The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses.

Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.

Oncostatin M-Enriched Small Extracellular Vesicles Derived from Mesenchymal Stem Cells Prevent Isoproterenol-Induced Fibrosis and Enhance Angiogenesis.

Myocardial fibrosis is a pathological hallmark of cardiac dysfunction. Oncostatin M (OSM) is a pleiotropic cytokine that can promote fibrosis in different organs after sustained exposure. However, OSM released by macrophages during cardiac fibrosis suppresses cardiac fibroblast activation by modulating transforming growth factor beta 1 (TGF-ß1) expression and extracellular matrix deposition. Small extracellular vesicles (SEVs) from mesenchymal stromal cells (MSCs) are being investigated to treat myocardial infarction, using different strategies to bolster their therapeutic ability. Here, we generated TERT-immortalized human MSC cell lines (MSC-T) engineered to overexpress two forms of cleavage-resistant OSM fused to CD81TM (OSM-SEVs), which allows the display of the cytokine at the surface of secreted SEVs. The therapeutic potential of OSM-SEVs was assessed in vitro using human cardiac ventricular fibroblasts (HCF-Vs) activated by TGF-ß1. Compared with control SEVs, OSM-loaded SEVs reduced proliferation in HCF-V and blunted telo-collagen expression. When injected intraperitoneally into mice treated with isoproterenol, OSM-loaded SEVs reduced fibrosis, prevented cardiac hypertrophy, and increased angiogenesis. Overall, we demonstrate that the enrichment of functional OSM on the surface of MSC-T-SEVs increases their potency in terms of anti-fibrotic and pro-angiogenic properties, which opens new perspectives for this novel biological product in cell-free-based therapies.

Transcriptome dynamics in developing testes of domestic cats and impact of age on tissue resilience to cryopreservation.

BACKGROUND: Fundamental knowledge of cellular and molecular mechanisms in developing testicular tissues is critical to better understand gonadal biology and responses to non-physiological conditions. The objective of our study was to (1) analyze transcriptome dynamics in developing testis of the domestic cat and (2) characterize age effects on the initial response of the tissue to vitrification. Tissues from adult and juvenile cats were processed for histology, DNA integrity, and RNA sequencing analyses before and after vitrification. RESULTS: Transcriptomic findings enabled to further characterize juvenile period, distinguishing between early and late juvenile tissues. Changes in gene expression and functional pathways were extensive from early to late juvenile to adult development stages. Additionally, tissues from juvenile animals were more resilient to vitrification compared to adult counterparts, with early juvenile sample responding the least to vitrification and late juvenile sample response being closest to adult tissues. CONCLUSIONS: This is the first study reporting comprehensive datasets on transcriptomic dynamic coupled with structural analysis of the cat testis according to the age and exposure to cryopreservation. It provides a comprehensive network of functional terms and pathways that are affected by age in the domestic cat and are either enriched in adult or juvenile testicular tissues.

Comparison of different intracellular cryoprotectants on the solid surface vitrification of red-rumped agouti (Dasyprocta Leporina Lichtenstein, 1823) ovarian tissue.

In contributing to the conservation of wild rodents, the aim of this study was to evaluate the use of distinct cryoprotectants, separately or in combination, for solid surface vitrification (SSV) of red-rumped agouti ovarian tissue. Ovarian cortex from nine females was recovered and fragmented. Fresh fragments (control) were used to analyse the pre-antral follicle (PF) morphology using a histologic procedure, viability using the Trypan blue test, cell proliferation by counting the argyrophilic nucleolar organizing regions (Ag-NORs technique) and DNA integrity using the TUNEL assay. The remaining fragments were vitrified using SSV method with 3 M or 6 M ethylene glycol (EG) or dimethyl sulfoxide (DMSO), or in combination (3 M EG/3 M DMSO), and further evaluated as reported for the fresh samples. All cryoprotectants were effective at preserving PFs morphology compared to the control group (80.7 ± 5.21%), except 6 M EG and 3 M DMSO that provoked a significant (p < .05) decrease on the values of morphologically normal primary (60.0 ± 19.0%) and primordial (44 ± 4.5%) follicles, respectively. Regarding viability, all cryoprotectants provided values similar to that verified for the control group (79.0%), but a significant decrease (p < .05) was observed with EG/DMSO combination (59%). Using Ag-NORs technique, the highest (p < .05) cell proliferative capacity was detected when using EG at each tested concentration. The TUNEL proved the preservation of DNA integrity regardless of the cryoprotectant. In summary, we suggest the use of 3 M EG for the solid surface vitrification of red-rumped agouti ovarian tissue.

