O que análises avançadas do sêmen podem contribuir de fato para a os resultados da IA em suínos? / What advanced semen analyzes can contribute to AI results in pigs?

O controle da qualidade seminal produzido por centrais e unidades de disseminação genética permite a maximização da produção de doses inseminantes por ejaculado, bem como organiza o acompanhamento da produtividade de cada macho. Este acompanhamento é crítico tanto quando o reprodutor jovem é introduzido na rotina de coleta de sêmen quanto na determinação do momento do descarte e reposição. Essencialmente, a avaliação do sêmen em suínos é a mesma há décadas, porém novas metodologias de análise individual da célula como a citometria de fluxo e de constituintes de tecidos e fluídos como a proteômica e metabolômica já estão trazendo avanços na andrologia animal. Na presente revisão, são abordados os aspectos técnicos, vantagens e limitações destas três análises avançadas aplicadas ao sêmen suíno, discutindo a sua implementação e impactos no manejo reprodutivo da espécie.(AU)

The quality control of the semen doses produced by artificial insemination centers and allows the maximization of the production of inseminating doses per ejaculate, as well as organizes the monitoring of the productivity of each male. This follow-up is critical when the young male is initiated in routine semen collection and in determining the moment of male replacement. Essentially, semen evaluation in pigs has been the same for decades, but new methodologies for individual cell analysis such as flow cytometry and tissue and fluid constituents such as proteomics and metabolomics are already bringing advances in animal andrology. In this review, the technical aspects, advantages, and limitations of these three advanced analyzes applied to swine semen are addressed, discussing their implementation and impacts on the reproductive management of the species.(AU)

Fertilidade e suscetibilidade à criopreservação espermática: avanços e desafios / Fertility and susceptibility to sperm cryopreservation: advances and challenges

Estudos sobre a fisiologia espermática demonstram que padrões de fertilidade e funcionalidade espermática pós-criopreservação possuem relação importante com a eficiência do metabolismo energético destas células, bem como com a sua capacidade de manter a homeostase oxidativa. Os conhecimentos sobre a relação entre perfil fisiológico de espermatozoides e fertilidade foram e, ainda estão sendo, aprimorados, com o uso de análises de perfis moleculares, com destaque para a metabolômica. As análises moleculares permitiram a identificação de classes de metabólitos importantes na fisiologia espermática, bem como de potenciais biomarcadores de fertilidade, inclusive em bovinos. No entanto, ainda não há disponível uma avaliação isolada capaz de estimar o padrão de fertilidade de amostras seminais. Há vários desafios a serem superados para a validação de biomarcadores de fertilidade, principalmente considerando-se as diferenças entre perfis metabólicos de raças distintas de touros e a heterogeneidade dos ejaculados. A superação destes desafios pode ser iniciada com um maior aproveitamento dos resultados já obtidos e futuros, com a aplicação de análises mais avançadas e com eficácia em elevado número de dados. Para tal, podem ser utilizados modelos estatísticos de inteligência artificial, cuja aplicação pode aumentar a acurácia das observações obtidas, bem como aproximá-las da aplicação pelo setor de produção animal.(AU)

Studies on sperm physiology demonstrate that fertility outcomes and sperm post-cryopreservation have an important relation with sperm energy metabolism efficiency and ability to maintain oxidative homeostasis. Knowledge on sperm physiology has been enhanced, continually, with the application of molecular profiling analysis, focusing on metabolomics. Molecular analysis allowed the identification of important classes of metabolites in sperm physiology, as well as potential fertility biomarkers, including in bovine. Despite all developments, there is still no isolated assessment available capable of estimating fertility on sperm samples. There are several challenges to be overcome for the validation of fertility biomarkers, especially considering the metabolic profiles differences between bull breeds and the ejaculate heterogeneity. Overcoming these challenges could start with the application of more advanced and effective data analysis from research database already obtained, as well as future ones. To this end, statistical models of artificial intelligence can be used, whose application can increase the accuracy of the observations obtained, as well as bringing them closer to the application by the animal production sector.(AU)

Plasma metabolite changes in anestrous dairy cows with negative energy balance identified using 1H NMR technology / [Alterações do metabólito plasmático em vacas leiteiras com balanço energético negativo identificado usando a tecnologia 1H NMR]

