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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]

ProAKAP4 is gaining increasing attention as a potential marker of semen quality in many species, but while there is a commercial kit for assessing proAKAP4 in the domestic cat, there are no publications about its use. The aim of this study was to evaluate the commercial proAKAP4 kit – Cat 4MID® Kit (SPQI – 4BioDx, Lille, France) for the assessment of feline semen. Semen was collected from 54 male cats by urethral catheterization. After a basic semen evaluation (subjective motility, CASA, viability, morphology), proAKAP4 levels in each sample were assessed using the Cat 4MID® Kit according to the manufacturer's protocol or with some modifications related to incubation time, sample storage conditions and number of spermatozoa used. Finally, the Spearman correlation of proAKAP4 concentration and sperm motility parameters was calculated. The most reliable results (acceptable intraassay coefficient of variation) were obtained with an optimized protocol of overnight incubation and isolation of proAKAP4 protein from 1 × 106 spermatozoa stored at −80°C. For fresh semen, there were no significant correlations between proAKAP4 concentration and sperm motility parameters, despite a strong correlation between motility parameters and sperm viability and morphology. A predominant effect of other sperm parameters and highly variable performance of lysis buffer question the usefulness of Cat 4MID® Kit for the assessment of feline semen. For frozen–thawed semen, there was a moderate, negative correlation between proAKAP4 concentration and two CASA parameters, VAP and VSL. As there were no correlations between proAKAP4 concentration in fresh semen and motility parameters in cryopreserved samples, proAKAP4 cannot be used as freezability marker in cats. More studies are needed to establish potential correlation with long‐lasting motility. [ABSTRACT FROM AUTHOR]

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.

Simple Summary: Veterinary regenerative medicine is an area of active research in which mesenchymal stem cells are applied. Mesenchymal stem cells (MSCs)are cells that can be obtained from various adult tissues; these cells have an extraordinary quality of being able to self-renew and develop into other cells. MSCs can be used to treat orthopaedic conditions in dogs, asthma, kidney disease, chronic gingivostomatitis, and inflammatory bowel disease in cats. Most studies have used adipose tissue-derived MSCs because they are easily obtainable and easy to work with. However, other stem cells from different tissues may be more suitable for treating certain diseases. In this manuscript, we report the generalities and the use of mesenchymal stem cells in cats and dogs, and we believe that the ongoing research in this field will eventually bring us to a point where stem cell treatments for currently untreatable diseases will become a reality. Finally, veterinary medicine now has access to new treatments, giving hope for a cure to illnesses in our furry friends. Mesenchymal stem cells (MSCs) are multipotent cells: they can proliferate like undifferentiated cells and have the ability to differentiate into different types of cells. A considerable amount of research focuses on the potential therapeutic benefits of MSCs, such as cell therapy or tissue regeneration, and MSCs are considered powerful tools in veterinary regenerative medicine. They are the leading type of adult stem cells in clinical trials owing to their immunosuppressive, immunomodulatory, and anti-inflammatory properties, as well as their low teratogenic risk compared with pluripotent stem cells. The present review details the current understanding of the fundamental biology of MSCs. We focus on MSCs' properties and their characteristics with the goal of providing an overview of therapeutic innovations based on MSCs in canines and felines. [ABSTRACT FROM AUTHOR]

Simple Summary: New cryopreservation methods for dog semen are constantly under investigation because the fertility results after insemination with frozen–thawed semen are still not satisfying. Most researchers do not investigate the fertilization ability of spermatozoa after the modification of cryopreservation protocols. In this study, we investigated whether spermatozoa membrane fluidity modification by use of cholesterol-loaded cyclodextrins (CLC) can improve kinematic parameters and also this method's effects on capacitation status and fertilization ability. The lowest concentration of CLC increased motility parameters and percentage of live spermatozoa without cholesterol efflux compared to the control. There was no change in capacitation status but the binding ability of the sperm to the oocyte was decreased. Overall, these data suggest that improvement of kinematic parameters does not necessarily improve the zona pellucida binding ability results of canine spermatozoa. Even though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen–thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa. [ABSTRACT FROM AUTHOR]