The Systemic Immune Response to Collagen-Induced Arthritis and the Impact of Bone Injury in Inflammatory Conditions.

Rheumatoid arthritis (RA) is a systemic disease that affects the osteoarticular system, associated with bone fragility and increased risk of fractures. Herein, we aimed to characterize the systemic impact of the rat collagen-induced arthritis (CIA) model and explore its combination with femoral bone defect (FD). The impact of CIA on endogenous mesenchymal stem/stromal cells (MSC) was also investigated. CIA induction led to enlarged, more proliferative, spleen and draining lymph nodes, with altered proportion of lymphoid populations. Upon FD, CIA animals increased the systemic myeloid cell proportions, and their expression of co-stimulatory molecules CD40 and CD86. Screening plasma cytokine/chemokine levels showed increased tumor necrosis factor-α (TNF-α), Interleukin (IL)-17, IL-4, IL-5, and IL-12 in CIA, and IL-2 and IL-6 increased in CIA and CIA+FD, while Fractalkine and Leptin were decreased in both groups. CIA-derived MSC showed lower metabolic activity and proliferation, and significantly increased osteogenic and chondrogenic differentiation markers. Exposure of control-MSC to TNF-α partially mimicked the CIA-MSC phenotype in vitro. In conclusion, inflammatory conditions of CIA led to alterations in systemic immune cell proportions, circulating mediators, and in endogenous MSC. CIA animals respond to FD, and the combined model can be used to study the mechanisms of bone repair in inflammatory conditions.

Identification of novel protein phosphatases as modifiers of alpha-synuclein aggregation in yeast.

Alpha-synuclein (aSyn) is a key player in a group of neurodegenerative diseases commonly known as synucleinopathies. Recent findings indicate phosphorylation in several aSyn residues can modulate its aggregation and subcellular localization, thereby affecting pathological processes. However, the precise molecular mechanisms governing aSyn phosphorylation are still unclear. Recent studies investigated the role of various families of protein kinases, such as the polo-like kinases, G protein-coupled receptor kinases or casein kinases. In contrast, our understanding of the phosphatases involved in the dephosphorylation of aSyn is rather limited. Here, we exploited the unique toolbox of the yeast Saccharomyces cerevisiae in order to identify novel phosphatases capable of modulating aSyn phosphorylation, inclusion formation and toxicity of human aSyn. In summary, given the association between aSyn phosphorylation and pathology in Parkinson's disease and other synucleinopathies, modulation of this post-translational modification may constitute an attractive target for therapeutic intervention.

Cryopreservation of collared peccary (Pecari tajacu L., 1758) epididymal sperm using extenders based on Tris and powdered coconut water (ACP®-116c).

SummaryThe aim of this study was to establish a functional freezing-thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen-thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.

OPNa Overexpression Is Associated with Matrix Calcification in Thyroid Cancer Cell Lines.

Osteopontin (OPN) spliced variants (OPN-SV: OPNa, OPNb, and OPNc) are aberrantly expressed in tumors and frequently associated with cancer progression. This holds true for papillary thyroid carcinoma (PTC), which is the most common type of thyroid cancer (TC). PTC often presents with desmoplasia and dystrophic calcification, including psammoma bodies (PB). This work aimed to investigate total OPN (tOPN) and OPN-SV expression and their association with the presence of PB in the PTC classical variants (cPTC), as well as the involvement of OPN-SV in matrix calcification of TC cell lines. We found that cPTC samples presenting PB showed higher OPN expression levels. In TC cell lines, OPNa overexpression promotes higher matrix calcification and collagen synthesis when compared to that of clones overexpressing OPNb or OPNc. In response to OPN knockdown, calcification was inhibited, paralleled with the downregulation of calcification markers. In conclusion, our data evidenced that OPN expression is associated with the presence of PB in cPTC samples. Among the OPN-SV, OPNa is the main contributor to matrix calcification in tested TC cells, providing clues to a better understanding on the biology and ethiopathogenesis of the calcification process in TC cells.

Characterisation and cryopreservation of the ovarian preantral follicle population from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831).

