Effect of mitochondrial potential of bovine sperm on in vitro embryo production

O uso de biotécnicas da reprodução vem sendo utilizado como ferramenta para melhorar tanto a eficiência reprodutiva quanto maximizar o uso de animais geneticamente superiores. O Brasil é responsável por 29% da produção in vitro de embriões bovinos (PIVE) no mundo. Apesar da extensa utilização desta biotécnica, existem pontos críticos que limitam a sua eficiência. Fatores relacionados à qualidade de oócitos, o ambiente in vitro (mais exposto ao O2 atmosférico) e efeito touro (atributos espermáticos). Os atributos espermáticos vêm sendo relacionados com a fertilidade. Estudos têm relacionado o potencial de membrana mitocondrial (ΔΨm) com o estresse oxidativo, sendo essa associação ora positiva ora negativa para a fecundação. Acredita-se que essa seja influenciada por outras características espermáticas associadas. Com isso, formulou-se a hipótese de que o ΔΨm é um atributo dependente dos demais atributos relacionados à qualidade espermática, sendo que o Alto Perfil espermático quando associado com o Maior ΔΨm, condiciona um ambiente mais ativo e oxidativo durante a fecundação in vitro (FIV). Esse ambiente mais exacerbado é deletério, diminuindo a taxa de blastocistos. No entanto, espermatozoides de Baixo Perfil associados a um Maior ΔΨm, proporcionam um ambiente menos ativo e menos exacerbado, portanto maiores taxa de blastocistos. O objetivo deste trabalho foi avaliar como o ΔΨm e os atributos relacionados ao perfil espermático interferem nas taxas de PIVE e na resposta antioxidante destes embriões. Portanto, foram realizadas manipulações de PIVE, análises dos parâmetros espermáticos por citometria de fluxo, cinética espermática (CASA), atividade mitocondrial (DAB), estresse oxidativo e detecção de óxido nítrico (NO. ), análise de expressão de genes antioxidantes nos embriões. Além disso, foram realizadas análises de compostos associados a mitocôndria (ADT, ATP, NADH, FADH2) e peroxidação lipídica (4-HNE) por cromatografia líquida em amostras de espermatozoides e meio de FIV, respectivamente. Os resultados demonstram que apesar de ter sido observado relação de dependência do ΔΨm com os demais atributos espermáticos, a relação encontrada entre o ΔΨm e o Perfil espermático foi contrária a esperada na hipótese proposta. Ou seja, o Alto Perfil associado ao Maior ΔΨm apresentou maiores taxas na PIVE. Assim como o Baixo Perfil com Maior ΔΨm apresentou as piores taxas na PIVE. O Perfil espermático influenciou mais na PIVE do que a diferença de ΔΨm. Quanto ao status oxidativo nos espermatozoides, não foram observadas diferenças entre os grupos estudados. As diferenças relacionadas ao metabolismo mitocondrial foram associadas à análise de DAB, e uma via compensatória foi observada no grupo de Menor ΔΨm. Os baixos níveis de NO. detectados no meio de FIV foram associados com maiores índices de fertilidade in in vitro. Para análise de expressão gênica, GPx4 foi mais expressa no grupo de Baixo Perfil espermático, demonstrando um desequilíbrio da resposta antioxidante no grupo de menor qualidade espermática. Não foi possível a detecção dos compostos relacionados ao metabolismo mitocondrial e peroxidação lipídica por cromatografia líquida. Portanto mais estudos, com metodologias mais sensíveis para essa detecção, devem ser realizados para que esses compostos sejam detectados e associados com o Δ Ψm, para melhor entendimento da função mitocondrial na PIVE.Reproductive biotechnologies have been used as a tool to improve both reproductive efficiency and maximize the use of genetically superior animals. Brazil is responsible for 29% of the in vitro production of bovine embryos (IVP) in the world. Despite the extensive use of this biotechnology, there are critical points that limit its efficiency. These factors relate to oocyte quality, in vitro environment (exposure to atmospheric O2) and the bull effect (sperm attributes). Sperm attributes have been associated with fertility. Studies have related the mitochondrial membrane potential (ΔΨm) with oxidative stress, with this association being either positive or negative for fertilization. This is believed to be influenced by other associated sperm characteristics. Thus, it is hypothesized that ΔΨm is an attribute dependent on the other attributes related to sperm quality, and the high sperm profile, when associated with the higher ΔΨm, leads to a more active and oxidative environment during in in vitro fertilization (IVF). This exacerbated environment is harmful, decreasing the rate of blastocyst formation. However, low profile sperm associated with a higher ΔΨm provides a less active and less exacerbated environment, therefore resulting in higher rates of blastocyst formation. The objective of this work was to evaluate how ΔΨm and attributes related to the sperm profile interfere in the PIVE rates and in the antioxidant response of these embryos. Therefore, IVP manipulations, analysis of sperm parameters by flow cytometry, sperm kinetics (CASA), mitochondrial activity (DAB), oxidative stress and detection of nitric oxide (NO. ), and analysis of expression of antioxidant genes in embryos were performed. In addition, analyses of compounds associated with mitochondria (ADT, ATP, NADH, FADH2) and lipid peroxidation (4-HNE) were performed by liquid chromatography on samples of sperm and IVF medium, respectively. The results demonstrate that although a relationship of dependence of ΔΨm with the other sperm attributes was observed, the relationship found between ΔΨm and the sperm profile was contrary to what was expected in the proposed hypothesis. In other words, the high profile sperm associated with higher Δ Ψm resulted in higher rates of IVP. The low profile with the Higher ΔΨm presented the worst rates of IVP. The sperm profile influenced IVP more than the ΔΨm differences. Regarding the oxidative status in sperm, no differences were observed between the groups studied. Differences related to mitochondrial metabolism were associated with the analysis of DAB, and a compensatory pathway was observed in the group of lower ΔΨm. Lower levels of NO detected in the IVF medium were associated with higher in in vitro fertility rates. For gene expression analysis, Gpx4 had higher expression in the low sperm profile group, demonstrating an imbalance of the antioxidant response in the group with lower sperm quality. It was not possible to detect compounds related to mitochondrial metabolism and lipid peroxidation by liquid chromatography. Therefore, more studies, with more sensitive methodologies for this detection, should be carried out and associated with ΔΨm, for a better understanding of mitochondrial function in IVP