Simple Summary: The work concerns upon the supplementation of linseed oil ethyl esters to boars in order to improve semen parameters. The study included two groups of animals each with n = 6 animals. The application of linseed oil ethyl esters at 3% to the complete feed mixture in the experimental group improved semen parameters. It significantly increased semen parameters in particular sperm viability (p < 0.001), higher semen volume (310 mL versus 216 mL, p < 0.001) and sperm concentration (331 versus 216 million per mL, p < 0.001). Higher semen quality parameters of normospermic boars may have a direct translation into prolonged use of animals, which will significantly reduce pig production costs. Numerous studies have shown that improvements in the sperm and semen quality of males of many species can be achieved with appropriate dietary supplements added to feed or fodder. Particularly promising seems to be the inclusion of omega polyunsaturated fatty acids in the diets of males. Among other things, it has been shown that linseed oil ethyl esters (EELO can be an excellent source of omega 3 polyunsaturated fatty acids in animal diets. These compounds are more durable and resistant to oxidation, epoxidation and resinification processes, and do not exhibit toxic properties in living organisms. At present, there is a lack of data in the literature on the enrichment of boar diets with EELO. The purpose of this study was to analyze the effects of the addition of EELO to boar diets on the properties of sperm in fresh semen. The study was conducted during the summer on semen collected from 12 boars of the line 990. Linseed oil ethyl esters were administered in each feeding at a rate of 3.0% (45 mL each) in basal diets for each boar on a daily basis for 16 weeks. Ejaculates were collected manually by the gloved-hand technique, at one-week intervals for eight-week periods, from the eighth week onwards after the start of feeding. Eight ejaculates were collected from each boar, totaling 96 samples. The addition of EELO to the diets of boars caused an increase in sperm viability (p < 0.001), semen volume (310 mL versus 216 mL, p < 0.001) and sperm concentration (331 versus 216 million per mL, p < 0.001). Furthermore, in the experimental animals, there was a decrease in the percentage of spermatozoa exhibiting DNA fragmentation. The experimental boars also showed an increase in the percentage of gametes without apoptosis and capacitation and an increase in the percentage of viable spermatozoa not showing lipid peroxidation membranes. Consequently, EELO nutritional supplementation resulted in the improved quality of the fresh semen of boars. [ABSTRACT FROM AUTHOR]

Simple Summary: Chemical signals are essential for communication between living organisms. Dogs possess two sensory organs enabling chemical communication: the main olfactory system and the vomeronasal organ (VNO). Additionally, contact chemoreception is also pertinent, by which non-volatile molecules, including but not limited to proteins, are recognized as chemical signals. However, non-volatile chemical signals have been sparsely studied in dogs. Therefore, we aimed to examine the urinary proteins of female domestic dogs during the estrus and anestrus phases to detect and identify such non-volatile chemical signals. The presence and identity of non-volatile chemical signals remain elusive in canines. In this study, we aim to evaluate the urinary proteins of female domestic dogs in the estrus and anestrus phases to evidence the presence of non-volatile chemical signals and to elucidate their identities. We collected urine samples from eight female dogs in the estrus and anestrus phases. A total of 240 proteins were identified in the urine samples using liquid chromatography–mass spectrometry (LC–MS analysis). The comparison of the proteins revealed a significant difference between the estrus and anestrus urine. We identified proteins belonging to the lipocalin family of canines (beta-lactoglobulin-1 and beta-lactoglobulin-2, P33685 and P33686, respectively), one of whose function was the transport of pheromones and which was present only in the estrus urine samples. Moreover, proteins such as Clusterin (CLU), Liver-expressed antimicrobial peptide 2 (LEAP2), and Proenkephalin (PENK) were more abundant in the estrus urine when compared to the anestrus urine. LEAP2 was recently described as a ghrelin receptor antagonist and implicated in regulating food intake and body weight in humans and mice. Proenkephalin, a polypeptide hormone cleaved into opioid peptides, was also recognized as a candidate to determine kidney function. As of yet, none of these have played a role in chemical communication. Clusterin, an extracellular chaperone protecting from protein aggregation implicated in stress-induced cell apoptosis, is a plausible candidate in chemical communication, which is a claim that needs to be ascertained further. Data are available via ProteomeXchange with the identifier PXD040418. [ABSTRACT FROM AUTHOR]

