Embryo development is impaired by sperm mitochondrial-derived ROS

BACKGROUND: Basal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development. RESULTS: We first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation. CONCLUSION: Collectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.

Preservação de fertilidade: cultivo de folículos ovarianos e novo dispositivo para criopreservação seminal / Fertility preservation: ovarian follicle culture and a new device for seminal cryopreservation

INTRODUÇÃO: A oncofertilidade tem o desafio de buscar estratégias para preservar a função reprodutiva. Este estudo explorou duas possibilidades como implementação para as técnicas de preservação da fertilidade feminina e masculina. OBJETIVO: Analisar a eficiência do cultivo de folículos pré-antrais de camundongos suplementado com lisado de plaquetas humanas e desenvolver um protótipo para criopreservação de sêmen humano. MATERIAIS E MÉTODOS: Os folículos pré-antrais foram isolados mecanicamente de ovários de fêmeas de camundongos e foram cultivados individualmente em sistema entre camadas de óleo mineral. Os folículos foram cultivados divididos em 4 grupos, sendo um controle, sem o uso do lisado de plaquetas e três grupos com diferentes concentrações de lisado de plaquetas humanas (PLTMax®). Foram avaliadas a sobrevivência celular, desenvolvimento folicular e características oocitárias. Para o segundo estudo foi desenvolvido e impresso em 3D com filamentos de acrilonitrilo butadieno-estireno (ABS) um protótipo que suporte 10 palhetas com amostras seminais no vapor de nitrogênio líquido (N2L), etapa essencial para criopreservação de sêmen humano. Para os testes foram utilizadas 40 amostras seminais. A temperatura ambiente e no interior das palhetas de envase das amostras foram medidas e estabelecida a curva de resfriamento. Os parâmetros de motilidade, vitalidade e fragmentação do DNA espermático foram avaliados antes do congelamento e após o descongelamento. Foram realizados dois testes, um de posicionamento das palhetas e outro comparativo entre o protótipo e um dispositivo com suporte em poliestireno expandido (EPS). RESULTADOS: O cultivo de 11 dias induziu um aumento no tamanho folicular em todas as condições, sendo maior no grupo controle, seguido do grupo com 10% de PLTMax®, mas com diferença significativa (p<0,001). O grupo controle apresentou maior número de oócitos intactos (>50%) em relação aos demais (<35%). Todos os 4 grupos apresentaram taxas de vitalidade celular acima de 70%. Quanto aos testes com o protótipo em ABS foi verificado que as curvas de refrigeração foram notavelmente reproduzíveis. O material do protótipo resistiu a inúmeros mergulhos (>300) no N2L, sem demonstrar danos ao material. Diferenças significativas (p<0,001) foram observadas para a taxa de recuperação média da motilidade e vitalidade espermática em relação aos dados da amostra 2 fresca em ambos os testes. A motilidade, a vitalidade e a fragmentação do DNA espermático antes do congelamento e após o descongelamento não mostraram diferenças em relação a posição das palhetas. Também não houve diferença quanto ao índice de fragmentação verificada das amostras criopreservadas com uso do protótipo em ABS e o suporte em EPS, mesmo após o cultivo, após 24 horas de cultivo. Contudo, houve diferença em relação a amostra fresca (p<00,1). Quanto a recuperação das taxas de motilidade e vitalidade não houve diferença entre o ABS e EPS após o descongelamento e 24 horas de cultivo. CONCLUSÃO: O PLTMax®, embora tenha apresentado menor desempenho que o HSA, é um candidato de suplementação para o cultivo de folículos pré-antrais que merece ser mais explorado. O protótipo em ABS demonstrou resistência, praticidade e segurança para criopreservação seminal de forma reprodutível e eficiente.

INTRODUCTION: Oncofertility has the challenge of seeking strategies to preserve reproductive function. This study explored two possibilities as implementations for female and male fertility preservation techniques. PURPOSE: To analyze the efficiency of mouse preantral follicle culture supplemented with human platelet lysate and to develop a prototype for human semen cryopreservation. MATERIAL AND METHODS: Preantral follicles were mechanically isolated from female mouse ovaries and were individually cultured using a mineral oil interlayer system. The follicles were cultured divided into 4 groups, one control, without the use of platelet lysate and three groups with different concentrations of human platelet lysate (PLTMax®). Cell survival, follicular development and oocyte characteristics were evaluated. For the second study, a prototype was developed and printed in 3D with acrylonitrile butadiene styrene (ABS) filaments to support 10 straws with seminal samples in liquid nitrogen (N2L) vapor, an essential step for human semen cryopreservation. For the tests 40 seminal samples were used. Ambient and internal temperatures inside the sample straws were measured and the cooling curve was established. The parameters of motility, vitality and sperm DNA fragmentation were evaluated before freezing and after thawing. Two tests were performed, one for positioning the straws and the other comparing the prototype and a device with expanded polystyrene (EPS) support. RESULTS: The 11-day culture induced an increase in follicular size in all conditions, being higher in the control group followed by the group with 10% PLTMax®, but with significant difference (p<0.001). The control group presented a higher number of intact oocytes (>50%) compared to the others (<35%). All 4 groups presented cell vitality rates above 70%. As for the ABS prototype tests, it was verified that the cooling curves were remarkably reproducible. The prototype withstood numerous dips (>300) in N2L without showing damage to the material. Significant differences (p<0.001) were observed for the mean recovery rate of sperm motility and vitality compared to the fresh sample data in both tests. Motility, vitality and sperm DNA fragmentation before freezing and after thawing showed no differences with respect to the position of the straws. There was also no difference in the fragmentation index verified for samples cryopreserved using the ABS prototype and the EPS support, even after 24 hours of culture. However, there was a difference compared to the fresh 4 sample (p<00.1). As for the recovery of motility and vitality rates there was no difference between ABS and EPS after thawing and 24 hours of culture. CONCLUSION: PLTMax®, although it showed lower performance than HSA, is a supplementation candidate for preantral follicle culture that deserves further exploration. The ABS prototype demonstrated strength, practicality and safety for seminal cryopreservation in a reproducible and efficient manner.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Dimethyl acetamide and dimethyl sulfoxide associated at glucose and egg yolk for cryopreservation of Pseudoplatystoma corruscans semen

