Evaluation of a commercial proAKAP4 kit for the assessment of fresh and frozen-thawed feline spermatozoa.

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.

Mito-Tempo improves acrosome integrity of frozen-thawed epididymal spermatozoa in tomcats.

Introduction: In tomcats, epididymal spermatozoa provide an additional source of male gametes available for cryopreservation. While this procedure is feasible, the survival rate and motility of epididymal cat spermatozoa are both low after thawing. Cryopreservation is known to induce oxidative stress in spermatozoa, with mitochondria and the plasma membrane being the two major generation sites, and an imbalanced presence of free radicals is a possible cause for this low survival rate. Different antioxidants have been tested before for their effect on cryopreserved cat spermatozoa quality, with varying results. Here, we used Mito-Tempo, which is a synthetic mitochondria-targeted antioxidant and a specific scavenger of the mitochondrial superoxide system. By supplementing Mito-Tempo with the freezing extender, we aimed to improve the sperm quality of frozen-thawed cat epididymal spermatozoa. Methods: Epididymal spermatozoa obtained from twelve tomcats were assessed for motility and concentration. Prior to freezing, samples were diluted in TRIS buffered extender with egg yolk and glycerol and divided into five aliquots supplemented with 0 (control), 0.5, 5, 50, and 1005M of Mito-Tempo. After thawing, sperm motility, concentration, morphology, plasma membrane integrity, acrosome integrity, and mitochondrial membrane potential were evaluated. A Friedman rank sum test with a Bonferroni post-hoc test was used to determine statistical in-between group differences in post-thaw semen parameters. Results and discussion: The results indicated a slight improvement in acrosome integrity across all groups that were supplemented with Mito-Tempo, with the group that received 55M of Mito-Tempo showing the greatest improvement [(median of 67.99%, IQR of 5.55) compared to the control group (median of 65.33%, IQR of 7.75; P = 0.05)]. For all other sperm parameters, no significant differences (P > 0.05) were detected between different Mito-Tempo concentrations. These findings highlight the protective effect of Mito-Tempo on acrosome integrity and suggest that 55M is the most effective concentration for maintaining acrosome integrity. Since Mito-Tempo has shown a positive effect on multiple sperm parameters in other species, such as men, boars, roosters, rams, and bulls, we need to conclude that species-specificity may play a role here.

The morphology, morphometry and functionality of fresh and cryopreserved wisent (Bison bonasus) epididymal spermatozoa.

Epididymal spermatozoa obtained post mortem are considered a valuable source of genetic material which is often irrevocably lost. This makes these gametes constitute a key element in protection and restitution programs. The wisent (Bison bonasus, Linnaeus 1758) is a species that survived in zoos after extinction from its natural habitat. This resulted in a narrowing of the genetic pool of the whole population, which is at present derived from only 12 ancestors. Currently, wisent protection programs are aimed at preserving the genetic diversity by establishing a germplasm bank. The objective of this study was to comprehensively characterize the morphology, morphometry and functionality of wisent epididymal spermatozoa and evaluate the effectiveness of their cryopreservation in extender based on Tris buffer and chicken egg yolk. The median total number of spermatozoa obtained from one individual was 1985.0 × 106 (62.5 × 106-7452.0 × 106). These gametes were characterized by median: 40.0% (0.5-70.0%) subjective motility, 69.8% (32.5-90.0%) viability and 54.3% (10.5-83.3%) normal morphology. The sperm head had a median size of 5.0 µm (3.5-6.7 µm) width, 8.5 µm (6.4-11.3 µm) length and 36.9 µm2 (23.7-48.6 µm2) surface area. The viable population of the obtained gametes was characterized by median values 53.2% (4.5-80.3%) of intact sperm membrane, 50.8 (26.0-76.6%) of intact acrosome, 0.4% (0-98.7%) of fragmented chromatin, 5.9% (0.0-88.8%) of cells with high mitochondrial potential and 42.1% (8.3-63.7%) without lipid peroxidation. The viable population of the frozen/thawed gametes was characterized by median values: 18.4% (2.4-57.9%) of intact sperm membrane, 35.1 (11.9-56.7%) of intact acrosome, 0.07% (0-89.2%) of fragmented chromatin, 12.8% (0.0-49.7%) of cells with high mitochondrial potential and 16.3% (2.2-53.6%) without lipid peroxidation. Due to the material originating from a relatively large number of wild individuals, the research presented here contributed to the description of certain species standards for the assessment of wisent epididymal spermatozoa. The presented effect of cryopreservation on these gametes justifies the use of an extender based on Tris buffer with the addition of chicken egg yolk. The obtained effects are satisfactory from the point of view of preserving valuable genetic material and their use in ART.

