperm fragmentation, with unchanged vitality parameters. Comparing the two incubation times, it was found that extending the incubation time to 45 minutes improved sperm concentration and spermatozoa with progressive motility. Nevertheless, prolonged incubation caused an increase in apoptotic spermatozoa that penetrate through a membrane with microchannels. It should be considered that sperm selection by microflow technique drastically reduces sperm concentration, which in practice translates into a significant reduction in the number of insemination doses which will increase costs significantly. However, this method can be successfully used for sperm selection for the ICSI procedure. [ABSTRACT FROM AUTHOR]

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.