Metabolomic signature of spermatozoa established during holding time is responsible for differences in boar sperm freezability†.

Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC-MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences.

CyanoMetDB, a comprehensive public database of secondary metabolites from cyanobacteria.

Harmful cyanobacterial blooms, which frequently contain toxic secondary metabolites, are reported in aquatic environments around the world. More than two thousand cyanobacterial secondary metabolites have been reported from diverse sources over the past fifty years. A comprehensive, publically-accessible database detailing these secondary metabolites would facilitate research into their occurrence, functions and toxicological risks. To address this need we created CyanoMetDB, a highly curated, flat-file, openly-accessible database of cyanobacterial secondary metabolites collated from 850 peer-reviewed articles published between 1967 and 2020. CyanoMetDB contains 2010 cyanobacterial metabolites and 99 structurally related compounds. This has nearly doubled the number of entries with complete literature metadata and structural composition information compared to previously available open access databases. The dataset includes microcytsins, cyanopeptolins, other depsipeptides, anabaenopeptins, microginins, aeruginosins, cyclamides, cryptophycins, saxitoxins, spumigins, microviridins, and anatoxins among other metabolite classes. A comprehensive database dedicated to cyanobacterial secondary metabolites facilitates: (1) the detection and dereplication of known cyanobacterial toxins and secondary metabolites; (2) the identification of novel natural products from cyanobacteria; (3) research on biosynthesis of cyanobacterial secondary metabolites, including substructure searches; and (4) the investigation of their abundance, persistence, and toxicity in natural environments.

The ideal holding time for boar semen is 24 h at 17 °C prior to short-cryopreservation protocols.

Boar semen cannot be immediately cryopreserved, it need be hold at 17 °C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17 °C for 24 h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 h) on boar spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8, 12, 24, 28 and 32 h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32 h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24 h.

Cryopreservation of boar semen in 0. 5 mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability / A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0, 5 mL em baixas concentrações espermáticas é melhor do que em altas concentrações

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.(AU)

Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.(AU)

Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation.

Nitric oxide (NO) is a reactive nitrogen species (RSN) that, over the years, has been shown to be integrated with biological and physiological events, including reproductive processes. NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O2.-). However, the role of NO in mammalian spermatozoa physiology seems paradoxical. The aim of this study was to investigate the effects of NO on motility, hyperactivation, membrane integrity, peroxidation, and capacitation in cryopreserved equine sperm. Ejaculates were collected and cryopreserved. After thawing, samples were centrifuged, suspended in an in vitro fertilization (IVF) medium and incubated with the following treatments: 1) C = control (IVF); 2) A = l-arginine (10 mM - In); 3) L = L-NAME (1 mM - Ih); 4) M = methylene blue (100 mM - Re); 5) AL = L-arginine + L-NAME (In + Ih); 6) AM = L-arginine + methylene blue (In + Re). The samples were evaluated for spermatic kinetics by CASA and other analyses [plasma and acrosomal membranes used the propidium iodide (PI) and Pisum sativum agglutinin (PSA), detection of tyrosine residues phosphorylation in the membrane (F0426), nitric oxide (DAF-2/DA), lipid peroxidation (C11-BODIPY581/591)] by flow cytometry. The l-arginine treatments reduced MOT, PROG, RAP and LIN only at time 0 min compared to the control and L-NAME. These treatments (MT and MP, VAP, VSL, LIN, RAP) also reduced the sperm movement characteristics but only at the beginning of the incubation period. After this period of incubation, motility recovered. NO removal by methylene blue almost completely inhibited sperm motility, but these treatments had the highest percentages of intact membranes. l-arginine treatments improved acrosome reactions and differed from M and AM. NO production, tyrosine phosphorylation and lipid peroxidation did not differ among treatments, except for M and AM, where a reduction in these variables was detected. Therefore, equine sperm capacitation and the acrosome reaction are part of an oxidative process that involves the participation of ROS, and NO plays an important role in the maintenance and regulation of motility, hyperactivation, induction of acrosome reaction and possibly in capacitation, which are indispensable processes for the fertility of equine sperm.

