Potassium Hydroxide Extraction of Polyphenols from Olive Leaves: Effect on Color and Acrylamide Formation in Black Ripe Olives.

Olive leaves are generated in large quantities in olive oil and table olive factories. This waste is currently used for multiple purposes, one of them being the extraction of bioactive substances, in particular phenolic compounds. The aims of this study were (i) to obtain a new polyphenolic extract from potassium hydroxide (KOH) -treated olive leaves; and (ii) to reduce acrylamide formation in black olives by using this extract. The results showed that olive leaves and leafless branches of the Manzanilla cultivar treated with 10 g/L KOH provide a solution that, concentrated under vacuum, had >6000 mg/L hydroxytyrosol and >2000 mg/L of hydroxytyrosol 4-glucoside. Moreover, the residual material generated after the treatment with KOH could be used for agronomic purposes, due to its high potassium content. The employment of this non-bitter extract during the darkening step of black ripe olive processing then resulted in darker fruits with higher potassium content. Likewise, the addition of the extract into the packing brine reduced the acrylamide formation by up to 32%, although this effect was batch-dependent. KOH olive extract could be useful for the reduction in acrylamide in black ripe olives along with the enrichment of this product in phenolic compounds and potassium.

Bioavailability profiling shows differences in OA, DTX1 and DTX2 toxins that justify their toxicity.

The marine toxins of the Okadaic acid (OA) group are natural compounds produced by dinoflagellates that enters the food chain by accumulating in seafood. They are responsible for Diarrhetic Shellfish Poisoning (DSP) events in humans over the world and therefore are also jointly named as Diarrhetic Shellfish Toxins (DSTs). The main objective of this study was to evaluate symptoms, toxicity, absorption, distribution, and elimination of OA, Dinophysistoxin-1 (DTX1), and Dinophysistoxin-2 (DTX2) at the sublethal dose of 90 µg toxin/kg bw administered through voluntary feeding to mice. The toxin comparison highlighted that OA and DTX1 induced more severe and specific symptoms such as diarrhea. After oral ingestion toxins were distributed through the entire organism being detected in liver, kidney, stomach, small and large intestine. Predominant excretion of the toxins was observed in feces, with OA exhibiting fast elimination, while DTX2 was showing prolonged excretion. The passage and accumulation of toxins in gastrointestinal organs instigated macroscopic damage in the stomach, small and large intestine that could persist up to 120 h. These findings highlight the importance of pharmacokinetic of sublethal doses of DSTs administered by voluntary feeding in their toxicity and their implication for public health.

Seminal plasma removal for medium-term preservation of ram sperm at 5 °C.

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

The Adaptation Time to the Extender as a Crucial Step for an Accurate Evaluation of Ram Sperm Quality during the Liquid Storage.

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.

The characterization of CellROX™ probes could be a crucial factor in ram sperm quality assessment.

Several authors have demonstrated that low levels of reactive oxygen species (ROS) are necessary for the physiological functions of sperm, such as capacitation, hyperactivation, acrosomal reaction and fertilization. However, high levels of ROS are associated with oxidative stress and detrimental effects on fertility. Consequently, deep characterization of ROS presence using different fluorescent probes could be crucial. In this sense, the study of intracellular ROS localization and the relationships between ROS and other conventional parameters could improve the characterization of sperm quality for semen preservation protocols in rams. In this work, a multiparametric study was carried out by analyzing four experimental groups of ram sperm with different initial qualities: fresh semen (from both breeding and nonbreeding seasons), frozen-thawed semen and, a positive control group treated with hydrogen peroxide (300 µM) as a marker of extreme damage. Sperm analyses, including viability, apoptosis, lipid peroxidation, motility and kinetic parameters, were applied to compare several experimental groups with different sperm qualities. After that, the signals from two different ROS probes: CellROX™ Deep Red (CRDR) and Green (CRG), were examined by flow cytometry (percentage of cells that express ROS) and fluorescence microscopy (intracellular ROS location). Comparing conventional parameters, fresh samples from the breeding season showed the highest sperm quality, while the positive control samples showed the worst sperm quality. Concerning the ROS probes, the CRDR levels were higher in fresh samples from the breeding season than in the positive control and cryopreserved samples. Surprisingly, CRG presented its highest level (P < 0.05) in the positive control group treated with peroxide by flow cytometry. CRDR and CRG presented opposite labeling patterns that were corroborated by fluorescence microscopy, which determined that the probes localized in different parts of sperm. CRDR was found in the sperm mitochondrial region, while CRG was observed in the cell nucleus, suggesting that ROS localization is an important factor. Finally, our study indicates that CRDR is correlated with proper viability and sperm motility, and could be associated with high mitochondrial activity, while CRG is associated with sperm damage.