Feline sperm head morphometry in relation to male pedigree and fertility.

Computer-assisted sperm morphometry analysis is an advanced tool which allows to precise measure sperm head parameters like length, width, area, and perimeter. On the basis of these and calculated parameters, morphometric subpopulations of spermatozoa can be distinguished. In many species, the distribution of subpopulation within the ejaculate is related to male fertility. There is no information about such a relation for domestic cats; therefore, the aim of this study was to evaluate whether spermatozoa from non-pedigree and purebred domestic cats differ in morphometric parameters. The second aim was to check if there is a relationship between sperm morphometry and fertility. Urethral semen was collected from 27 tomcats, divided into three study groups: non-pedigree cats of unknown fertility, purebred infertile cats and purebred fertile cats. The morphometric assessment was performed by CASMA, followed by principal component analysis and clustering. The results revealed huge intra- and inter-individual variation in sperm head morphometric parameters and three sperm-head morphometric subpopulations were identified in feline semen. Neither mean values of morphometric parameters nor the distribution of spermatozoa between morphometric subpopulations differ between non-pedigree cats of unknown fertility and purebred infertile and fertile cats. We hypothesize that other factors, especially abnormalities of the midpiece and tail, and overall worse quality of the semen of infertile males could have masked the effect of subtle changes in the sperm head morphometry.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa.

BACKGROUND: The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. RESULTS: Percoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. CONCLUSIONS: While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

Infertility In Toms: Clinical approach, experiences and challenges.

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.

How Can We Introduce ART into Wild Felid Conservation in Practice? Joint Experience in Semen Collection from Captive Wild Felids in Europe.

Although artificial reproductive techniques (ART) are considered to be a valuable tool for species conservation, information about their introduction into clinical practice for wild felids is limited. The aim of this paper was to jointly describe cases of non-experimental sperm collection from males of various species of wild felids, performed by three European centers focused on feline reproduction. In total, the article presents 22 attempts of semen collection in 12 species of wild felids. The reasons for semen collection were: fertility assessment (10 cases), artificial insemination (5 cases), sperm rescue (postmortem collection for cryopreservation, 5 cases), and sperm banking (in vivo collection for cryopreservation, 2 cases). Semen collection was successful (defined as at least 1 × 106 spermatozoa) in 15 cases. The failures in obtaining spermatozoa were most probably due to (1) male infertility, (2) wrong age/non-breeding season, or (3) recent multiple copulations. The cases presented here confirm that although ART have been introduced into clinical practice, they are mostly used in cases of infertility, not as routine breeding tools. Higher involvement of zoological gardens and private breeders is required, as many chances for preservation of valuable material are lost.

Hypo-Osmotic Swelling Test (HOST) for Feline Spermatozoa: The Simplified Procedure and the Aspect of Sperm Morphology.

Hypo-osmotic swelling test (HOST) is used to assess the functional integrity of sperm plasma membranes in many species. The primary aim of this study was to test a simplified HOST procedure for the evaluation of feline semen. The second objective was to check if sperm abnormalities can influence the results of this test. Urethral semen was collected from 19 male, domestic cats. In Exp. 1, HOST was performed in different media (50 mOsm/kg fructose or distilled water), temperature (37 °C or room temperature) and time (5 and 30 min). In Exp. 2, the potential effect of sperm abnormalities on HOST results was assessed by observing individual normal and abnormal spermatozoa microinjected into droplets of distilled water. The results showed no differences between the HOST results performed in different media, temperature and time. Viable abnormal spermatozoa were able to swell under hypo-osmotic conditions in the same manner as normal ones, except spermatozoa with distal droplets, which showed a higher frequency of 'despiralization'. In conclusion, HOST can be reliably performed at 0 mOsm/kg for 5 min at room temperature, which may contribute to a wider use of this test under clinical environments. Viable abnormal spermatozoa are able to swell under hypo-osmotic conditions; therefore, their presence in the ejaculate would not bias the results of HOST when total coiling is calculated.

A comparison of in vitro culture systems for cat embryos.

