Effects of Reactive Oxygen and Nitrogen Species on Male Fertility.

Significance: In recent decades, male fertility has been severely reduced worldwide. The causes underlying this decline are multifactorial, and include, among others, genetic alterations, changes in the microbiome, and the impact of environmental pollutants. Such factors can dysregulate the physiological levels of reactive species of oxygen (ROS) and nitrogen (RNS) in the patient, generating oxidative and nitrosative stress that impairs fertility. Recent Advances: Recent studies have delved into other factors involved in the dysregulation of ROS and RNS levels, such as diet, obesity, persistent infections, environmental pollutants, and gut microbiota, thus leading to new strategies to solve male fertility problems, such as consuming prebiotics to regulate gut flora or treating psychological conditions. Critical Issues: The pathways where ROS or RNS may be involved as modulators are still under investigation. Moreover, the extent to which treatments can rescue male infertility as well as whether they may have side effects remains, in most cases, to be elucidated. For example, it is known that prescription of antioxidants to treat nitrosative stress can alter sperm chromatin condensation, which makes DNA more exposed to ROS and RNS, and may thus affect fertilization and early embryo development. Future Directions: The involvement of extracellular vesicles, which might play a crucial role in cell communication during spermatogenesis and epididymal maturation, and the relevance of other factors such as sperm epigenetic signatures should be envisaged in the future.

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Indomethacin Induces Spermidine/Spermine-N1-Acetyltransferase-1 via the Nucleolin-CDK1 Axis and Synergizes with the Polyamine Oxidase Inhibitor Methoctramine in Lung Cancer Cells.

Indomethacin is a non-selective NSAID used against pain and inflammation. Although cyclooxygenase (COX) inhibition is considered indomethacin's primary action mechanism, COX-independent ways are associated with beneficial effects in cancer. In colon cancer cells, the activation of the peroxisome proliferator-activated receptor-γ (PPAR-γ) is related to the increase in spermidine/spermine-N1-acetyltransferase-1 (SSAT-1), a key enzyme for polyamine degradation, and related to cell cycle arrest. Indomethacin increases the SSAT-1 levels in lung cancer cells; however, the mechanism relying on the SSAT-1 increase is unclear. Thus, we asked for the influence of the PPAR-γ on the SSAT-1 expression in two lung cancer cell lines: H1299 and A549. We found that the inhibition of PPAR-γ with GW9662 did not revert the increase in SSAT-1 induced by indomethacin. Because the mRNA of SSAT-1 suffers a pre-translation retention step by nucleolin, a nucleolar protein, we explored the relationship between indomethacin and the upstream translation regulators of SSAT-1. We found that indomethacin decreases the nucleolin levels and the cyclin-dependent kinase 1 (CDK1) levels, which phosphorylates nucleolin in mitosis. Overexpression of nucleolin partially reverts the effect of indomethacin over cell viability and SSAT-1 levels. On the other hand, Casein Kinase, known for phosphorylating nucleolin during interphase, is not modified by indomethacin. SSAT-1 exerts its antiproliferative effect by acetylating polyamines, a process reverted by the polyamine oxidase (PAOX). Recently, methoctramine was described as the most specific inhibitor of PAOX. Thus, we asked if methoctramine could increase the effect of indomethacin. We found that, when combined, indomethacin and methoctramine have a synergistic effect against NSCLC cells in vitro. These results suggest that indomethacin increases the SSAT-1 levels by reducing the CDK1-nucleolin regulatory axis, and the PAOX inhibition with methoctramine could improve the antiproliferative effect of indomethacin.

Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR.

Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed equine spermatozoa into bovine oocytes using a piezoelectric system (Piezo-ICSI). Our results indicated that equine sex-sorting spermatozoa had a 97% and 94% certainty for X and Y sperm, respectively, while presumptive female and male equine/bovine hybrid embryos, generated by Piezo-ICSI, had an accuracy of 92% with respect to the desired sex. Therefore, it is concluded that the presented methodology is a reliable, cost-effective, and relatively simple option for standardizing sex-sorting of equine spermatozoa. This is supported by the results of the correct sexing of Piezo-ICSI heterologous embryos generated with the sexed spermatozoa, validating the correct sexing and viability of these gametes.