The objective of the present study was to investigate the different plasma metabolites between anestrus and estrus postpartum dairy cows and to provide a theoretical basis for prevention of anestrus in dairy farm cows. In the experiment, one hundred and sixty-seven Holstein dairy cows were selected with similar age and parity. According to the concentration of ß-hydroxybutyric acid, non-esterified fatty acids and glucose in plasma during 14 to 21 days in milk, all dairy cows were determined as having a status of energy balance. According to the results of clinical symptom, rectal and B ultrasound examination at 60 to 90 days postpartum, these cows were divided into twenty estrus and twenty-four anestrus group, other dairy cows were removed. 1H nuclear magnetic resonance technology was utilized to detect the plasma metabolites changes and screen different plasma metabolites between anestrus and estrus cows. Ten different metabolites including alanine, glutamic acid, asparagine, creatine, choline, phosphocholine, glycerophosphocholine, low-density lipoprotein, and very-low-density lipoprotein were significantly decreased in anestrous cows compared with estrous cows. Metabolic pathway analyses indicated that differential metabolites were primarily involved in amino acid and glycerophospholipid metabolism. These metabolites and their enrichment pathways indicate that reduced steroid hormone synthesis precursors result in lower levels of estradiol and progesterone and cause anestrus in negative energy balance. These data provide a better understanding of the changes that may affect estrus of postpartum dairy cows at NEB status and lay the ground for further research.(AU)

O objetivo do presente estudo foi investigar os diferentes metabolitos do plasma entre o cio e o cio pós-parto de vacas leiteiras e fornecer uma base teórica para a prevenção do cio de vacas em fazendas de leite. No experimento, foram selecionadas 127 vacas leiteiras Holstein com idade e paridade similares. De acordo com a concentração de ß- ácido hidroxibutírico, ácidos graxos não esterificados e glicose no plasma entre 14 e 21 dias no leite, todas as vacas leiteiras foram determinadas em estado de equilíbrio energético. De acordo com os resultados dos sintomas clínicos, do exame de ultra-som retal e B aos 60 a 90 dias pós-parto, estas vacas foram divididas em vinte cios e vinte e quatro grupos de cio, outras vacas leiteiras foram removidas. A tecnologia de ressonância magnética nuclear 1H foi utilizada para detectar as alterações dos metabólitos plasmáticos e para triar diferentes metabólitos plasmáticos entre as vacas do cio e do cio. Dez diferentes metabólitos incluindo alanina, ácido glutâmico, asparagina, creatina, colina, fosfocholina, glicerofosfocolina, lipoproteína de baixa densidade e lipoproteína de muito baixa densidade foram significativamente diminuídos nas vacas antróficas em comparação com as vacas estro. As análises da via metabólica indicaram que os metabólitos diferenciais estavam principalmente envolvidos no metabolismo de aminoácidos e glicerofosfolipídios. Estes metabólitos e suas vias de enriquecimento indicam que a redução dos precursores da síntese de hormônios esteróides resulta em níveis mais baixos de estradiol e progesterona e causa anestros no balanço energético negativo. Estes dados fornecem uma melhor compreensão das mudanças que podem afetar o cio das vacas leiteiras pós-parto no estado de NEB e preparam o terreno para mais pesquisas.(AU)

Evaluation of sample extracting methods of FCSM by Lactobacillus acidophilus based on a UPLC-Q-TOF-MS global metabolomics analysis

Abstract The study of metabolomics requires extracting as many metabolites as possible from a biological sample. This study aimed to determine the optimal method for the extraction of metabolites from solid-state fermented cottonseed meal (FCSM). The UPLC-Q-TOF-MS global metabolomics technology was used to detect the metabolites in FCSM, and the extraction quantity and extraction efficiency of seven different extraction methods, specifically the WA, 50MeOH, 50MeOHB, 50MeCNB, 80MeOHB, 80MeOH and AMF methods were evaluated. The results showed that the number of VIP metabolites extracted by AMF method are 196 and 184 in ESI+ and ESI- mode respectively, it is the largest number of all exacted methods; and the AMF methods also provided a higher extraction efficiency compared with the other methods, especially in indoleacrylic acid, dl-tryptophan and epicatechin (p 0.01). As a result, AMF/-4 °C method was identified as the best method for the extraction of metabolites from FCSM by Lactobacillus acidophilus. Our study establishes a technical basis for future metabolomics research of fermented feed.

