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With the exception of the domestic cat, all felid species (Felidae) are currently threatened with extinction in their natural habitat. To develop effective and optimal wild cat conservation programmes with assisted reproductive technology (ART) it is necessary to combine advances from different disciplines of science, starting from the biology of the species, through research into the population and habitat, assisted reproductive technologies, establishment of gene banks, developing bioinformatic systems, and ending with biodiversity and endangered species management. In the last few years knowledge of felid reproduction has expanded considerably thanks to comparative studies utilising the domestic cat as a research model for endangered wild cats. Basic reproductive techniques utilised in both domestic cat breeding and rescuing wild felid populations that are threatened with extinction include semen collection and cryopreservation, artificial insemination, oocyte collection, in vitro maturation, in vitro fertilisation, somatic cloning, and embryo transfer. The main directions in which assisted reproductive technologies are being developed in wild cat conservation implementations and the contribution of Polish research centres in advancing these methods are presented.

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]

Using passive hair traps to obtain genetic material from wild mammals is an effective form of testing the abundance of animal populations. Based on genetic analysis of collected hair, it is possible to estimate the number and sex of individuals present in a given area. The aim of this study was to confirm the success of the method of collecting hair from beavers in a non-invasive way, as well as to determine the effectiveness of using passive hair traps as sampling tools that can be applied in different regions. The study was carried out in the area of Stobrawa Landscape Park (southwestern Poland) between December 2017 and May 2018. For 17 control days, 12 samples were obtained, which gave the result of 0.7 samples per control day. This study shows that the proposed method, due to its simplicity and efficiency, could be a cost-effective way of collecting hair from free-ranging beavers without the need for capturing individuals. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

During the past decade the need for Artificial Reproductive Techniques in felids has greatly increased. Mostly, this is a result of growing expectations that these techniques may be applied in conservation biology and thereby contribute to saving wild felids from extinction. In this article we describe three most common methods of obtaining embryos in vitro in the domestic cat and its wild relatives: classic in vitro fertilisation, in vitro fertilisation by intracytoplasmic sperm injection and somatic cell nuclear transfer. Each of the methods provides a cleavage rate of around 50% and approx. 20% of embryos develop to the blastocyst stage. After the transfer of embryos produced by these methods, scientists obtained living offspring of the domestic cat, as well as several wild cats: the tiger, serval, fishing cat, caracal, ocelot, wild cat, sand cat, black-footed cat and the oncilla. These successes, in spite of the low efficiency of the discussed methods, are promising and suggest that biotechniques of reproduction will be valuable tools in the protection of wild species. Somatic cell nuclear transfer will allow to sustain the narrow gene pool in the critically endangered felids. For these reasons it is necessary to conduct further research on the optimization of artificial reproduction techniques in cats [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]

Non-invasive genetic sampling has been increasingly used in studies of wild animal populations, although poor quality and quantity of the DNA samples can require repeated amplifications to obtain reliable genetic profiles of each individuals. Consequently, we performed a pilot study to test whether using wire hair traps and DNA genotyping could be a useful tool in estimating the abundance of a low-density population of the European badger (Meles meles) in Poland. We located thirteen badger main setts in different regions of the Stobrawa Landscape Park (south-western Poland) and collected hair DNA samples using non-invasive hair-trapping devices. Traps were placed above the setts' entrances and on badger paths. Sampling was performed during two seasons: autumn (15 October to 22 November 2010) and spring (28 April to 27 June 2011). Trapping effort, defined as the number of trap-nights needed to obtain one hair sample, was 47.6 in autumn and 55.65 in spring. In total we collected 156 hair samples, of which 72 were collected in autumn and 84 in spring. [ABSTRACT FROM AUTHOR]

Simple Summary: This study was conducted with the aim of determining the morphological similarities and developmental potential of oocytes obtained from adult and prepubertal domestic cats (Felis catus) and wild cats. The results of our research showed that ovaries obtained from prepubertal felids may be a rich source of good quality oocytes that are competent for in vitro maturation and able to reach blastocyst stage after in vitro fertilization. The results are important in the context of the possibility of using oocytes from prepubertal felids threatened with extinction in conservation programs based on assisted reproductive techniques (ART). The aim of the study was to compare the morphology and developmental potential of oocytes obtained from adult and prepubertal domestic cats (Felis catus) and wild cats (Lynx lynx, Leptailurus serval, Felis manul, Panthera tigris altaica). The average number of oocytes obtained from an adult domestic cat was 23 ± 11, which was significantly lower than from kittens (43 ± 29). A similar number of oocytes was derived from adult Pallas's cats (28 ± 8), and serval (30). The lowest number of oocytes was collected from the lynx (5 ± 3). No oocytes were obtained from newborn Amur tiger while in the case of older domestic and Pallas's cat and lynx kittens (1–3 months) 43, 48 and 41 oocytes were collected, respectively. Significant differences (p < 0.001) were observed between the number of oocytes with dark cytoplasm from adult and prepubertal animals of all analyzed species. The diameter of oocytes from adult and prepubertal animals was similar in all species, and was on average 161 ± 4 µm for oocytes with dark cytoplasm and 150 ± 18 µm for oocytes with light cytoplasm. In all species, oocytes with light cytoplasm were significantly smaller (p < 0.05) than dark ones, and their population was more diverse. Results of in vitro maturation of the domestic and wild cat′s oocytes obtained from adult and prepubertal females were similar (47–52%). The cleavage rate after in vitro fertilization (IVF) was lower for prepubertal than adult domestic cats (42 vs. 51%; p < 0.05%). Moreover, we observed differences in the quantity (28 vs. 39%; p < 0.05) and quality of blastocysts and even greater problems with hatching blastocysts from prepubertal kittens (8 vs. 19%; p < 0.001). More blastomeres were detected in blastocysts of adult cats. They also demonstrated significantly higher number of inner cell mass (ICM) (p < 0.001) and higher number of trophoblast cells (TE) (p < 0.05). [ABSTRACT FROM AUTHOR]

Simple Summary: This study was conducted with the aim of determining the frequency of morphological defects in feline embryos, their competence to reach the blastocyst stage, and their ability to hatch. Embryonic morphological disorders affect development potential, and the use of time lapse monitoring (TLM) guarantees the precise observation of any changes that occur during in vitro embryo development. Some human, bovine, and mouse in vitro fertilized (IVF) embryos with morphokinetic abnormalities such as fragmentation, direct cleavage, and cytoplasmic vacuoles have the potential to reach the blastocyst stage, which is related to a high potential for implantation. The latest techniques of embryo development observation to enable the evaluation and selection of embryos are based on time lapse monitoring (TLM). The aim of this study was to determine the frequency of morphological defects in feline embryos, their competence to reach the blastocyst stage, and their ability to hatch. Oocyte-cumulus complexes were isolated after the scarification of ovaries and matured in vitro. Matured oocytes were fertilized in vitro by capacitated spermatozoa. Randomly selected oocytes were observed by TLM for seven-to-eight days. Out of 76 developed embryos, 41 were morphologically normal, of which 15 reached the blastocyst stage. Of 35 abnormally developed embryos, 17 reached the blastocyst stage, of which six had single aberrations and 11 had multiple aberrations. The hatching rate (%) was 15.6% in normally cleaving embryos, 6.25% in embryos with single aberrations, and 3.33% in those with multiple aberrations. The present study reports the first results, found by using TLM, about the frequency of the morphological defects of feline embryos, their competence to reach the blastocyst stage, and their ability to hatch. [ABSTRACT FROM AUTHOR]