The aim of the present study was to characterise the ovarian preantral follicle (PF) population and to establish a solid surface vitrification (SSV) process using dimethyl sulfoxide (DMSO) as a cryoprotectant for preservation of ovarian tissue from yellow-toothed cavies (Galea spixii). Ovaries were fixed for PF population analysis or were subjected to the SSV process. The mean (± s.e.m.) PF population per ovarian pair was estimated to be 416.0±342.8. There were 140.0±56.0 (63.4%) and 125.0±58.0 (64.0%) primary follicles on the right and left ovaries, respectively. The proportion of this follicle category was significantly greater than that of other follicle categories (P<0.05). The diameter of follicles (123.7±18.3µm), oocytes (50.1±5.0µm) and nuclei (14.27±2.01µm) was larger for secondary ones when compared with other PFs categories. Most PFs were morphologically normal (94.6%), with light microscopy identifying only a few atretic follicles (5.4%). After SSV, there was a reduction in the proportion of morphologically normal PFs compared with the non-vitrified group (69.5% vs 91.2%, respectively). Transmission electron microscopy revealed preservation of oocytes and granulosa cell membranes and the morphological aspect of follicles; the primary change observed in some vitrified PFs was the presence of vacuoles in the oocytes and granulosa cells cytoplasm and turgid mitochondria. In conclusion, the present study provides an estimative and characterization for the PF population in ovaries of G. spixii. Moreover, we report its PFs cryopreservation using an SSV process.

Phakic Intraocular Lens Implantation: Refractive Outcome and Safety in Patients with Anterior Chamber Depth between 2.8 and 3.0 versus ≥3.0 mm.

PURPOSE: To compare endothelial cell (EC) variation after anterior chamber phakic intraocular lens (AC-pIOL) implantation in highly myopic patients with a preoperative anterior chamber depth (ACD) between 2.8 and 3.0 versus ≥3.0 mm. METHODS: A total of 280 eyes submitted to primary AC-pIOL implantation were analyzed. Pre- and postoperative values for uncorrected distance visual acuity, corrected distance visual acuity, spherical equivalent, ACD (endothelial surface), and EC count were collected. The eyes were divided into 2 groups: group A - ACD between 2.8 and 3.0 mm; group B - ACD ≥3.0 mm. Mean global EC loss (ECL) and loss for each ACD group, according to pIOL type, were analyzed. RESULTS: Significant improvement of the spherical equivalent (-11.38 ± 4.57 vs. -0.49 ± 0.79; p = 0.000) and a significant decrease in EC density (2,810.95 ± 343.88 vs. 2,584.09 ± 374.88 cells/mm2; p = 0.000) were noted. The mean annual ECL was -2.19 ± 3.97%. Regarding group A (n = 80), a mean annual ECL of -2.06 ± 3.88% was registered, higher for the Acrysof Cachet® subtype, while group B (n = 200) showed -2.25 ± 4.01% ECL, higher for the Verisyse® subtype. There was no significant difference between the groups (p = 0.96). CONCLUSIONS: AC-pIOL implantation significantly improves the spherical equivalent in myopic patients. The mean annual ECL after pIOL implantation was higher in the larger ACD group, but this value was not statistically significant. A 2.8-mm ACD value seems to be a safe cutoff for AC-pIOL implantation.

Regioselectively Controlled Synthesis of N-Substituted (Trifluoromethyl)pyrimidin-2(1H)-ones.

A simple and regioselectively controlled method for the preparation of both 1,4- and 1,6-regioisomers of 1-substituted 4(6)-trifluoromethyl-pyrimidin-2(1H)-ones is described. Both regioisomers were synthesized from the cyclocondensation reaction of 4-substituted 1,1,1-trifluoro-4-methoxybut-3-en-2-ones: with nonsymmetric ureas for the 1-substituted 4-(trifluoromethyl)pyrimidin-2(1H)-ones (1,4-isomer) and with nonsymmetric 1-substituted 2-methylisothiourea sulfates for the synthesis of 1-substituted 6-(trifluoromethyl)pyrimidin-2(1H)-ones (1,6-isomer). Each method furnished only the respective isomer in very good yields. The structure of the products was assigned based on the (1)H and (13)C NMR as well as 2D HMBC spectral analysis.

Circulating extracellular vesicles: Their role in tissue repair and regeneration.

Extracellular vesicles (EVs) have been a growing interest of the scientific community in recent years due to the wide possibilities of their evaluation as biomarkers of disease, and their potential to be used as therapeutic agents or vehicles. EVs that circulate in plasma carry proteins and nucleic acids, potentially to distant locations in the body where they can interfere with several cellular processes. To aid understanding of this rapidly evolving field, circulating EVs, including immune cell-derived ones, are reviewed here. Their cellular origins and described functions are discussed in a perspective of their contribution to regenerative processes. Different techniques for EV engineering and examples of their application are reviewed as a strong future direction of EV research. A summary of important aspects yet to be addressed ties up this review.

Antitumoral activity of a trichloromethyl pyrimidine analogue: molecular cross-talk between intrinsic and extrinsic apoptosis.