This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.(AU)

Este estudo teve como objetivo desenvolver um protocolo para a criopreservação do sêmen de Pseudoplatystoma corruscans. Para tal, machos maduros foram induzidos hormonalmente com uma dose única de extrato de hipófise de carpa (5 mg/kg de peso vivo). O sêmen foi coletado e avaliado. Sendo testados para compor os diluentes, dois crioprotetores: dimetil acetamida (DMA) e dimetil sulfóxido (Me2SO), em duas concentrações (8% e 10%), + 5,0% glicose + 10% gema de ovo. O sêmen foi diluído na proporção 1: 4 (sêmen: extensor), embalado em palhetas de 0,5 mL e congelado em container dryshipper em vapores de nitrogênio líquido. Após o descongelamento, foram avaliados os aspectos cinéticos espermáticos, a morfologia espermática e a integridade do DNA dos espermatozoides criopreservados. Os machos de P. corruscans produziram sêmen com motilidade espermática > 80%. Todos os tratamentos proporcionaram após o descongelamento sêmen com motilidade espermática total > 40%, sem diferença significativa (P < 0,05) entre eles, como também entre os demais parâmetros cinéticos espermáticos avaliados. Os tratamentos com DMA proporcionaram uma menor fragmentação do DNA dos gametas. Malformações espermáticas foram identificadas, tanto no sêmen fresco, como no criopreservado, sendo identificado um aumento discreto dessas malformações nos espermatozoides das amostras de sêmen descongeladas de P. corruscans.(AU)

Cisplatin-induced azoospermia and testicular damage ameliorated by adipose-derived mesenchymal stem cells

BACKGROUND: The testes are highly susceptible to the adverse effects of chemotherapy and radiation at all stages of life. Exposure to these threats mainly occurs during cancer treatment and as an occupational hazard in radiation centers. The present study investigated the regenerative ability of adipose-derived mesenchymal stem cells (ADMSCs) against the adverse effects of cisplatin on the structure and function of the testes. METHODS: New Zealand white rabbits (N = 15) were divided into three groups of five: a negative control group (no treatment), a cisplatin group (single dose of cisplatin into each testis followed three days later by a PBS injection), and a cisplatin + ADMSCs group (cisplatin injection followed three days later by an ADMSC injection). On day 45 post-treatment, serum testosterone levels were evaluated, and the testes and epididymis were collected for histology, oxidative stress examination, and epididymal sperm analysis. RESULTS: Cisplatin caused damage to the testicular tissue and decreased serum testosterone levels, epididymal sperm counts, and oxidants. An antioxidant imbalance was detected due to increasing malondialdehyde (MDA) and reduced glutathione (GSH) levels in testicular tissue. The ADMSC-treated group displayed a moderate epididymal sperm count, adequate antioxidant protection, suitable hormone levels, and enhanced testicular tissue morphology. CONCLUSIONS: ADMSCs treatment repaired damaged testicular tissue, enhanced biochemical parameters, and modified pathological changes caused by cisplatin.

Detection of SARS-CoV-2 in semen samples: a scoping review / Detecção de SARS-CoV-2 em amostras de sêmen: revisão de escopo

Background: many doubts about the infection of SARS-CoV-2 were raised, such as sexual transmission, sterility, and changes in fertility procedures; however, information is not clearly stated and organized. Purpose: to review and summarize scientific evidence on detection of SARS-CoV-2 in semen samples of Covid-19 patients. Methods:literature search in PubMed, Web of Science, Scopus, Medline and Embase databases, and followed Scoping Review protocol defined by Joanna Briggs Institute (JBI) after the guiding question "Is it possible to detect SARS-CoV-2 in the semen of adult patients with a confirmed diagnosis of Covid-19?" Results: 287 studies were identified, and, after discerning analysis, 9 studies published in the English language were selected. Three researchers analyzed the studies for SARS-CoV-2 presence in the seminal fluid, patients' severity, days since the onset of disease, diagnosis confirmation, semen collection method, viral analysis method, and sample numbers. Conclusions: it was not possible to find strong evidence to confirm the presence or absence of Covid-19 in the semen of adult patients. New studies on the subject should be better designed, taking into account the possible anatomical and functional conditions and changes of the male reproductive system during and after the infection by SARS-CoV-2 (AU)

