Feline Wharton’s jelly-derived mesenchymal stem cells as a feeder layer for oocytes maturation and embryos culture in vitro

IntroductionDue to their capacity to release growth factors and cytokines, co-culture using mesenchymal stem cells has been considered a good alternative to promoting the maturation of the oocytes and the embryo’s development quality in vitro in different mammalian species. In this regard, we investigated the effect of feline Wharton’s jelly MSCs as feeders layer in oocyte maturation—consequently, the development of resulting embryos in co-culture.MethodsOocytes with dark cytoplasm and a few layers of cumulus cells were collected and subjected to in vitro maturation and embryo culture using commercial media with and without MSCs addition. The oocytes’ nuclear maturation and the degree of cumulus expansion in different groups were assessed after 24 h; the development of the embryo was evaluated every 12 h until day eight.ResultsAlthough MSCs increased the proportion of cumulus cells oocytes exhibiting cumulus expansion, there were no significant differences in the percentage of matured oocytes (metaphase II) among the groups (p > 0.05). However, the embryo development differs significantly, with a higher cleavage, morula, and blastocyst percentage in oocytes matured with MSC co-culture conditions than in commercial media alone (p < 0.05). Also, we observed higher morula and blastocyst rates in the embryos co-cultured with MSCs during the in vitro culture (p > 0.05).ConclusionBased on our results, the co-culture with MSCs during the oocyte maturation resulted in better embryo development, as well as the MSCs addition during embryo culture returned an increased number of morula and blastocysts. Further research is needed to fully understand and optimize the use of MSCs in oocyte maturation and embryo development

Influence of Single Layer Centrifugation with Canicoll on Semen Freezability in Dogs

Simple Summary Freezing dog semen is not always possible due to low quality sperm or poor survival during freezing. In order to make this assisted reproductive technique available to a larger number of dogs, this study investigated the benefit of selecting the best spermatozoa before freezing using single layer centrifugation (SLC). The results indicated that this technique was effective in separating spermatozoa according to their quality, although this resulted in losing some good quality spermatozoa. After thawing, spermatozoa centrifuged by SLC were of better quality than after standard centrifugation. However, spermatozoa from suboptimal quality semen did not survive freezing as well as spermatozoa from semen of optimal quality, even after SLC. Single layer centrifugation, therefore, makes it possible to obtain better quality spermatozoa after thawing but is not sufficient on its own to improve the inferior freezing ability of spermatozoa from suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile. This study evaluated how semen selection by single layer centrifugation (SLC) with Canicoll affects semen freezability in dogs. A total of eighteen ejaculates, collected from dogs with optimal and suboptimal semen quality (optimal: normal morphology (NM) >= 80%, n = 9; suboptimal: NM between 60 and 79%, n = 9), were divided into two aliquots and subjected to standard centrifugation or SLC before cryopreservation. Motility, NM, membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were improved in fresh samples after SLC, regardless of semen quality, but at the expense of some good quality spermatozoa. After thawing, NM and membrane integrity were improved in SLC-selected semen in both semen qualities. Interestingly, MMP was also higher but only in optimal quality semen. Still, spermatozoa from suboptimal quality semen did not survive freezing to the same extent as spermatozoa from optimal quality semen, even after selecting superior spermatozoa. Semen selection with Canicoll is, therefore, an effective technique to isolate a subpopulation of high-quality spermatozoa and obtain sperm samples of better quality after thawing, but is not sufficient to improve the intrinsic inferior freezability of suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile

Association between polymorphisms in the SOX9 region and canine disorder of sex development (78,XX; SRY-negative) revisited in a multibreed case-control study

Testicular or ovotesticular disorders of sex development (DSD) in individuals with female karyotype (XX) lacking the SRY gene has been observed in several mammalian species, including dogs. A genetic background for this abnormality has been extensively sought, and the region harboring the SOX9 gene has often been considered key in canine DSD. Three types of polymorphism have been studied in this region to date: a) copy number variation (CNV) in a region about 400 kb upstream of SOX9, named CNVR1; b) duplication of SOX9; and c) insertion of a single G-nucleotide (rs852549625) approximately 2.2 Mb upstream of SOX9. The aim of this study was thus to comprehensively analyze these polymorphisms in a large multibreed case-control cohort containing 45 XX DSD dogs, representing 23 breeds. The control set contained 57 fertile females. Droplet digital PCR (ddPCR) was used to study CNVR1 and the duplication of SOX9. Fluorescent in situ hybridization (FISH) was used to visualize copy numbers on a cellular level. The Sanger sequencing approach was performed to analyze the region harboring the G-insertion. We confirmed that CNVR1 is highly polymorphic and that copy numbers varied between 0 and 7 in the case and control cohorts. Interestingly, the number of copies was significantly higher (P = 0.038) in XX DSD dogs (mean = 2.7) than in the control females (mean = 2.0) but not in all studied breeds. Duplication of the SOX9 gene was noted only in a single XX DSD dog (an American Bully), which had three copies of SOX9. Distribution of the G-nucleotide insertion was similar in the XX DSD (frequency 0.20) and control (frequency 0.14) cohorts. Concluding, our study showed that CNVR1, located upstream of SOX9, is associated with the XX DSD phenotype, though in a breed-specific manner. Duplication of the SOX9 gene is a rare cause of this disorder in dogs. Moreover, we did not observe any association of G-insertion with the DSD phenotype. We assume that the genetic background of XX DSD can be different in certain breeds