Buenas Prácticas de Manufactura de carnicerías y prevalencia de Escherichia coli productor de toxina Shiga y Salmonella spp. aisladas en carne molida de carnicerías de Asunción, Paraguay

La inocuidad de la carne comercializada debe estar garantizada en la cadena de producción, para evitar enfermedades transmitidas por alimentos (ETA). Escherichia coli productor de toxina Shiga (STEC) y Salmonella spp. pueden encontrarse en el tracto gastrointestinal de los bovinos y contaminar la carne de consumo humano, pudiendo causar enfermedades en el hombre. Este trabajo tuvo como objetivo evaluar las condiciones higiénico-sanitarias de 52 carnicerías localizadas en Asunción y detectar la frecuencia de STEC y Salmonella spp. en muestras de carne molida. Las condiciones higiénico-sanitarias se evaluaron mediante la estimación del riesgo, utilizando una escala de clasificación por categorías. La detección de STEC y Salmonella spp. se realizó por PCR en tiempo real. En la evaluación inicial, se clasificaron a 33% de las carnicerías como de alto y moderado riesgo. Se detectó STEC no-O157 en un 50% (130/258) de las muestras y Salmonella spp. en un 11% (29/258). Se realizaron acciones de mejora. En la etapa post-intervención, no se detectaron carnicerías de alto riesgo. En el muestreo de seguimiento se detectó un 29% (66/237) de muestras positivas para STEC no-O157 y 7% (16 /237) para Salmonella spp. Este estudio permitió realizar recomendaciones específicas y detalladas a cada carnicería, lo que tuvo un efecto significativo en la mejora de sus condiciones. Esta situación resalta la importancia de continuar fortaleciendo la vigilancia multisectorial y multidisciplinaria. Es imperativo que los establecimientos que se dedican al rubro, implementen las Buenas Prácticas de Manufactura (BPM) como una medida para reducir los riesgos asociados.

The safety of marketed meat must be guaranteed in the production chain, to avoid foodborne illness. Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp. can be found in the gastrointestinal tract of cattle and contaminate meat for human consumption, potentially causing diseases in humans. This work aimed to evaluate the hygienic-sanitary conditions of 52 butcher shops located in Asunción and detect the frequency of STEC and Salmonella spp. in ground beef samples. Hygienic-sanitary conditions were evaluated by estimating risk, using a categorical classification scale. The detection of STEC and Salmonella spp. was performed by real-time PCR. In the initial evaluation, 33% of the butcher shops were classified as high and moderate risk. STEC non-O157 was detected in 50% (130/258) of the samples and Salmonella spp. in 11% (29/258). Improvement actions were carried out. In the post-intervention stage, no high-risk butcher shops were detected. In the follow-up sampling, 29% (66/237) of positive samples were detected for STEC non-O157 and 7% (16/237) for Salmonella spp. This study allowed specific and detailed recommendations to be made to each butcher shop, which had a significant effect on improving their conditions. This situation highlights the importance of continuing to strengthen multisectoral and multidisciplinary surveillance. It is imperative that establishments dedicated to the sector implement Good Manufacturing Practices (GMP) as a measure to reduce associated risks.

La gestión editorial de las revistas científicas como componente de la actividad de Ciencia, Tecnología e Innovación

Introducción: La actividad de Ciencia, Tecnología e Innovación constituye en la actualidad un pilar básico en el desarrollo de un país. Las revistas científicas son parte esencial de la investigación científica por ser el principal vehículo para la divulgación de los resultados. Objetivo: Realizar una valoración teórica sobre la concepción de la gestión editorial como una actividad científica. Método: Se utilizaron métodos de análisis documental mediante la selección de información obtenida de bases de datos científicas como SciELO, Scopus y Google Académico, con la utilización de criterios: gestión editorial; revistas científicas y actividad de ciencia y tecnología. Resultados: Se abordaron aspectos relacionados con la evolución de la comunicación científica a través de las revistas, sus enfoques tradicionalista, tecnológico y social; así como su papel en la comunicación científica de los resultados de la investigación científica y la gestión editorial para la publicación de las revistas científicas. Conclusiones: Los autores conciben la gestión editorial de las revistas científicas como un proceso científico-técnico con marcado carácter pedagógico que, a través de la evaluación rigurosa del resultado de investigación, depura y socializa el producto de la creación científica.