Objetivo: Revisar e resumir as evidências científicas em pesquisas realizadas para detectar a presença de SARS-CoV-2 em amostras de sêmen de pacientes com COVID-19. Métodos: A pesquisa de literatura foi conduzida nas bases de dados PubMed, Web of Science, Scopus, Medline e Embase. Seguiu o protocolo de revisão de escopo definido por Joanna Briggs Institute (JBI) e baseou-se na pergunta norteadora "É possível detectar SARS-CoV-2 no sêmen de pacientes adultos com diagnóstico confirmado de Covid-19?". Resultados: 287 estudos foram identificados, 9 estudos publicados em língua inglesa foram selecionados após análise minuciosa. Três pesquisadores analisaram os estudos em busca de presença de SARS-CoV-2 no fluído seminal, gravidade do paciente, dias desde o início da doença, confirmação diagnóstica, método de coleta de sêmen, método de análise viral e número de amostras. Conclusões: Não foi possível identificar fortes evidências para confirmar a presença ou ausência de COVID-19 no sêmen de pacientes adultos. Novos estudos sobre o tema devem ser melhor projetados, levando-se em conta as possíveis condições anatômicas e funcionais e mudanças no sistema reprodutor masculino durante e após a infecção por SARS-CoV-2 (AU)

Detection of SARS-CoV-2 in semen samples: a scoping review / Detecção de SARS-CoV-2 em amostras de sêmen: revisão de escopo

ABSTRAC: :Background: many doubts about the infection of SARS-CoV-2 were raised, such as sexual transmission, sterility, and changes in fertility procedures; however, information is not clearly stated and organized. Purpose: to review and summarize scientific evidence on detection of SARS-CoV-2 in semen samples of Covid-19 patients. Methods:literature search in PubMed, Web of Science, Scopus, Medline and Embase databases, and followed Scoping Review protocol defined by Joanna Briggs Institute (JBI) after the guiding question "Is it possible to detect SARS-CoV-2 in the semen of adult patients with a confirmed diagnosis of Covid-19?" Results: 287 studies were identified, and, after discerning analysis, 9 studies published in the English language were selected. Three researchers analyzed the studies for SARS-CoV-2 presence in the seminal fluid, patients' severity, days since the onset of disease, diagnosis confirmation, semen collection method, viral analysis method, and sample numbers. Conclusions: it was not possible to find strong evidence to confirm the presence or absence of Covid-19 in the semen of adult patients. New studies on the subject should be better designed, taking into account the possible anatomical and functional conditions and changes of the male reproductive system during and after the infection by SARS-CoV-2. (AU)

RESUMO:Objetivo: Revisar e resumir as evidências científicas em pesquisas realizadas para detectar a presença de SARS-CoV-2 em amostras de sêmen de pacientes com COVID-19. Métodos: A pesquisa de literatura foi conduzida nas bases de dados PubMed, Web of Science, Scopus, Medline e Embase. Seguiu o protocolo de revisão de escopo definido por Joanna Briggs Institute (JBI) e baseou-se na pergunta norteadora "É possível detectar SARS-CoV-2 no sêmen de pacientes adultos com diagnóstico confirmado de Covid-19?". Resultados: 287 estudos foram identificados, 9 estudos publicados em língua inglesa foram selecionados após análise minuciosa. Três pesquisadores analisaram os estudos em busca de presença de SARS-CoV-2 no fluído seminal, gravidade do paciente, dias desde o início da doença, confirmação diagnóstica, método de coleta de sêmen, método de análise viral e número de amostras. Conclusões: Não foi possível identificar fortes evidências para confirmar a presença ou ausência de COVID-19 no sêmen de pacientes adultos. Novos estudos sobre o tema devem ser melhor projetados, levando-se em conta as possíveis condições anatômicas e funcionais e mudanças no sistema reprodutor masculino durante e após a infecção por SARS-CoV-2. (AU)

Efecto del consumo de marihuana sobre los parámetros espermáticos humanos: Aproximación in vivo / Effect of Marijuana Use on Human Semen Parameters: In Vivo Approach

La evidencia sugiere que la exposición a sustancias psicoactivas se relaciona con alteraciones en la espermatogénesis que afectan la calidad espermática. El objetivo de este trabajo fue determinar los parámetros espermáticos en consumidores habituales de cigarrillos de marihuana. Se analizaron muestras seminales de 42 consumidores activos de cigarrillos de marihuana y de 16 voluntarios no consumidores de marihuana. Mediante un análisis de semen, se determinaron los parámetros seminales convencionales (viabilidad, movilidad, morfología, y concentración de los espermatozoides) siguiendo los lineamientos establecidos por la Organización Mundial de la Salud (OMS). Adicionalmente, se evaluó la capacidad antioxidante del plasma seminal mediante la determinación del porcentaje de inhibición del radical estable 1,1-difenil-2-picril-hidracilo. Los valores de la mediana de los consumidores respecto al grupo control fueron: volumen ­ 2,98 mL versus 3,95 mL (p = 0,0221); concentración total ­ 189 millones/mL versus 291,1 millones/mL (p = 0,0636); movilidad progresiva ­ 50% versus 56,5% (p = 0,0052); viabilidad ­ 65,3% versus 73,1% (p = 0,0732); y morfología normal ­ 5% versus 7% (p = 0,0167), respectivamente. Los resultados obtenidos en este estudio indican que el consumo de cigarrillos de marihuana afecta negativamente la movilidad progresiva, la morfología normal y la concentración total de espermatozoides; además, la concentración total de espermatozoides está afectada por la frecuencia del consumo de cigarrillos de marihuana.