Cellular and Molecular Consequences of Stallion Sperm Cryopreservation: Recent Approaches to Improve Sperm Survival.

Cryopreservation of stallion semen does not achieve the post-thaw quality or fertility results observed in other species like cattle. There are many reasons for this, but the membrane composition and intracellular changes in stallion sperm predispose them to low resistance to the cooling, freezing, and subsequent thawing process. Damage to the sperm results from different processes activated during cryopreservation, including oxidative stress, apoptosis, and structural modifications in the sperm membrane that increase the deleterious effect on sperm. In addition, significant individual variability is observed among stallions in the ability of sperm to survive the freeze-thaw process. Recent advances in genomics, transcriptomics, proteomics, metabolomics, and epigenetics are making it possible to advance our understanding of the cellular and molecular processes involved in the cryopreservation process, opening new possibilities for improvement. This review addresses the ongoing research on stallion semen cryopreservation, focusing on the cellular and molecular consequences of this procedure in stallions and discusses the new tools currently available to increase the tolerance of equine spermatozoa to freeze-thaw.

Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos.

Supplementation of the culture media for in vitro production (IVP) of bovine embryos with fetal bovine serum (FBS) is associated with inconsistent outcomes. The present study sought to replace FBS and BSA by insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). In Experiment 1, absence of FBS from maturation medium (MM) did not affect the rate of in vitro maturation, as assessed by the extrusion of the first polar body. However, when gonadotropins and FBS were removed from the MM, the maturation rate was significantly reduced even in the presence of growth factors. Therefore, gonadotropin-supplemented MM medium was established as the base medium for the defined maturation condition. In Experiment 2, the addition of growth factors to gonadotropin-supplemented MM medium supported similar maturation (~90%) compared to the undefined condition (FBS-carrying). In Experiment 3, the addition of growth factors to embryo culture medium showed similar in vitro competence compared to the undefined (FBS) control. In Experiment 4, completely defined conditions (absence of FBS and BSA during in vitro maturation and embryo culture) were tested. A higher cleavage was observed with FGF2 (86%) compared to EGF (77%) and the FBS control (77%), but similar blastocyst rates were observed for FGF2 (24%), EGF (19%) and the FBS control (25%). Embryo quality was similar among groups. Finally, post-thawing survival was higher for FGF2 (94%) compared to the FBS control (77%). Thus, we report a simple defined IVP system for bovine species that generates developmental outcomes and embryos of similar quality than those produced under conditions containing FBS.

Bovine ICSI: limiting factors, strategies to improve its efficiency and alternative approaches.

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique mainly used to overcome severe infertility problems associated with the male factor, but in cattle its efficiency is far from optimal. Artificial activation treatments combining ionomycin (Io) with 6-dimethylaminopurine after piezo-ICSI or anisomycin after conventional ICSI have recently increased the blastocyst rate obtained. Compounds to capacitate bovine spermatozoa, such as heparin and methyl-ß-cyclodextrin and compounds to destabilize sperm membranes such as NaOH, lysolecithin and Triton X-100, have been assessed, although they have failed to substantially improve post-ICSI embryonic development. Disulfide bond reducing agents, such as dithiothreitol (DTT), dithiobutylamine and reduced glutathione, have been assessed to decondense the hypercondensed head of bovine spermatozoa, the two latter being more efficient than DTT and less harmful. Although piezo-directed ICSI without external activation has generated high fertilization rates and modest rates of early embryo development, other studies have required exogenous activation to improve the results. This manuscript thoroughly reviews the different strategies used in bovine ICSI to improve its efficiency and proposes some alternative approaches, such as the use of extracellular vesicles (EVs) as 'biological methods of oocyte activation' or the incorporation of EVs in the in vitro maturation and/or culture medium as antioxidant defence agents to improve the competence of the ooplasm, as well as a preincubation of the spermatozoa in estrous oviductal fluid to induce physiological capacitation and acrosome reaction before ICSI, and the use of hyaluronate in the sperm immobilization medium.

