Induction of in vivo-like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells.

We recently developed re-differentiated equine oviduct epithelial cell (REOEC) monolayers demonstrating various in vivo morphological characteristics, but lacking secondary ciliation. In this study, we evaluated the effects of fetal bovine serum, reproductive steroid hormones, Wnt- and Notch ligands and inhibitors, and different EOEC seeding densities, in both conventional wells and on microporous membranes, on EOEC morphology and, in particular, secondary ciliation. REOEC monolayers were assessed by confocal microscopy after combined staining of nuclei, cilia and the cytoskeleton. Only Wnt ligands, Notch inhibitors and oviduct explant cell concentration affected EOEC morphology. Undesirable epithelial-mesenchymal transition was observed in REOEC monolayers exposed to Wnt3a containing medium and Wnt ligand CHIR 99021. With respect to secondary ciliation, only the combined effect of oviduct explant cell concentration and Notch inhibition steered REOEC monolayers to in vivo-like ciliation patterns. De-differentiated EOECs, formed 10 days after oviduct explant cell seeding, were reseeded on inserts; only at initial oviduct explant cell concentrations of 1 and 5 x106 cells per well was the formation of REOEC monolayers with a high rate of diffuse ciliation supported. Within 1 month after air-liquid interface introduction, >40% and > 20% of the REOECs showed secondary cilia, respectively. At higher oviduct explant cell seeding densities secondary ciliation was not supported after re-differentiation. Additionally, Notch inhibition helped boost secondary ciliation rates to >60% in REOEC monolayers with diffuse ciliation only. These monolayers demonstrated higher clathrin expression under follicular phase conditions. Overall, the ciliated REOEC monolayers better resemble in vivo oviduct epithelial cells than previous models.

Selection and application of small non-coding RNAs for normalizing RT-qPCR data of bovine preimplantation embryo conditioned medium.

Small non-coding RNAs (sncRNAs) present in the conditioned medium (CM) of bovine preimplantation embryos are potential noninvasive biomarkers for assessing embryo quality. Accurate quantification of sncRNA levels in the spent CM is of utmost importance in this regard. RT-qPCR is considered as the gold standard for quantifying RNA. In order to standardize RT-qPCR data in the sample type under investigation, the use of suitable stable sncRNAs is essential. Here, we selected 10 sncRNAs from small RNA sequencing of CM samples derived from both bovine blastocysts and degenerate embryos, and evaluated their expression stability together with that of cel-miR-39 as a spike and the often-used U6 small nuclear RNA at different embryo developmental stages. In CM of 2-cell embryos, rsRNA-1044 showed the most stable expression, while tDR-1:32-Gly-CCC-1 was the most stable expressed sncRNA in CM of the stages beyond the 2-cell stage. Next, tDR-1:32-Gly-CCC-1 was used for normalizing the RT-qPCR data from the CM of blastocysts and degenerate embryos. Bta-miR-155 and tDR-39:75-Arg-CCG-2 were found to be significantly up-regulated in the CM of blastocysts compared to that of the degenerated embryos (P = 0.028 and P = 0.017, respectively), suggesting their expression levels are related to embryo development stage. In conclusion, tDR-1:32-Gly-CCC-1 can serve as a suitable reference sncRNA for normalization of RT-qPCR data of the CM from bovine blastocysts.

Immunological defense mechanisms of ejaculates and the spread of viral infectious diseases through pig semen.

This review focuses on the mechanisms of immune tolerance and antimicrobial defense in the male genital tract of the pig. Sperm cells are foreign to the immune system and, therefore, they must be protected from the immune system. The blood-testis-barrier is mediated by a physical barrier between adjacent Sertoli cells, several cell types within the testis, and interactions between immunomodulatory molecules. The blood-epididymal-barrier is composed of a physical barrier that is lined with principal cells having a network of junctional complexes in their apical lateral membrane and completed by specific transporters. The seminal plasma (SP) contains many signaling agents involved in establishing a state of immune tolerance in the female genital tract, which is essential for successful fertilization. Specific SP-proteins, however, also have pro-inflammatory capacities contributing to transient uterine inflammation, supporting the removal of foreign cells, possible pathogens, and excessive spermatozoa. While many different proteins and other substances present in semen can damage sperm cells, they may also protect them against viral infections. A delicate balance of these substances, therefore, needs to be maintained. Related to this, recent studies have shown the importance of extracellular vesicles (EVs), as they contain these substances and convey immune signals. Yet, viruses may use EVs to interact with the male genital tract and circumvent immune responses. For this reason, further research needs to explore the role of EVs in the male reproductive tract, as it might contribute to elucidating the pathogenesis of viral infections that might be transmitted via semen and to developing better vaccines.