Evaluation of sample extracting methods of FCSM by Lactobacillus acidophilus based on a UPLC-Q-TOF-MS global metabolomics analysis

The study of metabolomics requires extracting as many metabolites as possible from a biological sample. This study aimed to determine the optimal method for the extraction of metabolites from solid-state fermented cottonseed meal (FCSM). The UPLC-Q-TOF-MS global metabolomics technology was used to detect the metabolites in FCSM, and the extraction quantity and extraction efficiency of seven different extraction methods, specifically the WA, 50MeOH, 50MeOHB, 50MeCNB, 80MeOHB, 80MeOH and AMF methods were evaluated. The results showed that the number of VIP metabolites extracted by AMF method are 196 and 184 in ESI+ and ESI- mode respectively, it is the largest number of all exacted methods; and the AMF methods also provided a higher extraction efficiency compared with the other methods, especially in indoleacrylic acid, dl-tryptophan and epicatechin (p < 0.01). As a result, AMF/-4 °C method was identified as the best method for the extraction of metabolites from FCSM by Lactobacillus acidophilus. Our study establishes a technical basis for future metabolomics research of fermented feed.(AU)

Applications of large-scale molecular profiling techniques to the study of the corpus luteum

The corpus luteum (CL) is vital for the establishment and maintenance of pregnancy. Throughout the history of luteal biology, cutting-edge technologies have been used to develop a thorough understanding of the functions of specific luteal cell types, the signaling pathways that result in luteal cell stimulation or demise, and the molecules that regulate specific functions of luteal cells. The advent of largescale profiling technologies such as transcriptomics, proteomics, and metabolomics, has brought with it an interest in discovering novel regulatory molecules that may provide targets for manipulation of luteal function or lifespan. Although the work to date is limited, transcriptomics have been effectively used to provide a global picture of changes in mRNA that relate to luteal development, steroidogenesis, luteolysis or luteal rescue. Some studies have been reported that profile microRNA (miRNA) and proteins, and although not yet published, metabolomics analyses of the CL have been undertaken. Thus far, these profiling studies seem to largely confirm earlier findings using targeted approaches, although previously unstudied molecules have also come to light as important luteal regulators. These molecules can then be studied using traditional mechanistic techniques. Use of profiling technologies has presented physiologists with unique challenges associated with analyses of big data sets. An appropriate technique for balancing the risks associated with type I (false discoveries) and type II (overlooking a real change) statistical error has not yet been developed and many big data studies may have potentially important differences that are overlooked. Also, it is imperative that attempts be made to integrate information from the various -omics studies before drawing conclusions based on expression of only one class of molecule, to better reflect the interdependency of molecular networks in cells. Currently, few analysis programs exist for such integrations. Despite challenges associated with these techniques, they have already provided new information about the biology of the CL, notably allowing identification of a key regulator of acquisition of luteolytic capacity and providing a big-picture view of the subtle changes that occur in the CL during early pregnancy. As these technologies become more accurate and less expensive, and as analysis becomes more userfriendly, their use will become much more widespread and many new discoveries will be made. This review will focus only on relevant studies in which these technologies were used to study the CL of ruminants.

Applications of large-scale molecular profiling techniques to the study of the corpus luteum

The corpus luteum (CL) is vital for the establishment and maintenance of pregnancy. Throughout the history of luteal biology, cutting-edge technologies have been used to develop a thorough understanding of the functions of specific luteal cell types, the signaling pathways that result in luteal cell stimulation or demise, and the molecules that regulate specific functions of luteal cells. The advent of largescale profiling technologies such as transcriptomics, proteomics, and metabolomics, has brought with it an interest in discovering novel regulatory molecules that may provide targets for manipulation of luteal function or lifespan. Although the work to date is limited, transcriptomics have been effectively used to provide a global picture of changes in mRNA that relate to luteal development, steroidogenesis, luteolysis or luteal rescue. Some studies have been reported that profile microRNA (miRNA) and proteins, and although not yet published, metabolomics analyses of the CL have been undertaken. Thus far, these profiling studies seem to largely confirm earlier findings using targeted approaches, although previously unstudied molecules have also come to light as important luteal regulators. These molecules can then be studied using traditional mechanistic techniques. Use of profiling technologies has presented physiologists with unique challenges associated with analyses of big data sets. An appropriate technique for balancing the risks associated with type I (false discoveries) and type II (overlooking a real change) statistical error has not yet been developed and many big data studies may have potentially important differences that are overlooked. Also, it is imperative that attempts be made to integrate information from the various -omics studies before drawing conclusions based on expression of only one class of molecule, to better reflect the interdependency of molecular networks in cells. Currently, few analysis programs exist for such integrations. Despite challenges associated with these techniques, they have already provided new information about the biology of the CL, notably allowing identification of a key regulator of acquisition of luteolytic capacity and providing a big-picture view of the subtle changes that occur in the CL during early pregnancy. As these technologies become more accurate and less expensive, and as analysis becomes more userfriendly, their use will become much more widespread and many new discoveries will be made. This review will focus only on relevant studies in which these technologies were used to study the CL of ruminants.(AU)

Understanding the uterine environment in early pregnancy in cattle: How have the omicsenhanced our knowledge?