Effect of cholestanol and cholesterol-loaded cyclodextrin on stallion sperm function and capacitation post-cryopreservation.

Cryopreservation of stallion semen is less efficient than other species such as bovine. This is mainly because of the greater susceptibility of stallion sperm to the freezing damage that generates oxidative stress and plasma membrane injury, resulting in DNA fragmentation and cell death. These data suggest the need to develop new strategies of sperm cryopreservation that can improve the efficiency of this technique in stallions by reducing or preventing membrane damage and cell death. The present study aimed to evaluate the effect of adding membrane stabilizers to the freezing medium and assess the quality and in vitro capacitation of stallion sperm after thawing. Semen samples from three stallions frozen with membrane stabilizers (cholesterol-loaded cyclodextrin and cholestanol-loaded cyclodextrin) were evaluated in two experiments: i) sperm quality and functional analysis after thawing, and ii) sperm quality and functional analysis after 4 h of post-thaw incubation in capacitating conditions. Plasma membrane integrity, mitochondrial membrane potential, membrane lipid disorder, intracellular Ca2+, tyrosine phosphorylation, acrosome reaction, DNA damage, sperm motility, and binding to the zona pellucida were assessed. The results showed that cholesterol-loaded cyclodextrin was the stabilizer that most efficiently reduced the membrane disruption and post-thaw cell damage. In addition, this stabilizer made it possible to obtain in vitro capacitated sperm showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, binding to the zona pellucida and better response to in vitro capacitating conditions.

Efectos de la COVID-19 sobre los servicios de salud en Santiago de Cuba, 2020 / Effects of COVID-19 on health services in Santiago de Cuba, 2020

Introducción: En Santiago de Cuba las acciones aplicadas para el control de la epidemia en el 2020 ocasionaron cambios y efectos negativos sobre muchos de los servicios de salud que se brindan de manera habitual en el territorio. Objetivo: Identificar el efecto de la epidemia de COVID-19 sobre los servicios de salud en la provincia Santiago de Cuba durante el año 2020. Métodos: Se realizó un estudio ecológico retrospectivo con series temporales como unidades de análisis comparadas en la provincia de Santiago de Cuba como único territorio geográfico para evaluar los efectos que tuvo la COVID-19 sobre la prestación de los servicios de salud durante el año 2020. Se seleccionaron algunos indicadores de servicios de salud de la serie de tiempo de 2015 a 2019. La fuente de datos fueron las estadísticas oficiales. Se estimó el porcentaje de cambio y se propuso una clasificación de los efectos de la COVID-19 según su magnitud y sentido. Resultados: Se produjeron cambios en la cantidad y tipos de servicios de salud en cuanto a las consultas médicas externas y de urgencia, los ingresos hospitalarios y promedio de estadía de las actividades de cardiología relacionados con ingresos, coronariografías realizadas, marcapasos implantados y operaciones ejecutadas, de las actividades quirúrgicas operaciones electivas y de urgencia, así como las ambulatorias y por mínimo acceso, los tratamientos rehabilitadores, los estudios para el diagnóstico mediante el uso de los laboratorios clínicos y de microbiología, estudios imagenológicos, en general, y los más afectados fueron los relacionados con la Estomatología, trasplantes renales y de córnea. Conclusiones: La epidemia de COVID-19 tuvo un efecto negativo en el funcionamiento de los servicios de salud al producirse cambios desfavorables en la cantidad de servicios prestados en los niveles de atención primario y secundario, lo que puede impactar en la salud de personas vulnerables si no se establecen estrategias alternativas(AU)