Acute lymphoblastic leukemia (ALL) is a malignant disorder caused by the proliferation of lymphoid progenitor cells and is the most common cancer in children. Cytotoxic nucleoside analogues are important chemotherapeutic agents, which are used in many cancers, including leukemias. In this study, we investigated the effects of the synthetic nucleoside analogue 1-(5,5,5-trichloro-2-methoxy-4-oxopenten-2-yl)-4-trichloromethyl-pyrimidin-2(1H)-one, named compound 3 or C3, on leukemia cell lines. The compound stimulated cell death by apoptosis, evidenced by DNA fragmentation, phosphatidylserine externalization, and caspase-3 activation. Compound 3 seemed to trigger several cell death pathways. The mitochondrial pathway was evidenced through a disturbance of mitochondrial membrane potential, strong cytochrome c liberation, decrease of antiapoptotic Bcl-2 protein expression, and caspase-9 activation. The C3 also induced caspase-8 and -12 activation, an increase in the intracellular calcium level, and an overproduction of reactive oxygen species. Increased caspase 8 activity suggests that the extrinsic pathway was activated and that the ROS production and enzyme activity alteration (glutathione S-transferase, glutathione peroxidase, catalase, and glutathione reductase) might be related to oxidative stress. Finally, the increase in calcium release, CHOP expression, and caspase-12 activity might characterize endoplasmic reticulum stress. Compound 3 was likewise cytotoxic to leukemic and melanoma human cell lines. Taken together, the results contribute to further understanding the new pyrimidine analogue as a potential chemotherapeutic drug or lead molecule.

Effect of Diluents and Storage Time on the Cryopreservation of Collared Peccary (Pecari tajacu) Semen after Cooling Storage in a Transport Container at 5 °C.

We verified the possibility of cooling peccary semen for 4, 24, and 48 h before cryopreservation, using different dilution media (TRIS + egg yolk (20%) and PRIMXcell Ultra). Ten ejaculates were divided equally into six aliquots and then diluted. Two aliquots were stored in a biological incubator (4 h), and the remaining aliquots were stored in a commercial container, the Botutainer® (24 and 48 h), both at 5 °C. The samples were cryopreserved and then evaluated for kinetic parameters, functionality, integrity, mitochondrial activity, morphology, and sperm binding capacity. After thawing, samples diluted in TRIS showed total motility of 43.4 ± 6.8%, 48.4 ± 6.2%, and 38.6 ± 5.0% after cooling for 4, 24, and 48 h before cryopreservation, respectively. Such results are significantly greater than those achieved with the use of PRIMXcell diluent for 4 (8.3 ± 2.8%), 24 (4.7 ± 1.4%), and 48 h (4.8 ± 2.9%) storage (p < 0.05). Furthermore, TRIS provided better preservation of sperm membrane integrity when samples were cooled for 24 h (44.5 ± 4.7%) before cryopreservation compared to those samples diluted in PRIMXcell Ultra stored for 24 (25.7 ± 4.0%) and 48 h (25.2 ± 4.0%) before freezing (p < 0.05). In summary, we suggest TRIS diluent + egg yolk (20%) as an effective option to allow semen to cool for 24 or 48 h in a transport container before cryopreservation.

In vivo phage display identifies novel peptides for cardiac targeting.

Heart failure remains a leading cause of mortality. Therapeutic intervention for heart failure would benefit from targeted delivery to the damaged heart tissue. Here, we applied in vivo peptide phage display coupled with high-throughput Next-Generation Sequencing (NGS) and identified peptides specifically targeting damaged cardiac tissue. We established a bioinformatics pipeline for the identification of cardiac targeting peptides. Hit peptides demonstrated preferential uptake by human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and immortalized mouse HL1 cardiomyocytes, without substantial uptake in human liver HepG2 cells. These novel peptides hold promise for use in targeted drug delivery and regenerative strategies and open new avenues in cardiovascular research and clinical practice.

Head-to-head comparison of relevant cell sources of small extracellular vesicles for cardiac repair: Superiority of embryonic stem cells.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.

Effect of Detergents Based on Sodium Dodecyl Sulfate on Functional Metrics of Frozen-Thawed Collared Peccary (Pecari tajacu) Semen.