How to beat the bull: Lycopene as a tool to improve in vitro fertilization efficiency in bulls with high polyspermy.

High levels of reactive oxygen species (ROS) derived from in vitro conditions compromise oocyte quality and subsequent polyspermy prevention by the zona and membrane block. Antioxidant supplementation, like lycopene, during in vitro maturation (IVM) mitigates ROS effects, yet, its efficacy in blocking polyspermy remains uncertain. This study aims to evaluate the effect of lycopene supplementation during IVM on oocyte maturation, fertilization, and developmental parameters. To this end, bovine oocytes were supplemented with 0.2 µM lycopene and fertilized with semen from three bulls. The three bulls showed different fertilization potential in vitro, with bull 1 showing the highest penetration and polyspermy rates and the lowest in vitro fertilization (IVF) efficiency. Interestingly, in bull 1, the treatment with lycopene improved IVF efficiency (p = 0.043) and reduced the polyspermy rate (p = 0.028). However, none of these effects were observed in bulls 2 and 3. Bulls with higher penetration rates exhibited better blastocyst rates although those rates did not seem to be associated with polyspermy or IVF efficiency. Oocyte mitochondrial distribution and activity and cortical granule migration and distribution were not influenced by lycopene. In conclusion, we demonstrated that lycopene addition during oocyte maturation had a positive impact on IVF efficiency by reducing polyspermy rates in a bull-dependent manner. The reduction in polyspermy rates was not caused by changes in cortical granule migration or oocyte mitochondrial distribution. Lycopene must therefore induce other changes in the oocyte that lower the in vitro penetration rates of specific bulls prone to polyspermy.

Comparative Evaluation of Diagnostic Methods for Subclinical Benign Prostatic Hyperplasia in Intact Breeding Male Dogs.

Benign prostatic hyperplasia (BPH) is an androgen-related non-neoplastic enlargement of the prostate gland that commonly affects both reproductive capabilities and the general health of intact dogs. The subclinical form of BPH can be challenging to diagnose due to a lack of clinical signs, even if rectal palpation is performed. Left untreated, this condition poses risks to the dogs' health and breeding status. This study, involving 65 male dogs, aimed to investigate subclinical BPH through rectal palpation, ultrasonography, and analysis of canine prostatic-specific esterase (CPSE). Of the participants, 35 had subclinical BPH, and 30 served as a healthy control group. Dogs suspected of subclinical BPH, as determined by examination results from ultrasonography and CPSE analysis, underwent fine needle aspiration (FNA) guided by ultrasound to enhance diagnostic precision. Findings revealed distinct differences in rectal palpation and ultrasonography between subclinical BPH and healthy dogs. This study established diagnostic thresholds based on prostatic volume and CPSE values and proposed new thresholds for subclinical BPH. Additionally, results showed that prostate gland volume depended on the weight and the age of the dog. In conclusion, early detection of this condition is possible through various examinations, such as changes in ultrasound features, CPSE levels, and rectal palpation. All together, these methods can aid practitioners in early detection of BPH and assist with scheduling screening programs for dogs, ultimately promoting their overall health and reproductive well-being. In conclusion, we advocate for routine, non-invasive prostate screenings in breeding males, underlining the effectiveness of a combination of various multiple techniques for early subclinical BPH detection.

Quantifying maternal investment in mammals using allometry.