Evidence suggests that exposure to psychoactive substances is related to spermatogenesis alterations that affect sperm quality. The objective of the present work was to determine sperm parameters in regular users of marijuana cigarettes. Seminal samples from 42 active consumers of marijuana cigarettes and 16 volunteer non-marijuana users were analyzed. Through a semen analysis, we identify conventional seminal parameters (viability, motility, morphology, and sperm concentration) according to the guidelines established by the World Health Organization (WHO). The antioxidant effect of the seminal plasma was evaluated through the determination of the percentage of inhibition of the stable radical 1,1-diphenyl-2-picrylhydrazyl. The median values of consumers with respect to the control group were respectively: volume ­ 2.98 mL versus 3.95 mL (p = 0.0221); total concentration ­ 189 million/mL versus 291.1 million/mL (p = 0.0636); progressive motility ­ 50% versus 56.5% (p = 0.0052); viability ­ 65.3% versus 73.1% (p = 0.0732); and normal morphology ­ 5% versus 7% (p = 0.0167). The results obtained in the present study indicate that the consumption of marijuana cigarettes negatively affects progressive motility, normal morphology, and total sperm concentration. In addition, the total sperm concentration is affected by the frequency of consumption of marijuana cigarettes

Utility of melatonin in mitigating ionizing radiation-induced testis injury through synergistic interdependence of its biological properties

BACKGROUND: Ionizing radiations (IR) have widespread useful applications in our daily life; however, they have unfavorable effects on reproductive health. Maintaining testicular health following IR exposure is an important requirement for reproductive potential. The current study explored the role of melatonin (MLT) in mitigating IR-induced injury in young adult rat testis. METHODS: Rats were given daily MLT (25 mg/kg) for 3 and 14 days after receiving 4 Gy γ-radiation. RESULTS: Serum MLT levels and other antioxidants, including glutathione content, and the activity of glutathione peroxidase and glutathione reductase in the testis of the irradiated rats were remarkably maintained by MLT administration in irradiated rats. Hence, the hydrogen peroxide level declined with remarkably reduced formation of oxidative stress markers, 4-hydroxynonenal, and 8-Hydroxy-2'-deoxyguanosine in the testis of irradiated animals after MLT administration. The redox status improvement caused a remarkable regression of proapoptotic protein (p53, Cyto-c, and caspase-3) in the testis and improved inflammatory cytokines (CRP and IL-6), and anti-inflammatory cytokine (interleukin IL-10) in serum. This is associated with restoration of disturbed sex hormonal balance, androgen receptor upregulation, and testicular cell proliferation activity in irradiated rats, explaining the improvement of sperm parameters (count, motility, viability, and deformation). Consequently, spermatogenic cell depletion and decreased seminiferous tubule diameter and perimeter were attenuated by MLT treatment post irradiation. Moreover, the testis of irradiated-MLT-treated rats showed well-organized histological architecture and normal sperm morphology. CONCLUSIONS: These results show that radiation-induced testicular injury is mitigated following IR exposure through synergistic interdependence between the antioxidant, anti-inflammatory, anti-apoptotic, and anti-DNA damage actions of MLT.

Utilización de la herramienta chroma key para el estudio de la morfología y la fisiología espermática en entornos virtuales / Use of the chroma key to study sperm morphology and physiology in virtual environments

RESUMEN: Uno de los retos en el uso de nuevas metodologías y tecnologías durante la crisis sanitaria causada por el virus SARS-CoV-2 ha sido mantener la motivación del alumnado en entornos virtuales. Por ello, el objetivo de este trabajo fue valorar la utilidad de materiales audiovisuales creados con chroma key en la metodología flipped classroom para impartir algunos conceptos teóricos en la asignatura de Biología del Desarrollo en el Grado en Biología de la Universidad de Alicante. Para ello, el profesorado de la asignatura elaboró vídeos utilizando la tecnología chroma key, los cuales fueron visualizados por parte del alumnado antes de las sesiones teóricas online. Durante dichas sesiones, el alumnado puso en práctica los conceptos comentados en los vídeos a través de la realización de actividades. La percepción del estudiantado sobre la metodología empleada se obtuvo mediante un cuestionario de opinión, en el cual el 90 % de los encuestados/as manifestaron que el uso combinado del flipped classroom con chroma key facilitaba el aprendizaje al adaptarse al ritmo y necesidades educativas de cada estudiante. Asimismo, destacaron que el uso de escenografía virtual con chroma key hizo más amena y atrayente la docencia online. En conclusión, el chroma key constituye una herramienta eficaz para realizar materiales educativos en flipped classroom que, además, resulta llamativo y motivador para el alumnado.