Bovine Vaccinia in dairy cattle and suspicion of vesicular disease on milkers in Brazil

ABSTRACT: Bovine vaccinia (BV) is a vesicular disease induced by the Vaccinia virus (VACV) that affects milk production and is an occupational zoonosis. This research had the following objectives: (i) detection of VACV by qPCR in cattle with clinical suspicion of vesicular disease; (ii) symptoms characterization in animals and milkers with clinical suspicion of the disease and virus detection in humans; and (iii) identification of risk factors for infections of VACV in herds from several Brazilian states. A total of 471 bovine epithelial samples from dairy farms, in 15 Brazilian states, were evaluated between 2007 and 2012. The samples were tested by quantitative PCR (qPCR) using SYBR Green® reagents, validated with a lower limit of detection of 100 TCID50/50µL (1.7x100 viral particles), and 45.1% of VACV positive samples were detected. Using official forms for epidemiological investigation (FORM-IN), the risk factors for VACV infections in cattle were determined to be farms with a lack of technological facilities (P=0.029) and the presence of rodents (P=0.001). There was an effect of seasonality in cattle with a higher occurrence of BV during the dry season. A total of 420 epidemiological questionnaires were applied at public health care centers, where 100% of the milkers had vesicular lesions on their hands (98.1%) and on their arms (6.9%). The most frequent clinical symptoms in humans were: local swelling (74.2%), headache (20.7%), fever (10.4%) and inguinal lymphadenopathy (74.2%). Only 19.98% of milkers aged between 39 and 58 years were seroreactive to VACV and were immunized with the human anti-smallpox vaccine. There was an increase in the frequency of BV in older individuals due to their natural decrease in specific immunity. It has been shown that the implementation of zootechnical management techniques and health planning are important for the prevention of BV in animals and humans

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)