Maternal investment influences the survival and reproduction of both mothers and their progeny and plays a crucial role in understanding individuals' life-history and population ecology. To reveal the complex mechanisms associated with reproduction and investment, it is necessary to examine variations in maternal investment across species. Comparisons across species call for a standardised method to quantify maternal investment, which remained to be developed. This paper addresses this limitation by introducing the maternal investment metric - MI - for mammalian species, established through the allometric scaling of the litter mass at weaning age by the adult mass and investment duration (i.e. gestation + lactation duration) of a species. Using a database encompassing hundreds of mammalian species, we show that the metric is not highly sensitive to the regression method used to fit the allometric relationship or to the proxy used for adult body mass. The comparison of the maternal investment metric between mammalian subclasses and orders reveals strong differences across taxa. For example, our metric confirms that Eutheria have a higher maternal investment than Metatheria. We discuss how further research could use the maternal investment metric as a valuable tool to understand variation in reproductive strategies.

The reproductive microbiome in dogs: Friend or foe?

The microbiome of the reproductive tract is an area of research in full development. Specifically, the microbiome may be involved in reproductive health, disease, and pregnancy outcomes, as has been shown in humans and animals, including dogs. The aim of the present review was to summarize current knowledge on the microbiome of the canine reproductive tract, to expose the controversial role that some bacterial agents may play in canine subfertility, and to highlight future research perspectives. This review discussed whether the use of antimicrobials in dogs is appropriate to increase reproductive performance and to treat subfertility without proper diagnosis, and the possible use of probiotics to modulate the reproductive canine microbiome. Finally, we indicate areas in which scientific knowledge is currently lacking, and could be promising directions for future research.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

STUDY QUESTION: Is pronuclear transfer (PNT) capable of restoring embryo developmental arrest caused by cytoplasmic inferiority of in vitro-grown (IVG) mouse oocytes? SUMMARY ANSWER: PNT to in vivo matured cytoplasm significantly improved embryo development of IVG mouse oocytes, leading to living, fertile offspring. WHAT IS KNOWN ALREADY: In vitro follicle culture has been considered as a fertility preservation option for cancer patients. Studies describing the culture of human follicles remain scarce, owing to low availability of tissue. Mouse models have extensively been used to study and optimize follicle culture. Although important achievements have been accomplished, including the production of healthy offspring in mice, IVG oocytes are of inferior quality when compared to in vivo-grown oocytes, likely because of cytoplasmic incompetence. STUDY DESIGN SIZE DURATION: The study was carried out from September 2020 to February 2022. In total, 120 15-day-old B6D2 mice were used to perform secondary follicle culture and assess the quality of IVG oocytes. In vivo-grown control oocytes were obtained from 85 8- to 12-week-old B6D2 mice, following ovarian stimulation. For sperm collection, four B6D2 males between 10 and 14 weeks old were used. For embryo transfer, 14 8- to 12-week-old CD1 females served as surrogate mothers and 10 CD1 vasectomized males 10-24 weeks old were used to generate pseudo-pregnant females. Finally, for mating, four B6D2 female mice aged 8-10 weeks and two B6D2 male mice aged 10 weeks old were used to confirm the fertility of nuclear transfer (NT)-derived pups. PARTICIPANTS/MATERIALS SETTING METHODS: Secondary follicles from 15-day-old B6D2 mice were isolated from the ovaries and cultured for 9 days, before a maturation stimulus was given. Following 16-18 h of maturation, oocytes were collected and evaluated on maturation rate, oocyte diameter, activation rate, spindle morphology, calcium-releasing ability, and mitochondrial membrane potential. For every experiment, in vivo-grown oocytes were used as a control for comparison. When cytoplasmic immaturity and poor embryo development were confirmed in IVG oocytes, PNT was performed. For this, the pronuclei from IVG oocytes, created following parthenogenetic activation and IVF, were transferred to the cytoplasm of fertilized, in vivo-grown oocytes. Genetic analysis and embryo transfer of the generated embryos were implemented to confirm the safety of the technique. MAIN RESULTS AND THE ROLE OF CHANCE: Following 9 days of follicle culture, 703 oocytes were collected, of which 76% showed maturation to the metaphase II stage. Oocyte diameters were significantly lower in IVG oocytes, measuring 67.4 µm versus 73.1 µm in controls (P < 0.001). Spindle morphology did not differ significantly between IVG and control oocytes, but calcium-releasing ability was compromised in the IVG group. An average calcium release of 1.62 arbitrary units was observed in IVG oocytes, significantly lower than 5.74 in control oocytes (P < 0.001). Finally, mitochondrial membrane potential was inferior in IVG compared to the control group, reaching an average value of 0.95 versus 2.27 (P < 0.001). Developmental potential of IVG oocytes was assessed following parthenogenetic activation with strontium chloride (SrCl2). Only 59.4% of IVG oocytes cleaved to two cells and 36.3% reached the blastocyst stage, significantly lower than 89.5% and 88.2% in control oocytes, respectively (P < 0.001 and 0.001). Both PNT and spindle transfer (ST) were explored in pilot experiments with parthenogenetically activated oocytes, as a means to overcome poor embryo development. After the added value of NT was confirmed, we continued with the generation of biparental embryos by PNT. For this purpose, IVG and control oocytes first underwent IVF. Only 15.5% of IVG oocytes were normally fertilized, in contrast to 45.5% in controls (P < 0.001), with resulting failure of blastocyst formation in the IVG group (0 versus 86.2%, P < 0.001). When the pronuclei of IVG zygotes were transferred to the cytoplasm of control zygotes, the blastocyst rate was restored to 86.9%, a similar level as the control. Genetic analysis of PNT embryos revealed a normal chromosomal profile, to a rate of 80%. Finally, the generation of living, fertile offspring from PNT was possible following embryo transfer to surrogate mothers. LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Genetic profiles of analysed embryos from PNT originate from groups that are too small to draw concrete conclusions, whilst ST, which would be the preferred NT approach, could not be used for the generation of biparental embryos owing to technical limitations. Even though promising, the use of PNT should be considered as experimental. Furthermore, results were acquired in a mouse model, so validation of the technique in human IVG oocytes needs to be performed to evaluate the clinical relevance of the technology. The genetic profiles from IVG oocytes, which would be the ultimate characterization for chromosomal abnormalities, were not analysed owing to limitations in the reliable analysis of single cells. WIDER IMPLICATIONS OF THE FINDINGS: PNT has the ability to overcome the poor cytoplasmic quality of IVG mouse oocytes. Considering the low maturation efficiency of human IVG oocytes and potential detrimental effects following long-term in vitro culture, NT could be applied to rescue embryo development and could lead to an increased availability of good quality embryos for transfer. STUDY FUNDING/COMPETING INTERESTS: A.C. is a holder of FWO (Fonds voor Wetenschappelijk Onderzoek) grants (1S80220N and 1S80222N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) 2018000504 (GOA030-18 BOF) funding. B.H. has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest.