SUMMARY: One of the challenges in the use of new methodologies and technologies during the health crisis caused by the SARS-CoV-2 virus has been to keep students motivated in virtual environments. Therefore, the objective of this work was to assess the usefulness of audiovisual materials created with chroma key in the flipped classroom methodology to teach some theoretical concepts in the subject of Developmental Biology in the Degree in Biology at the University of Alicante. For this, the teaching staff of the subject produced videos using chroma key technology, which were viewed by the students before the online theoretical sessions. During these sessions, the students put into practice the concepts discussed in the videos by carrying out activities. The students' perception of the methodology used was obtained through an opinion questionnaire, in which 90 % of the respondents stated that the combined use of the flipped classroom with chroma key facilitated learning by adapting to the rhythm and educational needs of each student. They also highlighted that the use of virtual scenery with chroma key made online teaching more enjoyable and attractive. In conclusion, the chroma key is an effective tool for creating educational materials in the flipped classroom that is also attractive and motivating for students.

Investigation of Tritrichomonas foetus in cryopreserved bovine semen by culture and polymerase chain reaction / Pesquisa por Tritrichomonas foetus em sêmen bovino criopreservado mediante isolamento e reação em cadeia da polimerase

Bovine Trichomoniasis (BT) is an infectious disease caused by Tritrichomonas foetus that can be transmitted either sexually or by fomites. In males, the disease is asymptomatic and permanent. T. foetus has been detected in semen samples where it is able to remain viable even when frozen. The objective of this study was to investigate the presence of T. foetus in 27 samples of commercial frozen bovine semen by culture and Polymerase Chain Reaction (PCR). Samples were thawed in water at 37°C. Part of the samples was inoculated in a test tube containing Diamond's medium and incubated at 35°C. Growth was evaluated every 24 hours via direct examination under a microscope. The other part was placed in an Eppendorf tube and frozen for later molecular analysis. After 10 days of culture, all samples were negative for T. foetus. The Quick-DNA Miniprep Kit (Zymo Research) without proteinase K was used for DNA extraction. The primers used in PCR were TRF3 and TRF4. PCR results were negative for all samples. In conclusion, bovine semen samples were negative for T. foetus in both diagnostic methods, according to the adopted methodology.(AU)

A tricomonose genital bovina (TGB), uma doença infectocontagiosa causada pelo Tritrichomonas foetus, é transmitida por via venérea e fômites contaminados. Em machos a doença é assintomática e permanente. O agente já foi encontrado em amostras de sêmen e é capaz de permanecer viável quando congelado. Este trabalho teve por objetivo investigar a presença de T. foetus em 27 amostras de sêmen bovino comercial congelado, por meio de cultivo e reação em cadeia da polimerase (PCR). As amostras foram descongeladas em água a 37ºC; parte foi inoculada em tubo de ensaio contendo meio Diamond, incubada a 35ºC com consequente avaliação de crescimento e avaliada a cada 24 horas, via exame direto em microscópio, e a outra parte foi diluída em PBS para análise molecular. Após 10 dias de cultivo, todas as amostras foram negativas. Para a detecção molecular foi utilizado o kit Quick-DNA Miniprep (Zymo Research) sem proteinase K para extração do DNA. Os iniciadores utilizados na PCR foram TRF3 e TRF4. O resultado da PCR foi negativo para todas as amostras. Conclui-se que as amostras utilizadas foram realmente negativas para a presença do patógeno em ambos os métodos diagnósticos, o que comprovou a inocuidade do sêmen testado.(AU)

The Effect of Glyphosate on Human Sperm: In Vitro Approximation / Efecto del glifosato sobre los espermatozoides humanos: una aproximación in vitro

Introduction Glyphosate is an herbicide used to eradicate illicit crops; however, its use is controversial due to different health problems associated with it. The present study aims to evaluate the effects of glyphosate on human sperm in vitro. Methods Twenty-two semen samples from healthy normozoospermic men were included; 11 semen samples were incubated with Panzer (INVESA S.A., Antiquia, Colombia) and 11 with Roundup (Monsanto Company, MO, USA). The changes in motility and viability were observed. Functional seminal parameters were evaluated as well. Results The samples exposed to glyphosate showed less motility and viability; a decrease in the potential of the mitochondrial membrane was observed, and an increase in the lipoperoxidation of the membrane was evidenced. Conclusion Based on the present results, we concluded that glyphosate has cytotoxic potential for exposed people and may affect their fertility.

Introducción El glifosato es un herbicida utilizado ampliamente para la erradicación de cultivos ilícitos; sin embargo, su uso es polémico debido a diferentes problemas de salud asociados con él. El objetivo del presente estudio fue evaluar los efectos del glifosato sobre los espermatozoides humanos in vitro. Métodos Se incluyeron 22 muestras de semen de hombres sanos normozoospérmicos, de las cuales 11 se incubaron con Panzer y 11 con Roundup, y se evaluaron los cambios en la movilidad y la viabilidad espermática, además de valorar los parámetros seminales funcionales. Resultados Las muestras expuestas al glifosato presentaron una menor movilidad y viabilidad, una disminución en el potencial de la membrana mitocondrial, y un aumento en la lipoperoxidación de la membrana. Conclusiones El glifosato es potencialmente citotóxico para las personas que estén expuestas, y puede afectar su fertilidad.