Effect of Fixatives and Fixation Period on Morphology and Immunohistochemistry of Feline Ovarian Tissue

Fixatives and fixation protocol have a profound effect on both the morphology and epitope sensitivity of ovarian tissue, which hampers accurate ovarian tissue evaluation. We aimed to establish the most suitable fixation protocol for feline (Felis catus) ovarian tissue. Fragments (1.5 mm diameter) were punched from 1 mm-thick feline ovarian tissue, divided into three groups then fixed with three different fixatives (Bouin, neutral buffered formalin [NBF] and form acetic acid [new compound fixative formulation for ovarian tissue composed of 5% acetic acid in NBF]) for five fixation periods. Subsequently, fragments were processed and evaluated for the morphology and intensity of immunohistochemical signals against three antigens (Ki-67, MCM-7 and activated caspase-3). Proportions of grade 1 or morphologically intact follicles were significantly lower in NBF when compared with Bouin and form acetic acid fixatives. However, Bouin fixative had the lowest mean DAB intensity (p p < 0.05) in Ki-67 and caspase-3, but in MCM-7, it was no different from form acetic acid. In conclusion, form acetic acid maintained ovarian tissue architecture with excellent follicular morphology in the same manner as Bouin fixative, and it also maintained reasonable DAB signals similar to NBF, thus providing a better alternative for feline ovarian tissue studies

The influence of Percoll (R) 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 spermatozoan. 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 (R) 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 (R) 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 (R) density gradient prior to the cryopreservation process

How can we introduce art into wild felid conservation in practice? Joint experience in semen collection from captive wild felids in Europe

Simple Summary Artificial reproductive techniques (ART), such as cryopreservation of sperm cells and artificial insemination, are useful tools for species conservation. However, there is relatively little information published about their introduction into clinical practice for wild felids. The aim of this paper was to describe how those techniques were applied by three European teams in various species of wild felids. In total, this article presents 22 semen collection attempts in 12 species of wild felids, 15 of which were successful and resulted in the collection of at least one million spermatozoa. 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 in the article confirm that although ART have been introduced into clinical practice, they are mostly used in cases of infertility, not as routine breeding tools. Greater involvement of zoological gardens and private breeders is required, as many chances for preservation of valuable material are lost. 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 x 10(6) 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

Serum Anti-Mullerian hormone : a potential semen quality biomarker in stud dogs?

Simple Summary Assessing semen quality in dogs requires experience and specialized equipment. Therefore, this study investigates the potential value of measuring the blood concentration of Anti-Mullerian hormone (AMH), a hormone produced by Sertoli cells, to predict semen quality in dogs. Forty-five healthy dogs were included in this study and their age as well as different semen parameters were correlated to blood AMH concentration. Moderate negative associations were found between AMH and semen motility and morphology indicating that high serum AMH may be a potential biomarker for identifying which dogs would require further semen investigation. Future research is however needed to confirm these preliminary results. Anti-Mullerian hormone (AMH) has been suggested to be involved in spermatogenesis. The aim of this study was to investigate the relationship between blood serum AMH concentration and semen quality in dogs. Moreover, this study sought to find the optimal cut-off point value of serum AMH with the greatest sensitivity and specificity to predict semen quality. Forty-five clinically healthy dogs were included in the study and their age as well as the following semen parameters were determined and correlated to serum AMH concentration: total sperm output, normal morphology, plasma membrane integrity, total motility, progressive motility, and velocity parameters. Statistical analysis for correlations were performed using Spearman's correlation coefficients. Moderate negative associations were found between serum AMH and semen total motility (r = -0.38, p = 0.01), progressive motility (r = -0.36, p = 0.01), and normal morphology (r = -0.36, p= 0.02). Based on these associations, an AMH concentration of 5.54 mu g/L was found to be the optimal cut-off point value to obtain the greatest summation of sensitivity (86%) and specificity (63%) to predict semen quality. The serum AMH assay may therefore be a potential hormonal marker to predict which dogs would require further semen analysis. Future research is however needed to confirm these preliminary results

Sperm gone smart : a portable device (iSperm®) to assess semen concentration and motility in dogs