Early pregnancy loss in cattle can be attributed to a myriad of sources. One key factor that can influence early pregnancy success or loss is the influence and interactions between the maternal environment and the developing embryo/conceptus. Recent advancesin high-throughput ‘omics' technologies coupled with improved bioinformatics capabilities represent a promising avenue for enhancing our understanding of fundamental developmental events– which would have direct agricultural, veterinary, and economic benefits. Thusly this review revolves around recent applications of advanced transcriptomic, proteomic, and metabolomic analyses within a bovine uterine secretomic and interactomic context, with an overriding aim to highlight the advantages of these emerging fields whilst identify ingareas for improvement, consideration, and further research and development.

Omics approaches in fish quality and safety / Procedimentos ômicos aplicados em qualidade e segurança de pescado

Omics is a new technology that uses genomics, proteomics, and metabolomics to investigate metabolites from foods. The global demand for fish has shown a progressive increase because it is a significant source of high quality protein, polyunsaturated fatty acids, especially omega-3, and essential minerals. However, there are barriers in the fishery production chain such as lack of standardization, knowledge, and technology transfer to industry. Moreover, fish effective monitoring is difficult due to restricted quality parameters and analytical methods determined by current Brazilian legislation. This review details the limiting chemical parameters and recent advances in analytical procedures for fish quality determination. To improve fish quality monitoring, total volatile basic nitrogen (TVB-N), trimethylamine (TMA), ammonia, pH, and biogenic amines values should be revised and established by fish category and/or type of fish product. On the other hand, protein carbonyl concentration, free fatty acids (FFAs), peroxide values (POV), and thiobarbituric acid reactive substances (TBARS) should be included in the national legislation. Simultaneously, the official authorities should take into account effective, practical, and low cost analytical methodologies, which lead to faster results in order to facilitate and enhance the quality control of the products from the fish production chain, ensuring the consumer's health. Moreover, analytical techniques for the identification of fish species must be introduced in the Brazilian legislation in order to avoid illegal substitutions and negative impacts to consumers.(AU)

Os procedimentos ômicos são uma nova tecnologia que utiliza a genômica, proteômica e metabolômica para avaliar metabólitos dos alimentos. A demanda mundial de pescado tem aumentado progressivamente devido à elevada qualidade de proteínas, minerais e ácidos graxos poli-insaturados, especialmente ômega-3. Todavia, a cadeia produtiva aquícola apresenta limitações como falta de padronização, ausência de conhecimento e transferência de tecnologia para as indústrias. Além disso, torna-se difícil garantir um monitoramento efetivo do pescado em decorrência das limitações dos parâmetros de qualidade atuais e dos métodos analíticos estabelecidos pela legislação nacional. O presente trabalho analisa os fatores limitantes relacionados aos parâmetros químicos, bem como os avanços recentes nos procedimentos analíticos, para determinação da qualidade do pescado. Levando-se em consideração a melhoria no controle de qualidade dessa matriz, os parâmetros de bases voláteis totais (BVT), trimetilamina (TMA), amônia, pH e aminas biogênicas deveriam ser revisados e estabelecidos por categorias de pescado e/ou por tipo de produto à base de pescado. Em contrapartida, parâmetros relacionados à concentração de carbonilas, ácidos graxos livres (AGLs), índice de peróxidos (IP) e malonaldeído (MDA) poderiam ser inseridos na legislação nacional. Simultaneamente, as autoridades oficiais devem levar em consideração metodologias analíticas que apresentem efetividade, praticidade, baixo custo e rapidez, facilitando e aprimorando o controle de qualidade de produtos de pescado e garantindo a saúde dos consumidores. Além disso, técnicas analíticas para identificação das espécies de peixes devem ser incluídas na legislação brasileira visando evitar substituições ilegais e impactos negativos aos consumidores.(AU)

Omics approaches in fish quality and safety / Procedimentos ômicos aplicados em qualidade e segurança de pescado