Introduction: In Santiago de Cuba province, the actions applied to control the epidemic in 2020 caused changes and negative effects on many of the health services that are usually provided in the territory. Objective: To identify the effect of the COVID-19 epidemic on health services in Santiago de Cuba province during 2020. Methods: A retrospective ecological study was conducted with time series as units of comparative analysis in Santiago de Cuba province as the only geographical territory to evaluate the effects of COVID-19 on the provision of health services during 2020. Some health service indicators from the time series from 2015 to 2019 were selected. The data source was official statistics. The percentage of change was estimated and a classification of the effects of COVID-19 according to its magnitude and meaning was proposed. Results: There were changes in the number and types of health services in terms of outpatient and emergency medical consultations, hospital admissions and average stay of cardiology activities related to admissions, coronary angiographies performed, pacemakers implanted and operations performed, elective surgical activities and emergency operations, as well as outpatient and minimal access ones, rehabilitative treatments, studies for diagnosis through the use of clinical and microbiology laboratories, imaging studies in general, and the most affected were those related to Stomatology, and kidney and corneal transplants. Conclusions: The COVID-19 epidemic had a negative effect on the functioning of health services as there were unfavorable changes in the number of services provided at the primary and secondary care levels, which can impact on the health of vulnerable people if alternative strategies are not established(AU)

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†.

The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

Effect of MnTBAP on in vitro capacitation of frozen-thawed stallion sperm.

In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 106 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating, and capacitating plus 150 mM MnTBAP. There were incubations for 4 h at 37.5 °C in a humidified air atmosphere. Sample analysis was performed immediately after thawing (0 h), and at the end of the incubation period (4 h), unless otherwise indicated. The following variables were evaluated for spermatozoa: plasma membrane integrity and fluidity, acrosome integrity, intracellular calcium concentrations, intracellular pH, tyrosine phosphorylation, ATP concentrations, motility and heterologous zona-binding assay, using flow cytometry, fluorescent microscopy and/or chemiluminescence, depending on the most appropriate procedure for the variable being evaluated. Results indicated that capacitation-like changes were synergistically induced by the cAMP agonists, phosphodiesterase inhibitor and bicarbonate. The presence of bovine serum albumin was harmful to the plasma membrane. The MnTBAP supplementation had a positive effect on viability-related markers (plasma membrane integrity, membrane fluidity, associated with greater intracellular pH) when there were capacitating conditions. In conclusion, the activity of MnTBAP contributes to improving the in vitro incubation conditions of frozen-thawed stallion sperm.

Cryopreservation of stallion semen: Effect of adding antioxidants to the freezing medium on sperm physiology.

Cryopreservation of stallion semen has not reached the level of efficiency and positive results described in other species. This is mainly due to the greater sensitivity of stallion sperm to the freezing process, showing higher rates of oxidative stress and plasma membrane damage, which trigger the activation of several cell damage pathways that ultimately culminate in DNA fragmentation and cell death. Therefore, finding molecules that improve the efficiency of this technique in stallion by preventing oxidative stress and cell damage is required. Thus, the aim of the present study was to evaluate the effect of adding three antioxidants (MnTBAP, NAC and FeTPPS) to the freezing medium on the quality and functional parameters of stallion sperm. Semen samples from three stallions frozen with the antioxidants were evaluated in two conditions: (a) adding the antioxidants before freezing, and (b) before and after freezing. Plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation, intracellular ROS levels, membrane lipid disorder, DNA damage, sperm motility and binding to the zona pellucida were assessed. The results showed that MnTBAP was the antioxidant treatment that best controlled the oxidative stress process and post-thaw cell damage, showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, number of spermatozoa bound to the zona pellucida of bovine oocytes and lower lipid disorder. Additionally, it was determined that a second post-thaw application of antioxidants is detrimental since induced higher cell damage and lower sperm motility, without showing any beneficial effect on the spermatozoa.

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation.

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.

Impact of post-thaw supplementation of semen extender with antioxidants on the quality and function variables of stallion spermatozoa.