Seropositivity for Bovine Viral Diarrhea and Enzootic Bovine Leukemia viruses in Blanco Orejinegro cattle in Colombia and infection associated-factors / Soropositividade para o vírus da diarréia viral bovina e leucemia bovina enzoótica em bovinos Blanco Orejinegro na Colômbia e fatores associados à infecção

ABSTRACT Serological controls for diseases of viral origin in animal production systems and the identification of factors associated with infections are decisive elements to establish prevention and control measures. The aim of this study was to establish the serological status for Bovine Viral Diarrhea (BVD) and Enzootic Bovine Leukemia (EBL) viruses in Blanco Orejinegro (BON) cattle from Colombia, and to identify the factors associated with seropositivity. A cross-sectional study was conducted with a total of 498 animals of all age groups and physiological states of the BON breed were selected, belonging to 14 herds located in 6 states of Colombia, in which a survey with 27 questions was conducted. By means of the chi-square test, possible factors associated with seropositivity against the 2 viruses were identified. A sample of 4 ml of blood was taken from each animal to extract plasma and make indirect Elisa tests to detect antibodies against both pathogens. General seropositivity of 27,1% was obtained for EBL, finding as factors associated with seropositivity the inadequate disposition of placental tissues after delivery of the cows and the non-performance of serological tests on new animals entering the herd. For BVD, seropositivity obtained was 50,6%, and the factors associated with seropositivity identified were having had a history of the disease in the herd, and using semen from bulls that are not known to be free for the infection. We suggest establishing control measures considering the factors associated with each viral infection to limit their expansion in the BON cattle production systems of Colombia.

RESUMO Os controles sorológicos para doenças de origem viral nos sistemas de produção animal e a identificação de fatores associados à infecção são elementos decisivos para estabelecer medidas de prevenção e controle. O objetivo deste estudo foi estabelecer o status sorológico dos vírus da Diarréia Viral Bovina (BVD) e da Leucemia Bovina Enzoótica (EBL) em bovinos Blanco Orejinegro (BON) da Colômbia e identificar os fatores associados à soropositividade. Foram selecionados 498 animais de todas as faixas etárias e estados fisiológicos da raça BON, de 14 rebanhos localizados em seis regiões da Colômbia, nos quais foi realizada uma pesquisa para identificar possíveis fatores associados à soropositividade contra os dois vírus. Foi retirada uma amostra de 4 ml de sangue de cada animal para extrair plasma e fazer testes ELISA indiretos para detectar anticorpos contra os dois patógenos. Obteve-se soropositividade geral de 27,1% para EBL, encontrando como fatores associados à soropositividade a disposição inadequada dos tecidos placentários após o parto das vacas e a não realização de testes sorológicos em novos animais que entraram no rebanho. Para a BVD, a soropositividade obtida foi de 50,6%, e os fatores associados à soropositividade identificados foram: histórico de doença no rebanho e uso de sêmen de touros que não são reconhecidos como livres da infecção. Sugerimos o estabelecimento de medidas de controle considerando os fatores associados a cada infecção viral para limitar sua expansão nos sistemas de produção de gado BON da Colômbia.

Scrotal thermographic profile and seminal characteristics of Mangalarga Marchador stallions bred in the Atlantic Rainforest biome / [Perfil termográfico escrotal e características seminais de garanhões Mangalarga Marchador criados no bioma Mata Atlântica]

The objective of this study was to determine the scrotal thermographic profile and to verify the influence of temperature and humidity of the humid tropical climate on testicular temperature and seminal quality of Mangalarga Marchador stallions. The thermal profiles of the proximal, middle, and distal zones of the testicles and total surface temperature (TSTT) were recorded using an FLIR E60bx thermal imager. The average air temperature (°C) and relative humidity (%) were obtained 1, 5, 9, 33, and 66 days before semen collection and showed a mean value of 26.5±2.4 and 80.4±6.0 respectively. The scrotal surface temperature was close to 34°C and there was no variation with the age of the stallion, reproductive activity, and characteristics of the ejaculate (P>0.05). The only significant correlations obtained were between TSTT and minor defects (R = 0.41; P<0.05), between TSTT and total defects (R = 0.46; P<0.01), and between TSTT and percentage of morphologically normal sperm (R = -0.46; P<0.05). It was concluded that the Mangalarga Marchador stallions maintained the testicular temperature within favorable conditions for spermatogenesis, demonstrating the efficiency of testicular thermoregulation mechanisms in the Atlantic Forest biome.

