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Miranda donkeys are an endangered autochthonous breed of Portugal. The HemoCue WBC DIFF and HemoCue Hb 201 portable analyzers, developed as a simplified alternative method for total and differential WBC count and hemoglobin measurement, respectively, may be relevant tools in veterinary practice. This study aimed to validate these instruments using Miranda donkey blood samples. For the HemoCue WBC DIFF, most parameters showed acceptable intra- and inter-assay precision with coefficients of variation (CV) below 5%, except for monocytes and eosinophils with higher CVs. The HemoCue Hb 201 showed CVs of 1.98% and 4.07%. Linearity correlation coefficients (r) ranged from 0.53 to 0.99 for HemoCue WBC DIFF and 0.99 for HemoCue Hb 201. Significant levels for neutrophils, lymphocytes, monocytes, eosinophils, and Hb measurements varied. Comparisons with ProCyte Dx showed an excellent correlation for WBC (r s = 0.96), neutrophils (r s = 0.91), lymphocytes (r s = 0.94), and eosinophils (r s = 0.90) but a poor correlation for monocytes and basophils. The HemoCue Hb 201 showed a correlation of r s = 0.96 with ProCyte Dx. In conclusion, both analyzers provided reliable results and are suitable for use in Miranda's donkey breed for WBC counts and Hb measurements.

Advances in tissue preservation techniques have allowed reproductive medicine and assisted reproductive technologies (ARTs) to flourish in recent years. Because radio- and chemotherapy procedures are often gonadotoxic, irreversible damage can preclude future gamete production and endocrine support. Accordingly, in recent years, the freezing and storage of gonadal tissue fragments prior to the first oncological treatment appointment and autologous transplantation post-recovery have been considered improved solutions for fertility recovery in cancer survivors. Nevertheless, the cryopreservation and transplantation of thawed tissues is still very limited, and positive outcomes are relatively low. This review aims to discuss the limitations of oncofertility protocols with a focus on the impacts of mitochondrial dysfunction, oxidative stress, and the loss of antioxidant defense in graft integrity., Competing Interests: The authors declare no conflicts of interest.

Age-related changes in the mitochondrial status of human cumulus cells (hCCs) impact oocyte quality; however, the relationship between hCC mitochondrial (dys)function and reproductive aging remains poorly understood. This study aimed to establish the interplay between hCC mitochondrial dysfunction and women's reproductive potential. In this investigation, 266 women were enrolled and categorized into two groups based on their age: a young group (<35 years old) and an advanced maternal age (AMA) group (≥35 years old). Comprehensive analysis of reproductive outcomes was conducted in our population. Various mitochondrial-related parameters were analyzed across distinct subsets. Specifically, mitochondrial membrane potential (∆Ψm) and mitochondrial mass were examined in 53 samples, mtDNA content in 25 samples, protein levels in 23 samples, bioenergetic profiles using an XF24 Extracellular Flux Analyzer in 6 samples, and levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in 39 and 43 samples, respectively. In our study, the reproductive potential of AMA women sharply decreased, as expected. Additionally, an impairment in the mitochondrial function of hCCs in older women was observed; however, no differences were found in terms of mitochondrial content. Regarding oxidative phosphorylation, metabolic profiling of hCCs from AMA women indicated a decrease in respiratory capacity, which was correlated with an age-dependent decrease in the ATP synthase (ATP5A1) protein level. However, intracellular ROS and ATP levels did not differ between groups. In conclusion, our study indicates that age-related dysfunction in hCCs is associated with impaired mitochondrial function, and, although further studies are required, ATP synthase could be relevant in this impairment.

The global trend of rising (male) infertility is concerning, and the unidentifiable causes in half of the cases, the so-called unknown origin male infertility (UOMI), demands a better understanding and assessment of both external/internal factors and mechanisms potentially involved. In this work, it was our aim to obtain new insight on UOMI, specifically on idiopathic (ID) and Unexplained male infertility (UMI), relying on a detailed evaluation of the male gamete, including functional, metabolic and proteomic aspects. For this purpose, 1114 semen samples, from males in couples seeking infertility treatment, were collected at the Reproductive Medicine Unit from the Centro Hospitalar e Universitário de Coimbra (CHUC), from July 2018-July 2022. Based on the couples' clinical data, seminal/hormonal analysis, and strict eligibility criteria, samples were categorized in 3 groups, control (CTRL), ID and UMI. Lifestyle factors and anxiety/depression symptoms were assessed via survey. Sperm samples were evaluated functionally, mitochondrially and using proteomics. The results of Assisted Reproduction Techniques were assessed whenever available. According to our results, ID patients presented the worst sperm functional profile, while UMI patients were similar to controls. The proteomic analysis revealed 145 differentially expressed proteins, 8 of which were specifically altered in ID and UMI samples. Acrosin (ACRO) and sperm acrosome membrane-associated protein 4 (SACA4) were downregulated in ID patients while laminin subunit beta-2 (LAMB2), mannose 6-phosphate isomerase (MPI), ATP-dependent 6-phosphofructokinase liver type (PFKAL), STAR domain-containing protein 10 (STA10), serotransferrin (TRFE) and exportin-2 (XPO2) were downregulated in UMI patients. Using random forest analysis, SACA4 and LAMB2 were identified as the sperm proteins with a higher chance of distinguishing ID and UMI patients, and their function and expression variation were in accordance with the functional results. No alterations were observed in terms of lifestyle and psychological factors among the 3 groups. These findings obtained in an experimental setting based on 3 well-defined groups of subjects, might help to validate new biomarkers for unknown origin male infertility (ID and UMI) that, in the future, can be used to improve diagnostics and treatments.