We evaluated the effects of detergents based on sodium dodecyl sulfoxide (SDS) on the functional parameters of collared peccary frozen-thawed sperm. Semen aliquots from ten individuals were diluted in a Tris-egg yolk-glycerol extender alone or with 0.5% Equex STM® paste or SDS (at 0.1%, 0.3% or 0.5% (v/v) concentration). Samples were fast frozen in liquid nitrogen with a post-thaw evaluation of motility, membrane functionality and integrity, mitochondrial activity, sperm binding ability and thermal resistance. The treatments without SDS (41.8 ± 3.5%) and those containing Equex (41.8 ± 4.4%) or 0.1% SDS (41.2 ± 5.5%) provided greater sperm motility (p < 0.05) than those containing SDS 0.3% (30.5 ± 4.7%) and 0.5% (31.2 ± 6.3%). Immediately after thawing, only treatments containing 0.1% SDS effectively preserved sperm straightness (STR) when compared to the negative control. All treatments preserved the amplitude of lateral head (ALH) and straightness (STR) during a thermal resistance test (p > 0.05), but SDS 0.5% impaired the membrane functionality and mitochondrial activity after thawing (p < 0.05). All treatments provided a similar recovery of sperm binding ability after thawing (p < 0.05). Our results showed that the addition of 0.1% SDS to the Tris-yolk-glycerol extender optimized the freeze-thaw recovery of peccary semen.

Changes in Sperm Morphology, Morphometry, and Motility from the Epididymis to the Vas Deferens in Rheas (Rhea americana, Linnaeus, 1758).

The objective was to characterize morphological, morphometric, and ultrastructural changes in rhea spermatozoa between the epididymis and the vas deferens. Sperm samples were collected from the reproductive tracts of seven adult individuals and evaluated for sperm characteristics using brightfield microscopy as well as ultrastructural features using scanning electron microscopy (SM). Mean sperm count tended to increase in the vas deferens (378.0 ± 135.0 × 106) compared to the epididymis (201.0 ± 77.4 × 106). Percentages of motile sperm grew from 37.0 ± 4.9% in the epididymis to 58.5 ± 7.7% in the vas deferens. The proportion of normal spermatozoa was 75.6 ± 1.8% and most common defects were bent tails (9.7 ± 0.9%). However, these proportions were not different between epididymis and vas deferens. SM analysis revealed further features of rhea spermatozoa. Normal rhea spermatozoa were threadlike with an acrosome (0.95 ± 0.0 µm), head (7.53 ± 0.01 µm), midpiece (2.08 ± 0.01 µm), and tail (30.7 ± 0.06 µm). Lengths of sperm acrosome, head, midpiece, and tail were longer in the vas deferens compared to the epididymis. Our findings suggest that rhea spermatozoa undergo a maturation process during the passage from the epididymis to the vas deferens.

Different Methods for Seminal Plasma Removal and Sperm Selection on the Quality and Fertility of Collared Peccary Sperm.

Methods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll® gradient densities (PG 45-90% and PG 35-70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45-90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35-70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45-90% was used for fertilization. PG 45-90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2-26.3%) and morula (8.1-10.5%) rates did not differ between the groups. Therefore, PG 45-90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes.

Medical residency in Portugal: a cross-sectional study on the working conditions.

Objectives: The current European crisis in human resources in health has opened the debate about working conditions and fair wages. This is the case with Resident doctors, which have faced challenges throughout Europe. In Portugal, they account for about a third of the doctors in the Portuguese National Health Service. No studies to date objectively demonstrate the working conditions and responsibilities undertaken. This study aims to quantify the residents' workload and working conditions. Methods: Observational, retrospective cross-sectional study which involved a survey on the clinical and training activity of Portuguese residents, actively working in September 2020. The survey was distributed through e-mail to residents' representatives and directly to those affiliated with the Independent Union of Portuguese Doctors. The descriptive analysis assessed current workload, and logistic regression models analyzed associations with geographical location and residency seniority. Results: There were a total of 2,012 participants (19.6% of invited residents). Of the residents giving consultations, 85.3% do so with full autonomy. In the emergency department, 32.1% of the residents work 24 h shifts and 25.1% work shifts without a specialist doctor present. Regarding medical training, 40.8% invest over EUR 1,500 annually. Autonomy in consultations was associated with being a Family Medicine resident (OR 4.219, p < 0.001), being a senior resident (OR 5.143, p < 0.001), and working in the Center (OR 1.685, p = 0.009) and South regions (OR 2.172, p < 0.001). Seniority was also associated with investing over EUR 1,500 in training annually (OR 1.235, p = 0.021). Conclusion: Residents work far more than the contracted 40 h week, often on an unpaid basis. They present a high degree of autonomy in their practice, make a very significant personal and financial investment in medical training, with almost no time dedicated to studying during working hours. There is a need to provide better working conditions for health professionals, including residents, for the sake of the sustainability of health systems across Europe.