Introduction: Science, Technology and Innovation activity currently constitutes a basic pillar in the development of a country. Scientific journals are an essential part of scientific research as they are the main vehicle for the dissemination of results. Objective: To carry out a theoretical assessment of the conception of editorial management as a scientific activity. Method: Documentary analysis methods were used by selecting information obtained from scientific databases such as SciELO, Scopus and Google Scholar, using criteria: editorial management; scientific journals and science and technology activity. Results: Aspects related to the evolution of scientific communication through magazines, their traditionalist, technological and social approaches were addressed; as well as its role in the scientific communication of the results of scientific research and the editorial management for the publication of scientific journals. Conclusions: The authors conceive the editorial management of scientific journals as a scientific-technical process with a marked pedagogical character that, through the rigorous evaluation of the research result, purifies and socializes the product of scientific creation.

Introdução: A atividade de Ciência, Tecnologia e Inovação constitui atualmente um pilar básico no desenvolvimento de um país. As revistas científicas são uma parte essencial da investigação científica, pois são o principal veículo de divulgação dos resultados. Objetivo: Realizar uma avaliação teórica da concepção de gestão editorial como atividade científica. Método: Foram utilizados métodos de análise documental selecionando informações obtidas em bases de dados científicos como SciELO, Scopus e Google Acadêmico, utilizando critérios: gestão editorial; revistas científicas e atividades de ciência e tecnologia. Resultados: Foram abordados aspectos relacionados à evolução da comunicação científica por meio de revistas, suas abordagens tradicionalistas, tecnológicas e sociais; bem como o seu papel na comunicação científica dos resultados da investigação científica e na gestão editorial da publicação de revistas científicas. Conclusões: Os autores concebem a gestão editorial das revistas científicas como um processo técnico-científico de marcado caráter pedagógico que, por meio da avaliação rigorosa do resultado da pesquisa, purifica e socializa o produto da criação científica.

New and Rapid Analytical Method Using HPLC-MS Detection for Acrylamide Determination in Black Ripe Olives.

The presence of acrylamide, a known human carcinogen, in various heated foods raises significant concerns among consumers. Therefore, the development of a good analytical method is of paramount interest to the scientific community. Keeping this in view, a rapid, simple, reliable, and low-cost analytical method was developed and validated for acrylamide quantification in black ripe olives. The method consisted of the water extraction of the compounds from crushed olives with the addition of (13C3)acrylamide as an internal standard. The quantification was performed using high-pressure liquid chromatography and mass detection with positive electrospray ionization. The limits of detection and quantification were determined to be 4 and 11 µg/kg, respectively. The developed method exhibited excellent results in terms of accuracy (98.4-104.8%) and intra- and inter-day precision limits, both less than 20%. This new method was carried out by analyzing 15 samples of Spanish commercial black ripe olives, revealing a wide range of values, from 79 to 1068 µg/kg of fruit. The new protocol reduces the analysis time to just one hour per sample versus the minimum 24 h required by gas chromatography and mass detection, meaning that it could be a good option for the routine analysis of acrylamide in black ripe olives, and may be extendable to the analysis of this compound in other foods.