tRNAGlu-derived fragments from embryonic extracellular vesicles modulate bovine embryo hatching.

Transfer RNA-derived small RNAs (tsRNAs) have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells. However, it is unknown whether tsRNAs also regulate embryo hatching. In this study, we mined the sequencing data of a previous experiment in which we demonstrated that the microRNA (miRNA) cargo of preimplantation embryonic extracellular vesicles (EVs) influences embryo development. We thus profiled the tsRNA cargo of EVs secreted by blastocysts and non-blastocysts. The majority of tsRNAs was identified as tRNA halves originating from the 5´ ends of tRNAs. Among the 148 differentially expressed tsRNAs, the 19 nt tRNA fragment (tRF) tDR-14:32-Glu-CTC-1 was found to be significantly up-regulated in EVs derived from non-blastocysts. RT-qPCR assays confirmed its significant up-regulation in non-blastocyst embryos and their conditioned medium compared to the blastocyst group (P < 0.05). Inhibition of tDR-14:32-Glu-CTC-1 by supplementing antagomirs to the conditioned medium improved embryo hatching (P < 0.05). Transcriptomic analysis of embryos treated with tDR-14:32-Glu-CTC-1 antagomirs further showed differential expression of genes that are associated with embryo hatching and implantation. In summary, tDR-14:32-Glu-CTC-1 is up-regulated in non-blastocyst embryos and their secretions, and inhibition of tDR-14:32-Glu-CTC-1 promotes embryo hatching, while influencing embryo implantation-related genes and pathways. These results indicate that embryonic EVs containing specific tRFs may regulate preimplantation embryo development.