The Lusitano horse is gaining popularity in the equestrian world, and as a result, the significance of applied sports medicine for this breed is growing. As cardiology plays a crucial role in this field, numerous studies have been conducted to establish electrocardiographic reference values in various breeds to ensure a more accurate evaluation. However, studies regarding healthy Lusitano horses are lacking. So, this study aimed to establish electrocardiographic reference values for Lusitano horses, utilizing a sample of 82 clinically healthy animals. The evaluation involved lead II and base-apex lead measurements, with a median heart rate of 39 beats per minute being recorded. The P wave demonstrated a predominantly bifid configuration, while the QRS complex exhibited various forms. The most common QRS configurations were QR and R in lead II, and RS in the base-apex lead. Additionally, most T waves displayed a biphasic shape in both methods. Furthermore, statistically significant differences were noted based on age and gender. Some of the electrocardiographic values obtained differed from those previously published for other breeds. Given the relevance of electrocardiogram in cardiovascular evaluation, these findings bring valuable insights regarding the specific parameters for Lusitano horse and emphasize the importance of obtaining breed-specific electrocardiographic reference values.

Nanomaterials offer a broad spectrum of applications in biomedicine. The shapes of gold nanoparticles could modulate tumor cell behavior. Spherical (AuNP sp ), stars (AuNP st ) and rods (AuNP r ) shapes of polyethylene glycol coated-gold nanoparticles (AuNPs-PEG) were synthesized. Metabolic activity, cellular proliferation, and reactive oxygen species (ROS) were measured and the impact of AuNPs-PEG in metabolic enzymes function was evaluated by RT-qPCR in PC3, DU145, and LNCaP prostate cancer cells. All AuNPs were internalized, and the different morphologies of AuNPs showed to be an essential modulator of metabolic activity. For PC3 and DU145, the metabolic activity of AuNPs was found to rank in the following order from lowest to highest: AuNP sp -PEG, AuNP st -PEG, and AuNP r -PEG. Regarding LNCaP cells, the AuNP st -PEG were less toxic, followed by AuNP sp -PEG and AuNP r -PEG, but it seems not to be dose-dependent. The proliferation was lower in AuNP r -PEG in PC3 and DU145 cells but was stimulated around 10% in most conditions (0.001-0.1 mM) in LNCaP cells (not statistically significant). For 1 mM, LNCaP cells showed a significant decrease in proliferation only for AuNP r -PEG. The outcomes of the current study demonstrated that different AuNPs conformations influence cell behavior, and the correct size and shape must be chosen considering its final application in the field of nanomedicine.

Follicular fluid (FF) is the microenvironment where a growing oocyte develops. Intrafollicular communication ensures oocyte competence and is carried out through paracrine signaling, the exchange of molecules via gap junctions, and the trafficking of extracellular vesicles (EVs). The study of FF-derived EVs is important for both translational and fundamental research in the female reproductive field. This study aimed to compare the efficacy and purity of two EV isolation methods: size-exclusion chromatography (SEC) and ultracentrifugation (UC). EVs isolated using SEC and UC were compared regarding their size and concentration using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA); protein contamination was assessed with microBCA; specific EV markers were detected with Western blot, and EV morphology was studied with transmission electron microscopy (TEM). Our results show that although both techniques isolated small EVs, a significantly increased yield in particle number was clear with UC compared with SEC. On the other hand, SEC generated purer EVs with fewer protein contaminants and aggregates. In conclusion, the selection of the most suited approach to isolate EVs must be conducted considering the degree of recovery, purity, and downstream application of the isolated EVs.

Over the last years, the importance of microRNAs (miRNAs) has increasingly been recognised. Each miRNA is a short sequence of non-coding RNA that influences countless genes' expression and, thereby, contributes to several physiological pathways and diseases. It has been demonstrated that miRNAs participate in the development of many cardiovascular diseases (CVDs). This review synopsises the most recent studies emphasising miRNA's influence in several CVDs affecting dogs and cats. It provides a concise outline of miRNA's biology and function, the diagnostic potential of circulating miRNAs as biomarkers, and their role in different CVDs. It also discusses known and future roles for miRNAs as potential clinical biomarkers and therapeutic targets. So, this review gives a comprehensive outline of the most relevant miRNAs related to CVDs in Veterinary Medicine.