Diagnóstico de vértigo periférico para el médico de atención primaria / Diagnosis of peripheral vertigo for primary care physicians / Diagnóstico de vertigem periférica para o médico de atenção primária

Resumen Para los médicos de atención primaria, constituye un desafío el paciente con sintomatologia compatible con síndromes vestibulares, debido a su amplia variedad de presentaciones y la dificultad en la diferenciación de las diversas patologías que ocasionan los síntomas. La necesidad de tener conceptos claros y actualizados en cuanto a los mecanismos patológicos del vértigo surge en el momento de evaluar al paciente. Para esto, es necesario realizar un adecuado interrogatorio y un examen físico neurotológico completo que permita un abordaje diagnóstico certero. El objetivo del presente artículo es presentar una revisión de la literatura actual, de las diferentes etiologías desencadenantes del vértigo periférico y, a partir de sus características clínicas, desarrollar un algoritmo diagnóstico que permita al profesional médico entender el mecanismo patológico del vértigo y proporcionar el tratamiento idóneo.

Summary For primary care physicians, a patient with symptoms compatible with vestibular syndromes is a challenge, due to its wide variety of presentations and the difficulty in differentiating the various pathologies that cause the symptoms. The need to have clear and up-to-date concepts regarding the pathological mechanisms of vertigo arises when evaluating the patient; for this, it is necessary to carry out an adequate interrogation and a complete neurological physical examination that allows for an accurate diagnostic approach. The objective of this article is to present a review of the current literature and of the different etiologies that trigger peripheral vertigo, as well as, based on the clinical characteristics, develop a diagnostic algorithm that allows the medical professional to understand the pathological mechanism of vertigo and provide appropriate treatment.

Resumo Para os médicos de atenção primária, constitui um desafio o paciente com sintomatologia compatível com síndromes vestibulares, devido a sua ampla variedade de apresentaçöes e a dificuldade na diferenciação das diversas patologias que ocasionam os sintomas. A necessidade de ter conceitos claros e atualizados em quanto aos mecanismos patológicos da vertigem surge no momento de avaliar ao paciente. Para isto, é necessário realizar um adequado interrogatório e um exame físico neurotológico completo que permita uma abordagem diagnóstica certeira. O objetivo do presente artigo é apresentar uma revisão da literatura atual, das diferentes etiologias desencadeantes da vertigem periférica e, a partir de sus características clínicas, desenvolver um algoritmo diagnóstico que permita ao profissional médico entender o mecanismo patológico da vertigem e proporcionar o tratamento idôneo.

Dietary inclusion of fish oil changes the semen lipid composition but does not improve the post-thaw semen quality of ram spermatozoa.

The aim of this study was to investigate the effects of dietary fish oil (FO) time-response on the fatty acid profile, cholesterol levels and sperm cryosurvival in ram semen. Criollo Araucano rams were randomly assigned to two groups (n=4) according to the type of supplementation: a control group without FO and a supplemented group fed a diet with 3% FO for 8 weeks. The semen lipid profile and post-thaw sperm quality were analyzed at weeks 0 (pre-supplementation), 4, 8, 12 and 16 (post-supplementation) to evaluate the effects of FO supplementation by time interaction. Post-thaw sperm quality was determined by CASA and flow cytometry. In spermatozoa, the supplemented group increased the linoleic acid (C18:2n6c) and docosahexaenoic acid (DHA; C22:6n3) with levels higher at week 16 (P<0.05). The effect of FO on cholesterol concentration in sperm was significant at the end of the experiment (week 16). In seminal plasma, statistical differences of butyric acid (C4:0), palmitic acid (C16:0), stearic acid (C18:0), eicosatrienoic acid (C20:3n3) and DHA were observed at week 12. The cholesterol concentration was not affected by dietary treatments (P>0.05). However, the post-thaw sperm quality of the FO treatment group decreased. Motility percentage decreased 50% and spermatozoa with permeable plasma membrane and reacted acrosome were higher (63%) at week 16 than the control group. These results showed that DHA was effectively incorporated into semen through dietary supplementation with FO, but evaluations of post-thaw sperm quality confirm alteration specificity related to the structure of the lipid bilayer.