Simple Summary: Flow cytometry (FC) is the recommended technique for assessing sperm quality. In comparison with fluorescence microscopy (FM), FC supports analyses of much larger sperm populations and generates more reliable results. However, FC is not always accessible, and sperm populations are often evaluated with the use of FM. In the present study, FC and FM were used to assess the functionality of various organelles in European red deer epididymal spermatozoa stored in liquid nitrogen. Spermatozoa were collected from the epididymides of hunter-harvested European red deer stags. The epididymides were stored in a refrigerator (2–4 °C) for 12 h before analysis. The study demonstrated that the refrigerated storage of the epididymides for 12 h had no significant effect on the sperm quality before cryopreservation, but it significantly influenced the percentage of early necrotic sperm after thawing. The results of FM and FC assays differed significantly, excluding in the assessment of the plasma membrane integrity. However, the results of both assays revealed significant correlations between the examined variables, except for mitochondrial activity. The study demonstrated that the spermatozoa from epididymides chill-stored for 12 h can be used for cryopreservation. Fluorescence microscopy and FC are equally reliable techniques, but FM was more useful for evaluating mitochondrial activity. Thawed spermatozoa, sampled post mortem from the fresh epididymides of European red deer and epididymides stored for up to 12 h at 2–4 °C, were evaluated by fluorescence microscopy (FM) and flow cytometry (FC). The sperm samples were extended and cryopreserved. The sperm motility (CASA), sperm viability (SYBR+/PI-), acrosome integrity, mitochondrial activity, apoptotic changes, and chromatin stability were assessed. Sperm were analyzed by FM before cryopreservation, and by FM and FC after thawing. Epididymal storage time (for 12 h) had no significant effect (p > 0.05) on the examined variables before cryopreservation. After thawing, the storage variants differed (p ˂ 0.05) in the percentage of apoptotic sperm (FM and FC) and DNA integrity (FC). The results of FM and FC differed (p ˂ 0.05) in all the analyzed parameters, excluding SYBR+/PI. Significant correlations (p ˂ 0.01) were observed between the sperm viability, acrosome integrity, and the percentage of non-apoptotic spermatozoa, regardless of the applied technique. In FM, the above parameters were also significantly correlated with mitochondrial activity. The study demonstrated that European red deer spermatozoa stored in the epididymides at 2–4 °C for 12 h can be used for cryopreservation. Both techniques were equally reliable, but FM was better suited for evaluating mitochondrial activity whereas FC was more useful in the evaluation of DNA fragmentation. [ABSTRACT FROM AUTHOR]

Sex pheromones are chemical substances secreted into the environment that affect the physiology and behavior of recipients. Females use these compounds during oestrus to attract males, which leads to attempts of mating. This study evaluates the influence of manual semen collection in male dogs, in the presence or absence of a female in estrus, on the blood concentrations of cortisol (CRT), oxytocin (OXT), prolactin (PRL) and testosterone (T), as hormones involved both in the physiology of reproduction and stress. Ten male dogs were used in Experiment 1 to measure the serum and plasma concentrations of the aforementioned hormones in the absence of semen collection. Subsequently in the same animals, the concentrations of these hormones were evaluated before and after semen collection in the presence (Exp. 2) or in absence of a female in estrus (Exp. 3). No significant changes in hormone concentration caused by the semen collection were found, either with, or without the presence of female in estrus. Obtained results suggest that the procedure of manual semen collection in dogs, probably due to its passive character, does not stimulate endocrine glands to secrete hormones, and the process of ejaculation is probably controlled by neural pathway. The lack of effect of semiochemical stimulation to the CRT, PRL, OXT and T level, could be caused by a short contact with female during semen collection. Further studies on involvement of the hormones during the process of natural mating, especially preceded by long courtships, similar to that observed under natural conditions, should shed a light on the physiology of mating and the connection between the endocrine system and semiochemical stimulation in dogs. [ABSTRACT FROM AUTHOR]

Cell cycle synchronization of donor cells is an important step in mammalian somatic cell nuclear transfer (SCNT). This study was designed to compare the efficiency of serum starvation (Ss) and contact inhibition (cI) on cell cycle synchronization of jaguarundi, manul, and domestic cat skin fibroblasts, in the production of G0/G1 cells suitable for SCNT in felids. Ss was performed after the growing (G) cells reached 40–50% (G50+Ss), 60–70% (G70+Ss) and full confluency (Fc), i.e. in association with cI (cI+Ss). Frozen-thawed cells were cultured to the given state of confluency (d0; controls), and subjected to Ss or cI for 1, 3, and 5 days (d). In manul, the effect of Ss on arresting fibroblasts in the G0/G1 phase was noted after just 1d of culture at G70 confluence, while G50+Ss and cI+Ss were effective after 5d of treatment. In jaguarundi, 1–5d of G50+Ss and 5d of G70+Ss increased the percentage of G0/G1 cells versus d0 (P<0.01), with 5d of G70+Ss producing more (P<0.05) quiescent cells than after the same period of G50+Ss, cI+Ss and cI. In the domestic cat, Ss was efficient only after 3 and 5d of G50+Ss. In all species, cI alone failed to increase the proportion of G0/G1 cells compared to d0, however in the domestic cat, 5d of cI was more efficient than the same period of G50+Ss. In jaguarundi, >93% of cells were already in G0/G1 phase at d0 of Fc, suggesting that culture to Fc could be also a valuable method for fibroblast cell cycle synchronization in this species. In contrast to cI, prolonged Ss generated cell loss and could induce apoptosis and/or necrosis. In conclusion, Ss was the more efficient method for skin fibroblast cell cycle synchronization at the G0/G1 phase in manul, jaguarundi and the domestic cat. The response of cells to the treatments was species-specific, depending on cell confluence and duration of culture. This research may find application in preparing donor karyoplasts for SCNT in felids. [ABSTRACT FROM AUTHOR]