Simple Summary Semen analysis can be subjective and time-consuming if automated instruments are not available. However, such devices are expensive and not transportable for on-field analyses. A portable device (iSperm(R)) is available for the evaluation of semen concentration and motility, but data on its reliability for canine semen analysis are still scarce. This study assessed the performances of the iSperm(R) on a large sample size (n = 224) by evaluating its correlation with a conventional computer-assisted sperm analyzer (ISAS(R)v1) for semen concentration and motility. The intra-assay variability of both the iSperm(R) and the ISAS(R)v1 and their ability to estimate semen concentration at a fixed value of 40 x 10(6)/mL were also investigated. Results showed that the intra-assay variability was lower for the ISAS(R)v1 compared to the iSperm(R). Hence, iSperm(R) results were more variable in-between fields. Both the iSperm(R) and the ISAS(R)v1 were not reliable in estimating semen concentration. Finally, the two devices were positively correlated, although providing different values for each parameter. Some improvements of the iSperm(R) software are therefore needed to make it a valid alternative to automated computerized systems for the analysis of canine semen. The iSperm(R) is a portable device for semen analysis. This study aimed to investigate its correlation with a conventional computer-assisted sperm analyzer (ISAS(R)v1) for the assessment of semen concentration and kinematic parameters in dogs (n = 224). The intra-assay variability of both devices and their ability to estimate semen concentration at a fixed value of 40 x 10(6)/mL were also investigated. Results showed that the intra-assay variability was lower for the ISAS(R)v1 for all parameters compared to the iSperm(R). Hence, iSperm(R) estimates were more variable in-between fields. Both the iSperm(R) and the ISAS(R)v1 were not reliable in estimating semen concentration (ISAS(R)v1: median 30 x 10(6)/mL, interquartile range (IQR) 12, p < 0.01; iSperm(R): median 35.12 x 10(6)/mL, IQR 11.11, p < 0.01). Finally, positive correlations were found between both devices with stronger correlations obtained when four fields were analyzed by the iSperm(R). However, the low number of spermatozoa analyzed per field and the inability to avoid artifacts are downsides that currently limit the reliability of the iSperm(R). Therefore, the software of iSperm(R) needs some improvement to make it a valid and practical alternative to automated computerized systems for the analysis of canine semen

Influence of single layer centrifugation with canicoll on semen freezability in dogs

Simple Summary Freezing dog semen is not always possible due to low quality sperm or poor survival during freezing. In order to make this assisted reproductive technique available to a larger number of dogs, this study investigated the benefit of selecting the best spermatozoa before freezing using single layer centrifugation (SLC). The results indicated that this technique was effective in separating spermatozoa according to their quality, although this resulted in losing some good quality spermatozoa. After thawing, spermatozoa centrifuged by SLC were of better quality than after standard centrifugation. However, spermatozoa from suboptimal quality semen did not survive freezing as well as spermatozoa from semen of optimal quality, even after SLC. Single layer centrifugation, therefore, makes it possible to obtain better quality spermatozoa after thawing but is not sufficient on its own to improve the inferior freezing ability of spermatozoa from suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile. This study evaluated how semen selection by single layer centrifugation (SLC) with Canicoll affects semen freezability in dogs. A total of eighteen ejaculates, collected from dogs with optimal and suboptimal semen quality (optimal: normal morphology (NM) >= 80%, n = 9; suboptimal: NM between 60 and 79%, n = 9), were divided into two aliquots and subjected to standard centrifugation or SLC before cryopreservation. Motility, NM, membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were improved in fresh samples after SLC, regardless of semen quality, but at the expense of some good quality spermatozoa. After thawing, NM and membrane integrity were improved in SLC-selected semen in both semen qualities. Interestingly, MMP was also higher but only in optimal quality semen. Still, spermatozoa from suboptimal quality semen did not survive freezing to the same extent as spermatozoa from optimal quality semen, even after selecting superior spermatozoa. Semen selection with Canicoll is, therefore, an effective technique to isolate a subpopulation of high-quality spermatozoa and obtain sperm samples of better quality after thawing, but is not sufficient to improve the intrinsic inferior freezability of suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile

Copy number variation in the region harboring SOX9 gene in dogs with testicular/ovotesticular disorder of sex development (78,XX; SRY-negative)

Although the disorder of sex development in dogs with female karyotype (XX DSD) is quite common, its molecular basis is still unclear. Among mutations underlying XX DSD in mammals are duplication of a long sequence upstream of the SOX9 gene (RevSex) and duplication of the SOX9 gene (also observed in dogs). We performed a comparative analysis of 16 XX DSD and 30 control female dogs, using FISH and MLPA approaches. Our study was focused on a region harboring SOX9 and a region orthologous to the human RevSex (CanRevSex), which was located by in silico analysis downstream of SOX9. Two highly polymorphic copy number variable regions (CNVRs): CNVR1 upstream of SOX9 and CNVR2 encompassing CanRevSex were identified. Although none of the detected copy number variants were specific to either affected or control animals, we observed that the average number of copies in CNVR1 was higher in XX DSD. No copy variation of SOX9 was observed. Our extensive studies have excluded duplication of SOX9 as the common cause of XX DSD in analyzed samples. However, it remains possible that the causative mutation is hidden in highly polymorphic CNVR1.ISSN:2045-232