Effectiveness of a 5-Week Virtual Reality Telerehabilitation Program for Children With Duchenne and Becker Muscular Dystrophy: Prospective Quasi-Experimental Study.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular diseases. DMD is the most prevalent in children. It affects dystrophin production, reducing the patient's mobility and quality of life. New technologies have become a part of physical therapy in DMD and BMD. During the COVID-19 pandemic, conducting telerehabilitation through virtual reality-based games could help these children maintain their physical abilities. Objective: This study examined if the use of a virtual platform in a multimodal intervention program changes the results of the 6-minute walk test (6MWT) in children with DMD and BMD. The main objective was to test whether children with DMD and BMD obtain different results on the 6MWT after completing 10 telerehabilitation treatment sessions. The secondary objective was to measure whether other specific motor scales also produce different results after the 10 defined sessions. Methods: This was a descriptive, open, and quasi-experimental study with a prospective A-B (control-intervention) design. A sample of 12 participants who fulfilled the inclusion criteria followed the program for 5 weeks with 10 telerehabilitation sessions. During the sessions, the participants used virtual reality glasses to train for the treatment goals. All participants were assessed in person before and after the intervention. Analysis was performed using R software according to the different functional assessments performed for each test. Results: The participants showed a 19.55-meter increase in the 6MWT. Motor function also remained stable according to other scales used to assess it. The North Start Ambulatory Assessment scores were stable in both treatment conditions (P=.20). Furthermore, the timed up and go test results were 0.1 seconds faster in the telerehabilitation condition, and the Motor Function Measure in all of the 3 dimensions showed no significant differences (P=.08). Finally, the Effort Perception Infant scale showed that during the training, fatigue increased in the middle and decreased by the end of the sessions, but the perception throughout the sessions was lower even as the exercise intensity increased. Conclusions: There were no differences between conventional and telerehabilitation treatments, so the telerehabilitation tool could be used without harming children with DMD and BMD, facilitating their access to therapies and stimulating learning to maintain their functional capacity. Therefore, telerehabilitation in general may be helpful in maintaining motor function in children with DMD and BMD. The learning effect helped reduce the feeling of fatigue in the children during the program.

Does Size Matter? Testicular Volume and Its Predictive Ability of Sperm Production in Rams.

Over the years, testicular volume has been used to evaluate the reproductive capacity of rams and the effects of different factors related to reproductive performance. The aim of this study was to determine the most suitable tool and formula to calculate testicular volume under field conditions to guarantee a more accurate determination of sperm production. First, testicles from 25 rams (n = 50) were measured in vivo and postmortem using calipers and ultrasonography during the breeding season (BS). The accurate testicular volume (ATV) was calculated through water displacement. In addition, the sexual status of donor rams was evaluated during a period of four years in a reproduction center, and the three most crucial groups in terms of genetic value and seminal collections were studied in the second part of this experiment: ER-NBS (Elite rams during the non-breeding season), ER-BS-S (Elite rams with a standard frequency of seminal collection), and ER-BS-O (Elite rams with a high frequency of seminal collection). The total testicular volume (TTV), testosterone (T), and total spermatozoa obtained from two consecutive ejaculates in the same day (SPERM) were measured, and the relationship between SPERM and TTV and T was analyzed to predict SPERM. Although all published formulas revealed statistically significant differences (p ≤ 0.05) from the ATV, our proposed formula (ItraULE) (Testicular volume = L × W × D × 0.61) did not show significant differences. In the second part of the study, in the ER as a model donor ram for its high genetic value and high demand from farmers, TTV and T showed strong positive correlations with SPERM (r = 0.587, p = 0.007 NBS; r = 0.684, p = 0.001 BS-S; r = 0.773, p < 0.0001 BS-O). Moreover, formulas were established to predict SPERM in these practical scenarios. In conclusion, the use of ultrasonography and a new formula adapted to rams could improve the prediction of SPERM considering crucial factors such as season and semen collection frequency.

Effectiveness of a 5 weeks telerehabilitation programme with virtual reality glasses in boys with Duchenne and Becker during the COVID-19: A clinical trial.