During cryopreservation procedures, the spermatozoa are exposed to physical and chemical stressors that generate an increase in the intracellular concentration of reactive oxygen species (ROS). If ROS concentrations are too great, this can lead to a state of oxidative stress that are detrimental to sperm quality. The aim of this study was to ascertain the profile the ROS production and assess the effects of post-thaw supplementation of a semen extender with different antioxidant compounds on the quality and function variables of frozen-thawed stallion spermatozoa incubated in vitro. Frozen-thawed stallion spermatozoa (2 × 106 cells/mL) were incubated with three different antioxidants (MnTBAP, NAC and FeTPPS) for 4 h at 38 °C. An untreated sperm suspension and a fresh sample were included as controls. Plasma membrane integrity (SYBR-14/PI), intracellular ROS concentration (DHE and ROS-ID™ total ROS/Superoxide Detection Kit), lipid peroxidation (BODIPY), DNA damage (TUNEL) and mitochondrial membrane potential (ΔΨm; TMRE/SYTOX) were evaluated by flow cytometry and fluorescence microscopy. In addition, sperm motility was evaluated using the ISAS system. Evaluations were performed at 0 and 4 h of incubation. The results indicate that superoxide anion is the main ROS produced by frozen-thawed stallion spermatozoa and that the use of MnTBAP improved sperm motility and viability, decreased the lipid peroxidation and DNA damage. In conclusion, this study provides relevant data to improve in vitro incubations conditions and to establish futures therapies using MnTBAP after thawing with the aim being to overcome the deleterious effects of semen cryopreservation and consequently preserve the stallion sperm quality through avoiding oxidative stress.

Improved expression of green fluorescent protein in cattle embryos produced by ICSI-mediated gene transfer with spermatozoa treated with streptolysin-O.

The ICSI-sperm mediated gene transfer (ICSI-SMGT) has been used to produce transgenic mice with high efficiency; however, the efficiency of this technique in farm animals is still less than desirable. Pretreatment of sperm with membrane destabilizing agents can improve the efficiency of ICSI in cattle. The objective of the present study was to evaluate streptolysin-O (SLO) as a novel treatment to permeabilize the bovine sperm membrane and assess its effect on efficiency of generating transgenic embryos by ICSI-SMGT. First, there was evaluation of the plasma membrane integrity (SYBR/PI), acrosome membrane integrity (PNA/FITC), DNA damage (TUNEL) and binding capacity of exogenous DNA (Nick Translation) in bull sperm treated with SLO. Subsequently, there was assessment of embryonic development and the efficiency in generating transgenic embryos with enhanced expression of the gene for green fluorescent protein (EGFP). Results indicate that SLO efficiently permeabilizes the plasma and acrosome membranes of bull spermatozoa and increases binding of exogenous DNA mostly to the post-acrosomal region and tail without greatly affecting the integrity of the DNA. Furthermore, treatment of bull spermatozoa with SLO prior to the injection of oocytes by ICSI-SMGT significantly increased the rate of embryo expression of the EGFP gene. Future experiments are still needed to determine the effect of this treatment on the development and transgene expression in fetuses and animals produced by ICSI-SMGT.

Cryopreservation induces mitochondrial permeability transition in a bovine sperm model.

When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 × 106 mL-1 and incubated for 4 h at 38 °C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca2+ level using FLUO3-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (ΔΨm) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by ΔΨm dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality.

Improved embryo development using high cysteamine concentration during IVM and sperm co-culture with COCs previous to ICSI in bovine.

In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O2 tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3 h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21 h with 1 (Cys 1) or 0.1 mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for ≥3 h with 16 × 106 sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI + Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O2 tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI + Co-cult and Cys 1 ICSI + Co-cult groups (n = 117, 92% and n = 116, 79%, respectively) compared to their controls (n = 132, 60% for Cys 0.1 ICSI and n = 108, 52% for Cys 1 ICSI) (p ≤ 0.05). Interestingly, the combined treatment (Cys 1 ICSI + Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI + Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p ≤ 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p ≤ 0.05). The relative abundance of mRNAs coding for INFτ, CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p ≤ 0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p ≤ 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI + Co-cult, except for HDAC1 (p ≤ 0.05). In conclusion, the use of 1 mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle.