Omics is a new technology that uses genomics, proteomics, and metabolomics to investigate metabolites from foods. The global demand for fish has shown a progressive increase because it is a significant source of high quality protein, polyunsaturated fatty acids, especially omega-3, and essential minerals. However, there are barriers in the fishery production chain such as lack of standardization, knowledge, and technology transfer to industry. Moreover, fish effective monitoring is difficult due to restricted quality parameters and analytical methods determined by current Brazilian legislation. This review details the limiting chemical parameters and recent advances in analytical procedures for fish quality determination. To improve fish quality monitoring, total volatile basic nitrogen (TVB-N), trimethylamine (TMA), ammonia, pH, and biogenic amines values should be revised and established by fish category and/or type of fish product. On the other hand, protein carbonyl concentration, free fatty acids (FFAs), peroxide values (POV), and thiobarbituric acid reactive substances (TBARS) should be included in the national legislation. Simultaneously, the official authorities should take into account effective, practical, and low cost analytical methodologies, which lead to faster results in order to facilitate and enhance the quality control of the products from the fish production chain, ensuring the consumer's health. Moreover, analytical techniques for the identification of fish species must be introduced in the Brazilian legislation in order to avoid illegal substitutions and negative impacts to consumers.(AU)

Os procedimentos ômicos são uma nova tecnologia que utiliza a genômica, proteômica e metabolômica para avaliar metabólitos dos alimentos. A demanda mundial de pescado tem aumentado progressivamente devido à elevada qualidade de proteínas, minerais e ácidos graxos poli-insaturados, especialmente ômega-3. Todavia, a cadeia produtiva aquícola apresenta limitações como falta de padronização, ausência de conhecimento e transferência de tecnologia para as indústrias. Além disso, torna-se difícil garantir um monitoramento efetivo do pescado em decorrência das limitações dos parâmetros de qualidade atuais e dos métodos analíticos estabelecidos pela legislação nacional. O presente trabalho analisa os fatores limitantes relacionados aos parâmetros químicos, bem como os avanços recentes nos procedimentos analíticos, para determinação da qualidade do pescado. Levando-se em consideração a melhoria no controle de qualidade dessa matriz, os parâmetros de bases voláteis totais (BVT), trimetilamina (TMA), amônia, pH e aminas biogênicas deveriam ser revisados e estabelecidos por categorias de pescado e/ou por tipo de produto à base de pescado. Em contrapartida, parâmetros relacionados à concentração de carbonilas, ácidos graxos livres (AGLs), índice de peróxidos (IP) e malonaldeído (MDA) poderiam ser inseridos na legislação nacional. Simultaneamente, as autoridades oficiais devem levar em consideração metodologias analíticas que apresentem efetividade, praticidade, baixo custo e rapidez, facilitando e aprimorando o controle de qualidade de produtos de pescado e garantindo a saúde dos consumidores. Além disso, técnicas analíticas para identificação das espécies de peixes devem ser incluídas na legislação brasileira visando evitar substituições ilegais e impactos negativos aos consumidores.(AU)

Understanding the uterine environment in early pregnancy in cattle: How have the omicsenhanced our knowledge?

Early pregnancy loss in cattle can be attributed to a myriad of sources. One key factor that can influence early pregnancy success or loss is the influence and interactions between the maternal environment and the developing embryo/conceptus. Recent advancesin high-throughput ‘omics' technologies coupled with improved bioinformatics capabilities represent a promising avenue for enhancing our understanding of fundamental developmental eventswhich would have direct agricultural, veterinary, and economic benefits. Thusly this review revolves around recent applications of advanced transcriptomic, proteomic, and metabolomic analyses within a bovine uterine secretomic and interactomic context, with an overriding aim to highlight the advantages of these emerging fields whilst identify ingareas for improvement, consideration, and further research and development.(AU)

Using new analytical tools to produce better embryos in vitro

The purpose of this review is to summarize what we know about preimplantation embryo metabolism, focusing on ruminant species, and to discuss how this knowledge informs our approach to culturing embryos in vitro. The important relationship between embryo metabolism and viability will be emphasized, and theories of metabolic networks in embryos presented. Methods that have historically been used to study embryo metabolism will be compared and contrasted to a new method of evaluating embryo metabolism; metabolomics. Finally, the advantages and disadvantages of using metabolomics technologies to study embryo metabolism will be critically evaluated. The application of metabolomics to assisted reproductive technologies, and specifically to embryo culture, will be highlighted. We conclude that use of metabolomics to study embryo physiology will enlighten our understanding of embryo metabolic pathways in the context of a complete media that enables good blastocyst production. This way of thinking about embryo metabolism as dynamic, complex and interrelated biochemical pathways, informed by metabolomics, will allow us to develop the next generation of embryo culture medium to support and manipulate metabolism to promote embryo viability, as well as to identify the most viable embryos for transfer.