BACKGROUND: Duchenne and Becker muscular dystrophy (from now "DMD" and "BMD" respectively) are the neuromuscular diseases with the most significant involvement in children. It affects dystrophin production, reducing the patient's mobility and quality of life. New technologies have become part of physical therapy in DMD and BMD. During the COVID-19 pandemic, telerehabilitation through virtual reality-based games could help these children to keep their abilities. OBJECTIVE: The purpose of this study is to know if the use of the virtual platform in a multimodal intervention program achieves changes in the results obtained in the six-minute walk test in children affected by DMD and BMD. To estimate the difference in mobility in patients with DMD and BMD, as measured with the six-minute walking test (6MWT), between 10 conventional and telerehabilitation treatment sessions. As secondary objectives, measuring other specific motor scales was proposed to see whether these had changed after receiving the 10 defined sessions. METHODS: Descriptive, open, quasi-experimental study with prospective A-B (control-intervention) design. Sample size of twelve participants who fulfilled the control criteria followed the program for five weeks, up to 10 telerehabilitation sessions. During the sessions, the participants used virtual reality glasses to train for the treatment goals. All sessions were in person, and participants were assessed before and after the intervention. Analysis was performed using R (R Core Team (2022) according to the different functional assessments performed for each test. RESULTS: The participants showed a 19.55 m increase in the 6MWT scale. The motor function was also kept stable according to other scales used to assess it. North Start result were kept stable in both treatments (P value = .199). Furthermore, Time up and go test was shorter in 0.1 seconds in telerehabilitation time and Motor Function Measure in all of the 3 dimensions shown no significant differences with a P value = .084. Finally, Infant effort (EPInfant) shown that during the training the fatigue increased in the middle and decreased by the end but the perception throughout the sessions, was lower even though the exercise intensity increased. CONCLUSIONS: There is no difference between a conventional and telerehabilitation treatment, so the telerehabilitation tool could be used without harming this type of children, facilitating their access to therapies and stimulating learning to maintain their functional capacity. Telerehabilitation may helpful maintain motor function in children with DMD and BMD. The learning effect helped to reduce the feeling of fatigue in children during the program. CLINICALTRIAL: This trial has the approval of the Andalucía Ethics Committee with PEIBA code 0107-N-20. The results of the research will be disseminated by the investigators to peer-reviewed journals. Trial registration no. NCT03879304.

Epidemiology of surgery associated acute kidney injury (EPIS-AKI): a prospective international observational multi-center clinical study.

PURPOSE: The incidence, patient features, risk factors and outcomes of surgery-associated postoperative acute kidney injury (PO-AKI) across different countries and health care systems is unclear. METHODS: We conducted an international prospective, observational, multi-center study in 30 countries in patients undergoing major surgery (> 2-h duration and postoperative intensive care unit (ICU) or high dependency unit admission). The primary endpoint was the occurrence of PO-AKI within 72 h of surgery defined by the Kidney Disease: Improving Global Outcomes (KDIGO) criteria. Secondary endpoints included PO-AKI severity and duration, use of renal replacement therapy (RRT), mortality, and ICU and hospital length of stay. RESULTS: We studied 10,568 patients and 1945 (18.4%) developed PO-AKI (1236 (63.5%) KDIGO stage 1500 (25.7%) KDIGO stage 2209 (10.7%) KDIGO stage 3). In 33.8% PO-AKI was persistent, and 170/1945 (8.7%) of patients with PO-AKI received RRT in the ICU. Patients with PO-AKI had greater ICU (6.3% vs. 0.7%) and hospital (8.6% vs. 1.4%) mortality, and longer ICU (median 2 (Q1-Q3, 1-3) days vs. 3 (Q1-Q3, 1-6) days) and hospital length of stay (median 14 (Q1-Q3, 9-24) days vs. 10 (Q1-Q3, 7-17) days). Risk factors for PO-AKI included older age, comorbidities (hypertension, diabetes, chronic kidney disease), type, duration and urgency of surgery as well as intraoperative vasopressors, and aminoglycosides administration. CONCLUSION: In a comprehensive multinational study, approximately one in five patients develop PO-AKI after major surgery. Increasing severity of PO-AKI is associated with a progressive increase in adverse outcomes. Our findings indicate that PO-AKI represents a significant burden for health care worldwide.

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile.

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.

First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing Molecular characterization of Escherichia coli O157 isolates from Paraguay / Primera caracterización molecular de aislamientos de Escherichia coli O157:H7 provenientes de muestras clínicas en Paraguay utilizando secuenciación de genoma completo

Abstract Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Resumen Escherichia coli O157:H7 es un patógeno transmitido por alimentos implicado en numerosos brotes en todo el mundo y es capaz de causar complicaciones extraintestinales en humanos. La sección de «Enteropatógenos¼ del Laboratorio Central de Salud Pública trabaja en mejorar la caracterización genómica de STEC, de modo de potenciar la vigilancia laboratorial y la investigación de brotes de enfermedades transmitidas por alimentos. La secuenciación de genoma completo (WGS, por sus siglas en inglés) se propone a nivel mundial como una herramienta de alta resolución para ser utilizada en el laboratorio de rutina, ya que permite obtener todos los resultados en un único proceso. El objetivo de este trabajo fue llevar a cabo, por primera vez, la caracterización genómica por WGS de nueve cepas STEC O157:H7 aisladas en Paraguay a partir de muestras de origen humano. Pudimos identificar los factores de virulencia, los mecanismos de resistencia, el subtipo MLST, e incluso pudimos establecer la relación filogenética entre los aislamientos. Además, detectamos que la mayoría de las cepas pertenecían al clado hipervirulento 8.