The aim of this study was to compare several culture systems for cat embryos. Domestic cat oocytes were matured in vitro (IVM), fertilized (IVF), and cultured individually or in groups in drops under oil (20 µL or 50 µL) and in 16 microwell dishes (Primo Vision®). Moreover, the effects of co-culture with a) uncleaved oocytes, b) homospecific and c) heterospecific co-culture with cat and sheep companion embryos were investigated using a time-lapse system. A higher proportion of blastocysts and hatching blastocysts was observed after culture in Primo Vision® dishes compared with the classical individual (p < 0.001) and group (p < 0.05) culture systems. Culture of presumptive zygotes 16 hpi and the presence of uncleaved oocytes did not reduce blastocyst development compared with culture of embryos 24 hpi without uncleaved oocytes. Co-culture with later-stage companion cator sheep embryos accelerated development of catembryos. The highest percentage of blastocysts was obtained in the group co-cultured with sheep embryos (54%). Moreover, the blastocyst cavity formed on average 10 h faster in this group than for the control group and for embryos co-cultured with cat embryos. The proportion of hatching blastocysts was similar in the co-cultures with cat and with sheep embryos (20% vs. 22%) and significantly (p < 0.05) than in the control group (12%).

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.

Fighting Like Cats and Dogs: Challenges in Domestic Carnivore Oocyte Development and Promises of Innovative Culture Systems.

In vitro embryo production in cats and dogs still presents some challenges, and it needs to be optimized to transfer efficient protocols to related wild, endangered species. While the chemical composition of culture media has been the focus of several studies, the importance of culture substrates for oocyte and embryo culture has often been neglected. Traditional in vitro systems, i.e., two-dimensional cultures, do not resemble the physiological environments where cells develop, and they may cause morphological and functional alterations to oocytes and embryos. More modern three-dimensional and microfluidic culture system better mimic the structure and the stimuli found in in vivo conditions, and they could better support the development of oocytes and embryos in vitro, as well as the maintenance of more physiological behaviors. This review describes the different culture systems tested for domestic carnivore reproductive cells along the years, and it summarizes their effects on cultured cells with the purpose of analyzing innovative options to improve in vitro embryo production outcomes.

Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System.

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21-22 hpi. Embryos that cleaved very early (17-18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1-3 or 3-5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2-3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127-167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142-150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

Immunolocalization of Adrenoceptors in the Reproductive Tract of Male Domestic Cats in Comparison to Rats.

Adrenoceptors mediate the action of the sympathetic nervous system, including the contraction of the epididymis and vas deferens. The aim of this study was to immunolocalize the adrenergic receptors in the reproductive tract of the male cat, as this information is not yet available. The epididymis and vas deferens of domestic cats and rats (the biological controls) were analyzed by immunohistochemistry to determine the localization of the α1A-, α1B-, α1D-, α2A-, α2B-, α2C-, ß1-, ß2-, and ß3-adrenoceptors. All the receptors were expressed in the peritubular smooth muscles of the cat, but the α1D-, α2C-, and ß1-adrenoceptors were not detected in this tissue in the rat. For the α2A-adrenoceptor, the intensity of immunostaining differed significantly between the caput epididymis (weakest staining) and the vas deferens (strongest staining). The presence of all the types of the receptors was also detected in the cytoplasm of the epithelial cells in all the regions of the reproductive tract. The strong expression of the α2A-adrenoreceptor suggests it has a leading role in the contraction of the reproductive tract in the cat. The presence of other adrenergic receptors in the smooth muscle of the epididymis and vas deferens indicates a potential clinical application for α1-mimetics in the optimization of pharmacological semen collection in felids.

Expression of Apoptosis-Related Genes in Cat Testicular Tissue in Relation to Sperm Morphology and Seasonality-A Preliminary Study.

Apoptosis is a crucial process in spermatogenesis, responsible for the elimination of abnormal sperm cells and testicular regression out of breeding season. The aim of this study was to assess if the expression of apoptosis-related genes in testicular tissue of domestic cats differed: (1) between normozoospermic and teratozoospermic donors, and (2) between reproductive and non-reproductive season. The expression of genes: BCL2L1, BCL2, BAX, BAD, FAS, FASLG, and caspases (CASP3, CASP8, CASP9, and CASP10) was analyzed by qRT-PCR in testicular tissue samples. During non-reproductive season significantly higher expression of two anti-apoptotic genes (BCL2L1 and BCL2) was observed. Additionally, there was a significant higher expression of CASP10 in teratozoospermic cats during non-reproductive than during reproductive season. No differences were noted between normozoospermic and teratozoospermic groups. Upregulation of some genes during the non-reproductive season indicates engagement of apoptotic mechanisms in the seasonal changes of semen quality in cats, however further studies on protein levels and analysis of changes on distinct testicular germinal layers are required. At the same time, teratozoospermia in the general population of cats seems to be not connected with dysregulation of apoptosis in the testes.