Despite the primary function of pioglitazone in antidiabetic treatment, this drug is a potent inducer of PPAR-γ, a crucial receptor that is involved in adipocyte differentiation. In this work, we propose an optimized methodology to enhance the differentiation of 3T3-L1 fibroblasts into adipocytes. This process is crucial for adipocyte secretome release, which is fundamental for understanding the molecular mechanisms that are involved in obesity for in vitro studies. To achieve this, a pioglitazone dose-response assay was determined over a range varying from 0 to 10 µM. Lipid accumulation was evaluated using Oil-Red-O. The results showed that 10 µM pioglitazone enhanced differentiation and increased secretome production. This secretome was then added into two cell lines: PC3 and RAW264.7. In the PC3 cells, an increase of aggressiveness was observed in terms of viability and proliferation, with the increase of anti-inflammatory cytokines. Conversely, in RAW264.7 cells, a reduction of viability and proliferation was observed, with a decrease in the overexpression of pro-inflammatory cytokines. Overall, the present work constitutes an improved method for adipocyte secretome production that is suitable for experimental biology studies and that could help with our understanding of the molecular mechanisms underlying adiposity influence in other cells.

This study aimed to evaluate maternal left ventricular (LV) systo-diastolic function using conventional and TDI echocardiography and included 10 healthy Saint-Bernard pregnant bitches. M-mode, peak transmitral flow velocities during early diastole (E) and atrial contraction (A), aortic and pulmonic flow, myocardial performance index (MPI), TDI studies (peak myocardial velocities during early diastole (E'), atrial contraction (A') and peak systole (S')), and blood pressure were measured at 21 to 28 (T1), 40 (T2) and 60 (T3) days of gestation and four to eight weeks postpartum (T4). Cardiac output and heart rate were 20% and 9% higher at T3, respectively, compared to T4 ( p < 0.01). Lateral S' was 36% higher at T3 than at T1 ( p < 0.05). Changes in diastolic function were demonstrated by 10% lower E wave and 15% A wave at T1, compared to T4 ( p < 0.05). E' and A' were 23% and 42% higher at T3 compared to T4 ( p < 0.01). Both lateral E/E' and E'/A' were 6% and 19% lower at T3 compared to T1 ( p < 0.01 and p < 0.05, respectively). At T3, MPI was 51% and 34% lower when compared to T1 or T2 ( p < 0.05). The echocardiographic evaluation of maternal cardiac function is important, as structural, and functional changes occur throughout pregnancy.

Type 2 Diabetes Mellitus (T2D) is defined as a chronic condition caused by beta cell loss and/or dysfunction and insulin resistance (IR). The discovering of novel biomarkers capable of identifying T2D and other metabolic disorders associated with IR in a timely and accurate way is critical. In this review, 2-hydroxybutyric acid (2HB) is presented as that upheaval biomarker with an unexplored potential ahead. Due to the activation of other metabolic pathways during IR, 2HB is synthesized as a coproduct of protein metabolism, being the progression of IR intrinsically related to the increasing of 2HB levels. Hence, the focus of this review will be on the 2HB metabolite and its involvement in glucose homeostasis. A literature review was conducted, which comprised an examination of publications from different databases that had been published over the previous ten years. A total of 19 articles fulfilled the intended set of criteria. The use of 2HB as an early indicator of IR was separated into subjects based on the number of analytes examined simultaneously. In terms of the association between 2HB and IR, it has been established that increasing 2HB levels can predict the development of IR. Thus, 2HB has demonstrated considerable promise as a clinical monitoring molecule, not only as an IR biomarker, but also for disease follow-up throughout IR treatment.

The pyrethroid insecticide cypermethrin is used for agricultural and public health campaigns. Its residues may contaminate soils and the beneficial soil organisms, like the earthworms, that may ingest the contaminated soil particles. Due to its ecological relevance, earthworms Eisenia andrei/fetida have been used in different ecotoxicological tests. The avoidance of soils treated with cypermethrin by compost worms Eisenia andrei was studied here as a bioindicator of the influence of treatment dosage and the pesticide formulation in three different agricultural soils indicated by the Brazilian environmental authorities for ecotoxicological tests. This earthworms' behavior was studied here as a first attempt to propose the test for regulation purposes. The two-compartment test systems, where the earthworms were placed for a two-day exposure period, contained samples of untreated soil alone or together with soil treated with technical grade or wettable powder formulation of cypermethrin. After 48 h, there was no mortality, but the avoidance was clear because all earthworms were found in the untreated section of each type of soil (p < 0.05). No differences were found by the Fisher's exact test (p ≤ 1.000) for each soil and treatment, demonstrating that the different soil characteristics, the cypermethrin concentrations and formulation, as well as the smaller amounts of soil and earthworms did not influence the avoidance behavior of the earthworms to cypermethrin. The number and range of treatments used in this study do not allow a detailed recommendation of the conditions applied here, but to the best of our knowledge, this is the first reported attempt to identify the avoidance of pesticide treated tropical soils by earthworms.