Manual versus deep learning measurements to evaluate cumulus expansion of bovine oocytes and its relationship with embryo development in vitro.

Cumulus expansion is an important indicator of oocyte maturation and has been suggested to be indicative of greater oocyte developmental capacity. Although multiple methods have been described to assess cumulus expansion, none of them is considered a gold standard. Additionally, these methods are subjective and time-consuming. In this manuscript, the reliability of three cumulus expansion measurement methods was assessed, and a deep learning model was created to automatically perform the measurement. Cumulus expansion of 232 cumulus-oocyte complexes was evaluated by three independent observers using three methods: (1) measurement of the cumulus area, (2) measurement of three distances between the zona pellucida and outer cumulus, and (3) scoring cumulus expansion on a 5-point Likert scale. The reliability of the methods was calculated in terms of intraclass-correlation coefficients (ICC) for both inter- and intra-observer agreements. The area method resulted in the best overall inter-observer agreement with an ICC of 0.89 versus 0.54 and 0.30 for the 3-distance and scoring methods, respectively. Therefore, the area method served as the base to create a deep learning model, AI-xpansion, which reaches a human-level performance in terms of average rank, bias and variance. To evaluate the accuracy of the methods, the results of cumulus expansion calculations were linked to embryonic development. Cumulus expansion had increased significantly in oocytes that achieved successful embryo development when measured by AI-xpansion, the area- or 3-distance method, while this was not the case for the scoring method. Measuring the area is the most reliable method to manually evaluate cumulus expansion, whilst deep learning automatically performs the calculation with human-level precision and high accuracy and could therefore be a valuable prospective tool for embryologists.

The dogma of the sterile uterus revisited: does microbial seeding occur during fetal life in humans and animals?

In brief: Opposing conclusions have been drawn regarding the presence of viable bacteria in the healthy pregnant uterus. Current evidence in humans and animals suggests that fetomaternal tissues present only traces of bacteria whose viability is still to be proven. Abstract: The debate about the pioneer colonization of the fetus is still open, being the 'in utero colonization' hypothesis versus the 'sterile womb paradigm' the two opposing sides. The seed in this field of research sprouted in human medicine in the last decade and became a central topic in other mammals as well. We aimed to review the literature on bacterial colonization of the healthy placenta, amniotic fluid, and meconium as representatives of the fetal environment. What emerges is that confirming the colonization of fetomaternal tissues by viable bacteria is challenging in humans as well as in animals. Contamination represents the major risk in this type of research as it can be related to different parts of the study design. Sampling at natural parturition or postpartum introduces risk for colonization by the vaginal microbiome of the mother or from the environment. Culture does not reveal the presence of unculturable microorganisms, and sequencing does not allow confirming bacterial viability, while also introducing the variability associated with the data analysis. Therefore, on the basis of the present review, we provide some guidelines on the best practices when performing this type of studies. What emerges from the current literature in humans and animals is that fetomaternal tissues are characterized by a very low biomass, that the viability of bacteria eventually present is still to be confirmed, while massive colonization happens at birth, priming the individual, regardless of the species.

Deep-Learning Based Quantification of Bovine Oocyte Quality From Microscopy Images.

The success rate of bovine in vitro embryo reproduction is low and highly dependent on the oocyte quality. The selection of the oocyte to be fertilized is done by the embryologists' visual examination of oocytes. It is time-consuming, subjective, and inconsistent between specialists in the area. In this paper, a semi-automatic solution is proposed to score the quality of an immature oocyte. It consists of a deep learning model to classify oocyte competence. The model was trained and tested with real data, composed of images of immature oocytes and their label of whether they developed into blastocysts after fertilization. To the best of our knowledge, automated bovine oocyte classification was not attempted before, but experimental results show that our proposed solution is more robust and objective than specialists' visual assessment and comparable with other works on human oocytes.Clinical relevance- This establishes a semi-automatic real-time method to score bovine immature oocytes, based on stereo-microscopy images. Our method will significantly reduce the time of in vitro embryo production and its success.

MicroRNA-146b negatively affects bovine embryo development and quality.