Practical relevance: An infertile tom used to be a relatively rare presentation in feline practice. However, as a result of breeding strategies targeting specific morphological/behavioural traits, and the influence of inbreeding (especially practised in rare breeds), among other factors, feline infertility has become a genuine clinical problem. Furthermore, increased interest among cat breeders and pet owners in diagnosing and treating an 'unsuccessful' tom (including using assisted reproductive techniques [ARTs]), has made such patients more commonplace in veterinary practice. Clinical challenges: A definitive cause for male infertility is often difficult to identify. One of the biggest challenges is the lack of fertility-based reference values for semen quality and hormone levels. Moreover, the literature in this field is scant and many reproductive tract conditions described in other species have not been reported in cats. The establishment of diagnostic tools and algorithms is essential to facilitate a reliable assessment of fertility potential. Global importance: There is a growing demand for appropriate veterinary care relating to feline reproduction. There is an expectation among breeders, particularly of pedigree cats of high breeding value, that the same options widely used for dogs (including ARTs) should also be available for cats. Equipment and technical skills: Equipment required for investigating male infertility includes a microscope and ultrasound machine; a blood analyser may also be useful, but samples can instead be sent to a laboratory. The skills required are often already performed by veterinarians; for example, catheterising the urethra, performing ultrasonography and blood sampling. Evidence base: There are some reports of fertility problems in male cats in the literature, but this area of feline reproduction is still relatively undeveloped. This review draws on the comprehensive knowledge developed and shared by specialists in this field, and is supported by the authors' own clinical experience. [ABSTRACT FROM AUTHOR]

Simple Summary: Benign prostatic hyperplasia affects over 80% of old, non-castrated male dogs. In many cases, the severity of the symptoms means that treatment is required. The treatment of choice is surgical castration, which is unacceptable for the owners of stud dogs who are interested in therapy that does not exclude their animals from breeding. In those cases, treatment with deslorelin acetate or osaterone acetate offers an alternative to surgery. The aim of our study was to compare the changes in semen parameters over time. The deslorelin acetate implant gradually, during the course of its action, led to a full prevention the stud dogs from mating. However, in the first week following implant placement, in some individuals we observed an increase in sperm concentration (flare up effect), offering the possibility to obtain sperm in valuable dogs before the action of deslorelin acetate is completely established, precluding semen collection. On the other hand, osaterone acetate allowed for the continuous reproductive use of a stud dog, as it only affected some semen parameters, and mainly caused a temporary reduction in semen volume with the subsequent increase in semen concentration without altering the libido. Benign prostatic hyperplasia (BPH) is one of the most common problems in older male dogs that often has a huge impact on their health and welfare. This article presents a comparison between osaterone acetate (Ypozane®; Virbac®)(OA) and deslorelin acetate (Suprelorin®; Virbac®)(DA), medications that are the main therapeutic alternative to castration in dogs with BPH. Forty dogs were divided into four groups: I—negative control (five dogs without BPH); II—positive control (10 individuals diagnosed with BPH); III—15 dogs treated with DA, and IV—10 individuals treated with OA. Semen fractions were collected on days 0 (day of treatment), 7, 14, and 21, and weeks 8, 12, 16, and 20. Macroscopic, microscopic and CASA analyses were performed. Both DA and OA significantly affected the properties of the canine ejaculate. The DA lead to the lack of libido and had lesser effects to the sperm function before it caused azoospermia, whereas OA had no effect on libido and only temporary reduction in seminal plasma volume was observed, which resulted in temporary deterioration in the percentage of motile and progressive spermatozoa. [ABSTRACT FROM AUTHOR]

Simple Summary: The article summarizes the current knowledge on factors related to pregnancy, parturition, and newborns that affect the health status of a puppy and determine its chances for survival and development. The detailed information is provided in terms of breed predispositions, objectives of pregnancy monitoring, potential sources of complications, and veterinary advances in care and treatment of perinatal conditions. Successful pregnancy outcomes still pose challenges in veterinary neonatology; thus, publications presenting the current state of knowledge in this field are in demand. The perinatal period has a critical impact on viability of the newborns. The variety of factors that can potentially affect the health of a litter during pregnancy, birth, and the first weeks of life requires proper attention from both the breeder and the veterinarian. The health status of puppies can be influenced by various maternal factors, including breed characteristics, anatomy, quality of nutrition, delivery assistance, neonatal care, and environmental or infectious agents encountered during pregnancy. Regular examinations and pregnancy monitoring are key tools for early detection of signals that can indicate disorders even before clinical signs occur. Early detection significantly increases the chances of puppies' survival and proper development. The purpose of the review was to summarize and discuss the complex interactions between all elements that, throughout pregnancy and the first days of life, have a tangible impact on the subsequent fate of the offspring. Many of these components continue to pose challenges in veterinary neonatology; thus, publications presenting the current state of knowledge in this field are in demand. [ABSTRACT FROM AUTHOR]