Comparison of the Morphology and Developmental Potential of Oocytes Obtained from Prepubertal and Adult Domestic and Wild Cats.

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).

Infertility in purebred cats - A review of the potential causes.

Although purebred cat breeding is growing in popularity in European countries, most research and publications concern dog reproduction. Infertility in queens has been poorly studied. It may not come as a surprise as common domestic shorthair cats are well known for their excellent fertility. However, even in the latter, the infertility rate is around 20%. Only recently, published articles have analyzed the reproductive performances in different breeds, for example in UK, Sweden, France and Italy, and found similar figures. In cats, infertility may arise from many factors such as mismating, prolonged anestrus or silent heats, hormonal, nutritional, genetical or chromosomal causes. Also, infectious diseases, probably frequent in breeding facilities, include pathological agents that are well recognized as having a negative effect on pregnancy or other pathogens for which the involvement in fertility problems is unclear. However, analyzing the literature, it appears that the most prevalent causes of infertility in pedigree cats may well be, in females, uterine pathology (sub-clinical endometritis, cystic endometrial hyperplasia, or mucometra), and in males, teratospermia, which may be "permanent" and linked to reduced heterozygosity or "transient" in tomcats that present normal quantitative spermatogenic parameters. The influence of the breed is unknown. There is an urgent need to study the origin of infertility in purebred cats in order to improve the diagnosis and to develop treatments that may restore normal fertility in subfertile or infertile animals.

The frequency of collapse as a predictor of feline blastocyst quality.

Domestic cats are frequently used as a research model for felid species that are threatened with extinction. Until now, the development of feline embryos has been evaluated using both classical observation methods and time-lapse monitoring (TLM). Blastocyst collapse observed using time-lapse cinematography is used as a predictor of blastocyst quality and is closely related to implantation potential. The aim of this study was to determine the relationship between the quality of domestic cat blastocysts obtained after in vitro fertilization and the frequency and duration of collapse, and of hatching. There was a significant difference in the average number of collapses and weak contractions between good and poor quality blastocysts. There was no significant difference between hatching and non-hatching blastocysts in terms of blastocyst cavity formation time or average number and duration of collapse. These results showed that the time of cavity formation was not related to blastocyst quality. The number of collapses and the occurrence of hatching were positively related to blastocyst quality, and poor quality blastocysts have, as a consequence, a reduced potential for implantation. TLM plays a significant role in cat embryo evaluation.

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification.

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.

Proteome of cat semen obtained after urethral catheterization.

The binding of seminal plasma (SP) proteins by spermatozoa plays an important role in the regulation of sperm epididymal maturation, motility gaining in female reproductive tracts and sperm-egg interaction. The aim of the study was to analyze the SP and sperm extracts proteome of cat (Felis catus) semen. The seminal plasma and spermatozoa were obtained by urethra catheterization from 10 male cats. Proteins were extracted using RIPA buffer and separated by electrophoresis (SDS-PAGE). The gels were analyzed using MultiAnalyst software. The proteins were subsequently analyzed using NanoUPLC-Q-TOF/MS. UniProt database-supported identification resulted in 106 proteins identified in the cat SP and 98 proteins in the extracts of spermatozoa. Based on a gene ontology analysis, dominant molecular functions of feline SP proteins were binding, catalytic, and antioxidant activity (56%, 33%, and 11% of cases, respectively). The molecular functions of sperm extracts proteins were mainly involved in catalytic activity (41%) and binding (23%). The proteins present in both, the SP and spermatozoa's extracts, were: serum albumin (ALB), semenogelin 2 (SEMG 2), clusterin (CLU), lactoferrin (LTF), prostatic acid phosphatase (ACPP), prolactin inducible protein (PIP), negative elongation factor E (NELF-E) and ectonucleotide pyrophosphatase (ENPP3). Protein-protein interactions analysis showed significant connection for 12 proteins in the cat semen. The seminal plasma proteins which, with high probability score, participate in important metabolic pathways are: glutathione peroxidases (GPx5 and 6), prostatic acid phosphatase (ACPP), ß-hexosaminidase (HEXB), polymeric immunoglobulin receptor (pIgR) and serpin family F member 1 (SERPINF1). For sperm protein extracts it were: pyruvate dehydrogenase (PDHB), succinate-CoA-ligase (SUCLA2), malate dehydrogenase (MDH2), ATP synthase F1 subunit alpha (ATP5F1A) and tubulin beta (TUBB).

Using Time Lapse Monitoring for Determination of Morphological Defect Frequency in Feline Embryos after In Vitro Fertilization (IVF).

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.