O objetivo deste estudo foi determinar o perfil termográfico escrotal e verificar a influência da temperatura e da umidade do clima tropical úmido na temperatura testicular e na qualidade seminal de garanhões Mangalarga Marchador. Os perfis térmicos das zonas proximal, média e distal dos testículos e a temperatura da superfície total (TSTT) foram registrados usando-se um termovisor FLIR E60bx. A temperatura média do ar (° C) e a umidade relativa (%) foram obtidas um, cinco, nove, 33 e 66 dias antes da coleta de sêmen e apresentaram valor médio de 26,5 ± 2,4 e 80,4 ± 6,0, respectivamente. A temperatura da superfície escrotal foi próxima a 34°C, e não houve variação com a idade do garanhão, a atividade reprodutiva e as características do ejaculado (P>0,05). As únicas correlações significativas obtidas foram entre TSTT e defeitos menores (R=0,41; P<0,05), entre TSTT e defeitos totais (R=0,46; P<0,01), e entre TSTT e porcentagem de espermatozoides morfologicamente normais (R=-0,46; P<0,05). Concluiu-se que os garanhões Mangalarga Marchador mantiveram a temperatura testicular dentro de condições favoráveis para a espermatogênese, demonstrando a eficiência dos mecanismos de termorregulação testicular no bioma Mata Atlântica.

Efecto de la suplementación del medio de incubación con extracto de Berberis microphylla (calafate) sobre la funcionalidad espermática en semen bovino descongelado / Effect of supplementation of incubation medium with Berberis microphylla (calafate) extract on sperm functionality in thawed bovine semen

RESUMEN: En el semen criopreservado, los procesos de congelación/descongelación y posterior manipulación, dañan las células espermáticas provocando disminución de la capacidad fecundante de los espermatozoides descongelados. Estos procesos han sido asociados con el estado de estrés oxidativo (EO) inducido por altos niveles de especies reactivas de oxígeno (EROS), causando daño a la función y estructura espermática. Los espermatozoides descongelados pueden ser protegidos de este daño, con la adición de antioxidantes (AO) al medio de incubación. El fruto de Calafate (Berberis microphylla G. Forst.) posee una alta capacidad antioxidante, lo que hace interesante investigar el efecto de sus componentes antioxidantes en estos procesos biotecnológicos especialmente postdescongelación. El objetivo de este estudio fue determinar el efecto de la suplementación de extracto liofilizado de fruto de Calafate (ELC), sobre la calidad espermática post-descongelación. Previamente se caracterizó el ELC, determinando la actividad antioxidante y metabolitos como fenoles y antocianinas; posteriormente, espermatozoides de bovino descongelados fueron incubados en un medio base suplementado con diferentes concentraciones de ELC. Post-incubación se evaluó la motilidad progresiva; la viabilidad e integridad de la membrana plasmática (SYBR14- PI) y acrosomal (FITC-PNA/PI) y la peroxidación lipídica (BODIPY) por citometría de flujo. La caracterización de ELC demostró que tanto la actividad antioxidante como los fenoles y antocianinas incrementan concomitante con el aumento de la concentración de ELC. La adición de ELC al medio de incubación, dependiendo de la concentración y tiempo de incubación, sería eficaz en proteger la motilidad, viabilidad e integridad de la membrana plasmática y disminuir la lipoperoxidación en los espermatozoides de bovino descongelados.

SUMMARY: In cryopreserved semen, the freezing/thawing process following of manipulation, damage the sperm cell, decreasing the fertilizing capacity of the thawed sperm; being one of the main factors of this damage the oxidative stress. The sperm once thawed can be protected from this damage, with the addition of antioxidants to the incubation medium. The Calafate fruit (Berberis microphylla G. Forst.) has a high antioxidant capacity, making it an interesting resource for investigating the effect of its antioxidant components on biotechnological processes. The objective of this study was to determine the effect of supplementation of Calafate fruit lyophilized extract (ELC) on sperm quality. The lyophilized extract of the Calafate fruit was characterized, determining the antioxidant activity and metabolites such as phenols and anthocyanins; subsequently, thawed bovine sperm were incubated in a medium supplemented with different concentrations of ELC. Post-incubation, progressive motility was evaluated. By flow cytometry, the viability and integrity of the plasma (SYBR14-PI), and acrosomal (FITC-PNA / PI), as well as lipid peroxidation (BODIPY), was determined. The characterization of Calafate fruits lyophilized extract indicated that antioxidant activity, phenols and anthocyanins increased concomitantly with the increase of dose extract used. The addition of ELC to the incubation medium, depending on the concentration and incubation time, would be effective to protect motility, viability and integrity of the plasma membrane and decreased lipid peroxidation in thawed bovine sperm.

The association of seminal oxidation reduction potential with sperm parameters in patients with unexplained and male factor infertility