Using new analytical tools to produce better embryos in vitro

The purpose of this review is to summarize what we know about preimplantation embryo metabolism, focusing on ruminant species, and to discuss how this knowledge informs our approach to culturing embryos in vitro. The important relationship between embryo metabolism and viability will be emphasized, and theories of metabolic networks in embryos presented. Methods that have historically been used to study embryo metabolism will be compared and contrasted to a new method of evaluating embryo metabolism; metabolomics. Finally, the advantages and disadvantages of using metabolomics technologies to study embryo metabolism will be critically evaluated. The application of metabolomics to assisted reproductive technologies, and specifically to embryo culture, will be highlighted. We conclude that use of metabolomics to study embryo physiology will enlighten our understanding of embryo metabolic pathways in the context of a complete media that enables good blastocyst production. This way of thinking about embryo metabolism as dynamic, complex and interrelated biochemical pathways, informed by metabolomics, will allow us to develop the next generation of embryo culture medium to support and manipulate metabolism to promote embryo viability, as well as to identify the most viable embryos for transfer.(AU)

PERFIL METABOLÔMICO DO PLASMA SEMINAL DE TOUROS GUZERÁ (Bos taurus indicus) COM DIFERENTES PARÂMETROS DE CONGELABILIDADE DO SÊMEN

O plasma seminal, além de ser um importante meio carreador dos espermatozoides ejaculados, relaciona-se com diversos metabólitos que exercem significativo papel em vários eventos biológicos e têm sido vinculados a possíveis biomarcadores, auxiliando a predizer o potencial reprodutivo de animais selecionados. Dessa forma, o presente estudo objetivou avaliar os metabólitos do plasma seminal de touros Bos taurus indicus da raça Guzerá e suas associações com a congelabilidade do sêmen. O sêmen de nove touros adultos foi colhido por eletroejaculação e, em seguida, foi realizado o exame andrológico e a criopreservação. Posteriormente, o sêmen foi descongelado e os parâmetros seminais foram avaliados pelo CASA. As amostras do plasma seminal dos touros foram preparadas e, porteriormente, analisadas com a utilização de cromatografia gasosa aliada à espectrometria de massas (CG-EM) e análises multivariadas e univariadas dos dados obtidos. Os metabólitos do plasma seminal e as suas respectivas vias foram identificados por bioinformática a partir dos programas Xcalibur e Cytoscape com o plug-in Metscape e da plataforma MetaboAnalyst. Sessenta e dois metabólitos foram identificados no plasma seminal dos touros. O 1,3-Dioxan-5-ol, ácido esteárico, miristato de colesterila, ácido 2-etilheptanoico e isobutilamina foram os metabólitos mais abundantes enquanto que o ácido heptadecanoico, pirrolidina, ácido não-adecanoico, 1-etil-1-nitrosoureia e o lactato de etila foram os metabólitos menos abundantes no plasma seminal. A análise estatística multivariada indicou uma separação distinta entre os touros de alta e baixa congelabilidade. Os metabólitos com maior pontuação de importância variável na projeção (VIP > 1,0) incluíram o ácido propanoico, ribose, glicina, ácido heptadecanoico e ácido undecanoico. Entre os compostos, o ácido propanoico (VIP = 3,5) e a ribose (VIP = 2,3) foram mais abundantes no grupo de alta congelabilidade (AC) do que no grupo de baixa congelabilidade (BC). A análise do teste t sugeriu que a concentração de ribose foi a única significativa (P = 0,014). Este metabólito pode influenciar na redução do estresse oxidativo, na reparação de danos ao DNA e na prevenção da apoptose celular, além de servir como possível fonte de energia para o organismo. Desse modo, a ribose pode ser considerada um potencial biomarcador de congelabilidade seminal em touros. Até onde sabemos, nosso estudo é o primeiro a conduzir uma avaliação dos metabólitos no plasma seminal de touros da raça Guzerá relacionados com diferentes fenótipos de congelabilidade. As descobertas deste estudo servirão de auxílio para o desenvolvimento de novas pesquisas e de uma melhor compreensão sobre os mecanismos de criopreservação espermática.