MicroRNAs (miRNAs), which can be carried inside extracellular vesicles (EVs), play a crucial role in regulating embryo development up to the blastocyst stage. Yet, the molecular mechanisms underlying blastocyst development and quality are largely unknown. Recently, our group identified 69 differentially expressed miRNAs in extracellular vesicles (EVs) isolated from culture medium conditioned by bovine embryos that either developed to the blastocyst stage or did not (non-blastocysts). We found miR-146b to be more abundant in the EVs derived from media conditioned by non-blastocyst embryos. Using RT-qPCR, we here confirmed the upregulation of miR-146b in non-blastocyst (arrested at 2-4 cell and morula stage) embryos compared to blastocysts (p<0.005), which coincides with the upregulation of miR-146b in EVs derived from the medium of these non-blastocysts. To evaluate a functional effect, bovine embryo culture media were supplemented with miR-146b mimics, resulting in significantly decreased embryo quality, with lower blastocyst rates at day 7 and lower total cell numbers, while the opposite was found after supplementation with miR-146b inhibitors, which resulted in reduced apoptosis rates (P < 0.01). Transcriptomic analysis of embryos treated with miR-146b mimics or inhibitors showed differential expression (P < 0.01) of genes associated with apoptosis, cell differentiation, and the RNA Pol II transcription complex, including WDR36, MBNL2, ERCC6l2, PYGO1, and SNIP1. Overall, miR-146b is overexpressed in non-blastocyst embryos and in EVs secreted by these embryos, and it regulates genes involved in embryo development and apoptosis, resulting in decreased embryo quality.

A comparative study of canine epididymal sperm collection techniques and cryopreservation.

Introduction: An optimized collection method and freezing protocol for preservation of epididymal spermatozoa remains a topic of interest to many scientists. The current study focused on the collection and preservation of canine epididymal spermatozoa. During the process of collection of canine epididymal spermatozoa, blood content can occur, which may affect sperm cryopreservation in a negative way. Here, we compared first two epididymal sperm collection techniques [epididymal mincing (EM) and single incision epididymal sperm aspiration (SESA)]; and next we tried to solve the issue of blood content using an erythrocyte lysis buffer (ELB). Methods: Hence spermatozoa were collected after weighing the epididymides, either by EM or SESA, and sperm quality assessed prior to and post freezing (concentration, total sperm output (TSO), motility, viability and morphology). Next, new sperm samples were collected from eight epididymides by EM and subjected either to a standard freezing protocol or to an ELB treatment freezing protocol. Post-thaw sperm parameters (concentration, TSO, motility, viability and morphology), including intracellular reactive oxygen species (ROS) and lipid peroxidation were assessed. The correlation between the weight of the epididymis and the TSO was evaluated based on the collection technique, and differences in sperm parameters were detected both within different collection techniques and between different pre-freezing treatment protocols. Results: There was a very strong correlation between the weight of the epididymis and the TSO for the EM technique (p = 0.002, R2 = 0.6), along with an increased sperm motility with EM compared to SESA (median 80%, inter-quartile range (IQR) 88-65 and median 67.5%, IQR 72.5-52.5, respectively; (p = 0.002). Post-thaw samples subjected to ELB treatment freezing protocol had lower motility and higher intracellular ROS compared to the standard freezing protocol (motility: median 56.25%, IQR 60-48.75 and median 70%, IQR 72.5-63, respectively; p = 0.01; ROS: median 78.5%, IQR 81.25-75.5 and median 70%, IQR 70.5-68.75, respectively; (p = 0.04). Discussion: The results indicated that EM is a better technique to harvest epididymal spermatozoa despite the presence of some blood content. Furthermore, the ELB treatment should not be implemented to remove those red blood cells prior to cryopreservation of epididymal spermatozoa in dogs.

Hsa-miR-665 Is a Promising Biomarker in Cancer Prognosis.