ABSTRACT Purpose: Understanding the effects of high oxidation reduction potential (ORP) levels on sperm parameters will help to identify patients with unexplained and male factor infertility who may have seminal oxidative stress and determine if ORP testing is needed. This study aimed to evaluate the association between seminal ORP and conventional sperm parameters. Materials and Methods: A total of 58 patients who provided a semen sample for simultaneous evaluation of sperm parameters and ORP between January and September 2019 were enrolled in this retrospective study. To identify normal and high ORP levels, a static ORP (sORP) cut-off value of 1.36mV/106sperm/mL was used. Sperm parameters were compared between infertile men with normal sORP (control group, n=23) and high sORP values (study group, n=35). Results: Men with sORP values >1.36mV/106sperm/mL had significantly lower total sperm count (TSC) (p <0.001), sperm concentration (p <0.001) and total motile sperm count (TMSC) (p <0.001). In addition, progressive motility (p=0.04) and fast forward progressive motility (p <0.001) were significantly lower in the study group. A negative correlation was found between sORP and TSC (r=-0.820, p <0.001), sperm concentration (r=-0.822, p <0.001), TMSC (r=-0.808, p <0.001) and progressive motility (r=-0.378, p=0.004). Non-progressive motility positively correlated with sORP (r=0.344, p=0.010). Conclusions: This study has shown that TSC, sperm concentration, progressive motility and TMSC are associated with seminal oxidative stress, indicated by a sORP cut-off of 1.36mV/106sperm/mL. Presence of oligozoospermia, reduced progressive motilty or low TMSC in sperm analysis should raise the suspicion of oxidative stress and warrants seminal ROS testing.

Classification of the sperm chromatin compaction alterations in bulls (Bos taurus) and its correlation with the efficiency of the in vitro production of embryos

This paper proposes to classify the sperm chromatin compaction alterations in bulls, according to the affected area location and its objective is evaluating the correlation of the intensity, the heterogeneity and these kinds of chromatin decompaction with the rates of cleavage and the formation of blastocysts of in vitro production of embryos (IVPE). It was used several subfertile animals sperm samples, which were evaluated using the toluidine blue staining and computer image analysis, making possible the categorization of the chromatin decompaction according to their location. The percentages of chromatin decompaction and heterogeneity were also evaluated. IVPEs were done and the rates of cleavage and of blastocysts were correlated with the chromatin characteristics. It made possible the classification of the chromatin decompaction according to the head affected part in at least four types: base decompaction, basal half decompaction, central axis decompaction, total decompaction. Based on the correlation, it can be implicated that each type of classification has different influences on the bull fertility. It made possible understanding that sperms amount with 5% or more of chromatin decompaction intensity interferes in the bull fertility and this condition can be featured as an uncompensable defect, while the heterogeneity of chromatin is not an important factor in the IVPE results.

Efeito da adição do ácido palmítico e da vitamina E ao diluidor Tris-gema na criopreservação de sêmen canino / Effect of addition of palmitic acid and Vitamin E to the Tris-egg yolk diluter in canine semen cryopreservation

Canine sperm is a very delicate cell that is quite susceptible to oxidative stress since the cytoplasm is restricted and features little antioxidant reserves. Furthermore, the sperm membrane has some polyunsaturated fatty acids sensitive to lipid peroxidation, which makes it important to addition antioxidant substances to the diluter aiming at decreasing such stress to the sperm cell, particularly during seminal cryopreservation. Several antioxidants have been used in this process in some domestic animal's species, however, the use of palmitic acid has been little reported in works on cryopreservation of semen of the canine species. Hence, this study aimed to assess the effect of addition antioxidants palmitic acid and vitamin E to the Tris-egg yolk diluter on the semen quality of dogs after thawing. Samples were collected from the ejaculates of 4 adult dogs, apparently healthy, of the American Pit Bull Terrier breed of kennels in the city of Teresina, PI, places where the pre-freezing procedures of the dog's semen were performed. The samples were diluted in Tris citric acid fructose (3.28 g Tris-hydroxymethyl-aminomethane, 1.78 g citric acid monohydrate and 1.25 g D-fructose), dissolved in 100 mL distilled water, and added 20% egg yolk and 6% glycerol, at the concentration of 100x106 sptz/mL. The semen samples were divided into 3 mL aliquots to form 3 experimental groups: G1 - Only Tris-egg yolk (Control group); G2 - Tris-egg yolk + 100 µM palmitic acid; and G3 - Tris-egg yolk + 116 µM vitamin E. Semen was collected weekly over a period of little over 2 months. After thawing, thermorresistance test (TTR) was carried out at 0, 30, 60, and 90 min to assess spermatics motility and vigor, in addition to analysis of integrity of plasma membrane, acrosomal membrane and mitochondrial activity of the sperm, using fluorescent probes. These assessments were performed out at the Animal Reproduction Biotechnology Laboratory (LBRA/UFPI). In the TTR, G2 and G3 didn't exhibit significant results for spermatics motility or vigor when compared with the control group. The palmitic acid and vitamin E also had no significant effects on the parameters of acrosomal membrane integrity or mitochondrial activity. However, sperm cryopreserved with the addition of palmitic acid exhibited significant differences for plasma membrane integrity, providing greater protection to the sperm cells in G2. The palmitic acid is one of the most saturated fatty acids in human semen, with reports of great proportions also in the seminal plasma of dogs. Its main role is to protect the plasma membrane from external damage, improving viability and fertility of the sperm after cryopreservation. Data is scarce in the literature on the composition of fatty acids in canine semen and regarding the use of palmitic acid as a seminal antioxidant in that species, which grants further studies aiming to investigate such valuable information for canine reproduction. It is concluded that addition palmitic acid at 100µM concentration to the Tris-egg yolk diluter was able to preserve the integrity of the plasma membrane during the process of cryopreservation of canine semen.(AU)