First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing.

Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Interlaboratory Evaluation of Multiple LC-MS/MS Methods and a Commercial ELISA Method for Determination of Tetrodotoxin in Oysters and Mussels.

BACKGROUND: Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods. OBJECTIVE: The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods. METHODS: Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores. RESULTS: Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC-MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC-MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC-MS/MS. CONCLUSION: The results from this study demonstrate that current LC-MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC-MS/MS protocol to further improve interlaboratory precision. HIGHLIGHTS: Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

Mecanismos plasmídicos de resistencia a quinolonas, betalactámicos y colistina en Salmonella enterica. Paraguay 2020-2021

Las bacterias son capaces de desarrollar mecanismos de resistencia a los antimicrobianos, aquellos adquiridos y transmisibles son los más significativos debido al potencial de diseminación. La aparición de Salmonella enterica con resistencia a C3aG, quinolonas y a colistina representa una amenaza progresiva. El objetivo fue determinar la resistencia a los antimicrobianos y la presencia de los mecanismos de resistencia plasmídicos a quinolonas, ß-lactámicos y colistina en aislados de Salmonella provenientes de la vigilancia integrada de enteropatógenos. Fueron estudiadas 501 cepas de Salmonella spp. colectadas entre los años 2020 y 2021, por la red de enteropatógenos del Laboratorio Central de Salud Pública. Se investigó la resistencia a las C3aG, quinolonas y colistina, en aislamientos de humanos, alimentos, animales de consumo y ambiente. Las cepas estudiadas exhibieron resistencia a tetraciclina (32,5%), ácido nalidíxico (29%), ampicilina (13,2%), nitrofurantoína (11,6%), C3aG (7,2%), cotrimoxazol (5,8%), ciprofloxacina (2,2%). El 18% (90/501) presentaron resistencia trasferible por plásmidos, fueron detectados 111 genes (71 cepas con un gen, 17 cepas dos genes y 2 cepas tres genes diferentes). Qnr B: 41,1% (37/90), mcr-1: 38,9% (35/90), CMY: 23,3% (21/90), CTX-M: 16,7% (15/90) y Qnr S: 3,3% (3/90). Heidelberg fue el serovar predominante en muestras de pollo y el mayor portador de genes de resistencia de tipo CMY y mcr-1. La detección de genes en alimentos y animales de consumo, que pueden transmitirse fácilmente al ser humano es motivo de alerta y resalta la importancia de continuar fortaleciendo la vigilancia multisectorial y multidisciplinaria.

Bacteria can develop antimicrobial resistance mechanisms, those acquired and transmissible being the most significant due to the potential for dissemination. The emergence of Salmonella enterica with resistance to third-generation cephalosporins, quinolones, and colistin represents a progressive threat. The objective was to determine antimicrobial resistance and the presence of plasmid resistance mechanisms to quinolones, ß-lactams, and colistin in Salmonella isolates from integrated surveillance of enteropathogens. Five hundred and one strains of Salmonella spp. collected between 2020 and 2021 were studied by the enteropathogen network of the Laboratorio Central de Salud Publica (Central Public Health Laboratory). Research was conducted on the resistance to third-generation cephalosporins, quinolones, and colistin, isolated from humans, foodstuffs, animals for consumption, and the environment. The strains studied exhibited resistance to tetracycline (32.5%), nalidixic acid (29%), ampicillin (13.2%), nitrofurantoin (11.6%), third-generation cephalosporins (7.2%), cotrimoxazole (5.8%), and ciprofloxacin (2.2%). Eighteen percent (90/501) presented plasmid-transferable resistance, 111 genes were detected (71 strains with one gene, 17 strains with two genes, and 2 strains with three different genes). Qnr B: 41.1% (37/90), mcr-1: 38.9% (35/90), CMY: 23.3% (21/90), CTX-M: 16.7% (15/90), and Qnr S: 3.3% (3/90). Heidelberg was the predominant serovar in chicken samples and the largest carrier of CMY and mcr-1 resistance genes. The detection of genes in foodstuffs and animals for consumption, which can be easily transmitted to humans, is a cause for alarm and highlights the importance of continuing to strengthen multisectoral and multidisciplinary surveillance.