Biomarkers are biomolecules used to identify or predict the presence of a specific disease or condition. They play an important role in early diagnosis and may be crucial for treatment. MicroRNAs (miRNAs), a group of small non-coding RNAs, are more and more regarded as promising biomarkers for several reasons. Dysregulation of miRNAs has been linked with development of several diseases, including many different types of cancer, and abnormal levels can be present in early stages of tumor development. Because miRNAs are stable molecules secreted and freely circulating in blood and urine, they can be sampled with little or no invasion. Here, we present an overview of the current literature, focusing on the types of cancers for which dysregulation of miR-665 has been associated with disease progression, recurrence, and/or prognosis. It needs to be emphasized that the role of miR-665 sometimes seems ambiguous, in the sense that it can be upregulated in one cancer type and downregulated in another and can even change during the progression of the same cancer. Caution is thus needed before using miR-665 as a biomarker, and extrapolation between different cancer types is not advisable. Moreover, more detailed understanding of the different roles of miR-665 will help in determining its potential as a diagnostic and prognostic biomarker.

Scientific and technical assistance on welfare aspects related to housing and health of cats and dogs in commercial breeding establishments.

This Scientific Report addresses a mandate from the European Commission according to Article 31 of Regulation (EC) No 178/2002 on the welfare of cats and dogs in commercial breeding establishments kept for sport, hunting and companion purposes. The aim was to scrutinise recent recommendations made by the EU Platform on Animal Welfare Voluntary Initiative on measures to assist the preparation of policy options for the legal framework of commercial breeding of cats and dogs. Specifically, the main question addressed was if there is scientific evidence to support the measures for protection of cats and dogs in commercial breeding related to housing, health considerations and painful procedures. Three judgements were carried out based on scientific literature reviews and, where possible a review of national regulations. The first judgement addressed housing and included: type of accommodation, outdoor access, exercise, social behaviour, housing temperature and light requirements. The second judgement addressed health and included: age at first and last breeding, and breeding frequency. Judgement 3 addressed painful procedures (mutilations or convenience surgeries) and included: ear cropping, tail docking and vocal cord resections in dogs and declawing in cats. For each of these judgements, considerations were provided indicating where scientific literature is available to support recommendations on providing or avoiding specific housing, health or painful surgical interventions. Areas where evidence is lacking are indicated.

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.

Challenging the Hypothesis of in Utero Microbiota Acquisition in Healthy Canine and Feline Pregnancies at Term: Preliminary Data.

At present, there are no data on the presence of bacteria in healthy canine and feline pregnancies at term. Here, we investigated the uterine microbiome in bitches (n = 5) and queens (n = 3) undergoing elective cesarean section in two facilities. Samples included swabs from the endometrium, amniotic fluid, and meconium, and environmental swabs of the surgical tray as controls. Culture and 16S rRNA gene sequencing were used to investigate the presence of bacteria. Culture was positive for 34.3% of samples (uterus n = 3, amniotic fluid n = 2, meconium n = 4, controls n = 0), mostly with low growth of common contaminant bacteria. With sequencing techniques, the bacterial abundance was significantly lower than in environmental controls (p < 0.05). Sequencing results showed a species-specific pattern, and significant differences between canine and feline bacterial populations were found at order, family, and genus level. No differences were found in alpha and beta diversities between feto-maternal tissues and controls (p > 0.05). Dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria in different proportions based on tissue and species. Culture and sequencing results suggest that the bacterial biomass is very low in healthy canine and feline pregnancies at term, that bacteria likely originate from contamination from the dam's skin, and that the presence of viable bacteria could not be confirmed most of the time.

Development of a Microfluidic Chip Powered by EWOD for In Vitro Manipulation of Bovine Embryos.

Digital microfluidics (DMF) holds great potential for the alleviation of laboratory procedures in assisted reproductive technologies (ARTs). The electrowetting on dielectric (EWOD) technology provides dynamic culture conditions in vitro that may better mimic the natural embryo microenvironment. Thus far, EWOD microdevices have been proposed for in vitro gamete and embryo handling in mice and for analyzing the human embryo secretome. This article presents the development of the first microfluidic chip utilizing EWOD technology designed for the manipulation of bovine embryos in vitro. The prototype sustains the cell cycles of embryos manipulated individually on the chips during in vitro culture (IVC). Challenges related to the chip fabrication as well as to its application during bovine embryo IVC in accordance with the adapted on-chip protocol are thoroughly discussed, and future directions for DMF in ARTs are indicated.