Seminal plasma, in addition to being an important carrier medium for ejaculated sperm, is connected to several metabolites that play a significant role in many biological events and have been linked to possible biomarkers, helping to predict the reproductive potential of selected animals. The present study aimed to evaluate the seminal plasma metabolites of Bos taurus indicus bulls of the Guzerá breed and their associations with semen freezability. Semen from nine adult bulls was collected by electroejaculation, followed by andrological examination and cryopreservation. Afterwards, the semen was thawed and the seminal parameters were evaluated by CASA. Seminal plasma samples from bulls were prepared and then analyzed using gas chromatography combined with mass spectrometry (GC-MS) and multivariate and univariate analyzes. The seminal plasma metabolites and their respective pathways were identified by bioinformatics from Xcalibur and Cytoscape programs and the MetaboAnalyst platform. Sixty-two metabolites were identified in the seminal plasma of the bulls. 1,3-Dioxan-5-ol, stearic acid, cholesteryl myristate, 2-ethylheptanoic acid and isobutylamine were the most abundant metabolites, while heptadecanoic acid, pyrrolidine, nonadecanoic acid, 1-ethyl-1-nitrosourea and ethyl lactate were the least abundant metabolites in the seminal plasma. The multivariate statistical analysis indicated a distinct separation between high and low freezability bulls. The metabolites with the highest score of variable importance in the projection (VIP> 1.0) included propanoic acid, ribose, glycine, heptadecanoic acid and undecanoic acid. Among the compounds, propanoic acid (VIP = 3.5) and ribose (VIP = 2.3) were more abundant in the high freezability group (HF) than in the low freezability group (LF). The t-test analysis suggested that only the concentration of ribose was significant (P = 0.014). This metabolite can influence the reduction of oxidative stress, the repair of DNA damage and the prevention of cellular apoptosis, in addition to serving as a possible source of energy for the body. Thus, ribose can be considered a biomarker of seminal freezability in bulls. As far as it is known, this is the first study to conduct an evaluation of the metabolites in the seminal plasma of Guzerá bulls related to different freezability phenotypes. The findings of this study will serve to assist the development of new research and a better understanding of the mechanisms of sperm cryopreservation.

Metabolism in the pre-implantation oocyte and embryo

An understanding of oocyte and embryo metabolism is critical to understanding and developing in vitro culture systems. In the last 60-70 years there has been a constant evolution in the way metabolism studies have been conducted. This includes a change from studying the metabolism of the oocyte alone vs. as a whole cumulus oocyte complex. The study of in vivo environments has lead to the creation of defined sequential culture systems, resulting in overcoming developmental blocks and improved embryo development. And techniques for studying metabolism have evolved from the use of radiolabelled isotopes to increasingly specific fluorescence probesand metabolomics, allowing for large, integrative profiles. Metabolism is a potential diagnostic for selecting the most likely embryos to implant. We envisage the future of metabolism will involve the ability to measure ‘more-in-less (more substrates, less volumes) and allow for a holistic approach to understanding the relationship between metabolism and developmental competence, as it is unconceivable that a single metabolic output will be able to assess health and/or quality.(AU)

Metabolism in the pre-implantation oocyte and embryo

An understanding of oocyte and embryo metabolism is critical to understanding and developing in vitro culture systems. In the last 60-70 years there has been a constant evolution in the way metabolism studies have been conducted. This includes a change from studying the metabolism of the oocyte alone vs. as a whole cumulus oocyte complex. The study of in vivo environments has lead to the creation of defined sequential culture systems, resulting in overcoming developmental blocks and improved embryo development. And techniques for studying metabolism have evolved from the use of radiolabelled isotopes to increasingly specific fluorescence probesand metabolomics, allowing for large, integrative profiles. Metabolism is a potential diagnostic for selecting the most likely embryos to implant. We envisage the future of metabolism will involve the ability to measure ‘more-in-less’ (more substrates, less volumes) and allow for a holistic approach to understanding the relationship between metabolism and developmental competence, as it is unconceivable that a single metabolic output will be able to assess health and/or quality.

Assinaturas metabolômicas da influência do holding time sobre o aumento da criotolerância dos espermatozoides suíno