Application of ultrasound technique to evaluate the testicular function and its correlation to the sperm quality after different collection frequency in rams.

The frequency of semen collection is a crucial factor to consider in the rams performance inside breeding centers workout. To evaluate this factor, ram Breeding Soundness Evaluation could include sperm quality evaluation and new predictive and non-invasive tools such as ultrasound technique. In this work, an advanced ultrasonography technology, analyzing the testicular volume, echotexture, and vascular function, was used in three different frequencies of semen collection (abstinence frequency, AF; standard frequency, SF; and intensive frequency, IF). Semen samples were cooled (15°C, 6 h) and evaluated in terms of production, motility, viability, apoptosis, and content of reactive oxygen species. Correlation coefficients were calculated between ultrasonography measurements of echotexture and blood flow and sperm quality parameters. Our results showed an increase in the testicular echotexture when the frequency of semen collection was intensified. Doppler parameters (PSV, RI, PI, TABF) increased (P ≤ 0.05) when the frequency of semen collection was intensified. The sperm motility and functionality decreased in the samples of IF (P ≤ 0.05), evidencing the frequency of semen collection's influence. Moreover, moderate positive correlations were established among echotexture and different Doppler parameters with motility parameters in SF. Furthermore, the influence of abstinence days on AI success was analyzed in a field assay. The highest fertility rates were obtained when males had two to five abstinence days. To conclude, frequency of semen collection could be influenced in terms of semen quantity and sperm quality, showing changes in parenchyma echotexture and testicular vascularization. The standard semen collection frequency was the most adequate option. In addition, ultrasonography may be a predictive tool for estimating variations in the sperm quality of donor rams subjected to different frequencies of semen collection in reproduction centers.

An optimized centrifugation protocol for ram sperm ensuring high sample yield, quality and fertility.

The optimization and implementation of artificial insemination (AI) in sheep is necessary to increase the livestock productivity through enhanced control of reproductive function. Sperm centrifugation is a common procedure in the ejaculate handling in AI and other assisted reproductive technologies (ART), as part of new methods of sperm analysis, selection or preservation. However, our research group previously established that this simple procedure might cause a large sperm loss and induce deleterious effects on the sperm function of the ovine species when high centrifugation forces are employed. To our knowledge, there are no studies on combined effect of extender and different centrifugal forces on ram sperm yield and quality. Furthermore, evidence of in vivo fertility rate using sperm obtained with various centrifugation forces is also lacking in this species. Thus, the objective of this work was to define the ideal conditions for ram semen centrifugation that will achieve the best quantity and quality sample to ensure unaffected fertilization ability of centrifuged ram sperm. The Experiment 1 evaluated the effect of the centrifugation procedure of two extenders (INRA 96 and Tyrode's) and two cooling protocols (Rapid and Slow Refrigeration -35 °C to 15 °C-) on sperm recovery rate and quality (motility and kinetic parameters, viability, apoptosis and mitochondrial activity). INRA 96 combined with Slow Refrigeration and Tyrode's at room temperature registered the highest sperm recovery and quality values (P ≤ 0.05). In Experiment 2, the influence of three centrifugal forces (600, 1200 and 6000×g for 10 min) was assessed immediately after centrifugation on the technical performance and sperm functionality in diluted samples with INRA 96 and Tyrode's at the conditions set out in Experiment 1. The lowest pellet weight (P ≤ 0.05) without harmful effect on sperm physiological status (P > 0.05) was achieved at 1200×g, since 6000×g induced sperm motility damage (P ≤ 0.05) with both extenders. Finally, to ensure the total safety of the centrifugation protocol, Experiment 3 tested in a combined in vitro and in vivo test the effect of these three centrifugal forces on ram sperm quality after dilution (INRA 96) and liquid storage (6-8 h at 15 °C). The damage produced by 6000×g on sperm motility (P ≤ 0.05) was maintained over time, coinciding with a lower fertility (P ≤ 0.05). In conclusion, ram sperm can be centrifuged in INRA 96 extender up to 1200×g for 10 min at 15 °C as secure values with high recovery rates and without detrimental effects on sperm quality and fertility.