Características clinicoepidemiológicas de la enfermedad diarreica aguda por Vibrio cholerae en pacientes de hasta 10 años / Clinical epidemiological characteristics of the acute diarrheal disease due to Vibrio cholerae in patients up to 10 years

Se realizó una investigación observacional, descriptiva y transversal de 95 niños en las edades de 0 a 10 años, con diagnóstico de enfermedad diarreica aguda a causa del Vibrio cholerae, atendidos en el Hospital Infantil Norte Dr Juan de la Cruz Martínez Maceira de Santiago de Cuba, durante el 2016, a fin de caracterizarles según algunas variables clínicas y epidemiológicas. Entre los principales resultados se obtuvo que el grupo etario más afectado fuera el de 0 a 11 meses y el municipio con mayor número de casos el de Santiago de Cuba, los que correspondieron fundamentalmente a las áreas de salud de los policlínicos Frank País García, José Martí Pérez y Josué País García. Asimismo se evidenció que la principal manifestación del proceso infeccioso fue la diarrea líquida y la complicación más frecuente, la deshidratación isotónica moderada. Todos los niños egresaron vivos, lo cual demuestra la eficacia de la atención médica en el territorio suroriental de Cuba

An observational, descriptive and cross-sectional investigation of 95 children aged 0 to 10, with diagnosis of acute diarrheal disease due to Vibrio cholerae, assisted in Dr Juan de la Cruz Martínez Maceira Northern Children Hospital in Santiago de Cuba, was carried out during 2016, in order to characterize them according to some clinical and epidemiological variables. Among the predominant results there were the 0 to 11 months age group as the most affected and the presence of a higher number of cases in Santiago de Cuba municipality, that corresponded mainly to the health areas of Frank País García, José Martí Pérez and Josué País García polyclinics. Also it was evidenced that the main manifestation of the infectious process was the liquid diarrhea and the most frequent complication, the moderate isotonic dehydration. None of the children died, which demonstrates the effectiveness of medical care in the southeastern territory of Cuba

Características clinicoepidemiológicas de la enfermedad diarreica aguda por Vibrio cholerae en pacientes de hasta 10 años / Clinical epidemiological characteristics of the acute diarrheal disease due to Vibrio cholerae in patients up to 10 years

Se realizó una investigación observacional, descriptiva y transversal de 95 niños en las edades de 0 a 10 años, con diagnóstico de enfermedad diarreica aguda a causa del Vibrio cholerae, atendidos en el Hospital Infantil Norte Dr Juan de la Cruz Martínez Maceira de Santiago de Cuba, durante el 2016, a fin de caracterizarles según algunas variables clínicas y epidemiológicas. Entre los principales resultados se obtuvo que el grupo etario más afectado fuera el de 0 a 11 meses y el municipio con mayor número de casos el de Santiago de Cuba, los que correspondieron fundamentalmente a las áreas de salud de los policlínicos Frank País García, José Martí Pérez y Josué País García. Asimismo se evidenció que la principal manifestación del proceso infeccioso fue la diarrea líquida y la complicación más frecuente, la deshidratación isotónica moderada. Todos los niños egresaron vivos, lo cual demuestra la eficacia de la atención médica en el territorio suroriental de Cuba(AU)

An observational, descriptive and cross-sectional investigation of 95 children aged 0 to 10, with diagnosis of acute diarrheal disease due to Vibrio cholerae, assisted in Dr Juan de la Cruz Martínez Maceira Northern Children Hospital in Santiago de Cuba, was carried out during 2016, in order to characterize them according to some clinical and epidemiological variables. Among the predominant results there were the 0 to 11 months age group as the most affected and the presence of a higher number of cases in Santiago de Cuba municipality, that corresponded mainly to the health areas of Frank País García, José Martí Pérez and Josué País García polyclinics. Also it was evidenced that the main manifestation of the infectious process was the liquid diarrhea and the most frequent complication, the moderate isotonic dehydration. None of the children died, which demonstrates the effectiveness of medical care in the southeastern territory of Cuba(AU)

Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT.

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.