Resumo: A criopreservação do sêmen suíno ainda é um grande desafio, no que diz respeito a qualidade do sêmen congelado, independente da sua qualidade pré-criopreservação. Isso ocorre em decorrência das diferenças nos potenciais de criotolerância do ejaculados. O holding time é uma alternativa utilizada nos protocolos de criopreservação para melhorar a qualidade do sêmen suíno descongelado, e é caracterizado pela refrigeração do sêmen à 17 °C anterior à criopreservação. O presente trabalho teve como objetivo 1) verificar qual o período de holding time ideal para criopreservação; 2) verificar se o potencial de congelabilidade dos ejaculados é afetada pelo uso ou não do holding time; e 3) averiguar os efeitos do holding time e congelabilidade dos ejaculados no metaboloma dos espermatozoides e do plasma seminal (PS) de suínos. No primeiro estudo o sêmen suíno foi criopreservado utilizando sete diferente período de holding time. Os resultados do sêmen descongelado mostraram que para a motilidade total e progressiva e a integridade das membranas plasmática e acrossomal os melhores resultados foram obtidos com 24 horas de holding time. Para o segundo estudo foram utilizados 27 ejaculados de 27 cachaços criopreservados com (24 horas) e sem (0 horas) o uso de holding time, em cada período (0 e 24 horas) o PS e os espermatozoides foram separados para posterior análise do metaboloma. Do total de ejaculados cinco foram classificados como de alta (EAC) e de baixa (EBC) congelabilidade. Foi observado interação entre o uso de holding time e a congelabilidade dos ejaculados para algumas características da fisiologia espermática; ou seja, os EAC somente são capazes de demostrar esse potencial se criopreservados após 24 horas à 17 °C. Ainda, foram realizadas as análises metabolômicas do plasma seminal e dos espermatozoides antes e após o holding time dos EAC e EBC. Com essas análises foi possível observar que as alterações de abundância de alguns metabolitos nos espermatozoides suínos são decorrentes das diferenças de congelabilidade inerentes ao ejaculado; enquanto as alterações na abundância dos metabólitos do PS são decorrentes das diferenças intrínsecas ao holding time. Com isso, é possível que essa interação entre as origens das diferenças dos metabólitos pode ser responsável por explicar os resultados de interação observados na fisiologia dos espermatozoides descongelados.

Abstract: Cryopreservation of boar semen is still a major challenge for science regarding the quality of frozen-thawed semen, regardless of its in-natura quality. It is due to differences in cryotolerance among ejaculates. Holding time is an alternative used in boar semen cryopreservation protocols to improve the quality of frozen-thawed semen. This period is characterized by refrigeration of semen at 17 ° C prior to cryopreservation. The present work aimed to 1) verify the ideal holding time period for boar semen cryopreservation; 2) verify if the freezing potential of ejaculates is affected by the use or not of the holding time, and 3) investigate the effects of holding time and freezability of ejaculates on boar sperm and seminal plasma metabolome. In the first study, boar semen was cryopreserved using seven different holding time periods. The results of frozen-thawed semen showed that for total and progressive motility and the integrity of the plasma and acrosomal membranes the best results were obtained with 24 hours holding time. In the second study, 27 ejaculates from 27 boars were cryopreserved with (24 hours) and without (0 hours) holding time, and in each period (0 and 24 hours) seminal plasma and spermatozoa were separated for further metabolome analysis. From the total of ejaculates five were classified as high (EAC) and low (EBC) freezability. It was observed interaction between the use of holding time and the ejaculate freezability for some characteristics of sperm physiology; that is, EACs are only able to demonstrate this potential if cryopreserved after 24 hours at 17 ° C. Also, seminal plasma and spermatozoa metabolomic analyses were performed before and after holding time on the EAC and EBC. With these analyses it was possible to observe that the changes in abundance of some metabolites in boar spermatozoa are due to the differences in freezing inherent to the ejaculate; whereas changes in seminal plasma metabolite abundance are due to differences intrinsic to the holding time. Thus, this interaction between the origins of metabolite differences may be responsible for explaining the interaction results observed in thawed sperm physiology.

ANÁLISE PROTEÔMICA E METABOLÔMICA DOS FLUIDOS DO TRATO REPRODUTIVO E CÉLULAS ESPERMÁTICAS DE CARNEIROS MORADA NOVA

O objetivo do estudo foi identificar por meio da proteômica e da metabolômica as moléculas presentes no plasma seminal, fluidos das glândulas sexuais acessórias e epidídimos, e membranas espermáticas de carneiros Morada Nova. No estudo 1 foram coletadas amostras de sêmen de seis carneiros (utilizando-se vagina artificial) para obter o plasma seminal e espermatozoides. Posteriormente, os animais foram vasectomizados e o fluido das glândulas acessórias coletado por vagina artificial. Em seguida, os animais foram abatidos e os epidídimos, vesículas seminais e glândulas bulbouretrais foram coletados para a recuperação dos fluidos reprodutivos e espermatozoides epididimários. Por meio de eletroforese bidimensional, espectrometria de massas e ferramentas de bioinformática foi avaliado o proteoma das secreções obtidas do trato reprodutivo e membranas espermáticas. As proteínas mais abundantes no plasma seminal foram binder of sperm 1 (BSP1), binder of sperm 5 (BSP5), bodesina-2 e espermadesina Z13. Proteínas do fluido das glândulas sexuais acessórias que contribuem para a composição do plasma seminal incluem BSPs, bodesinas, 72kDa colagenase tipo IV, BPI fold-containing family A member 1, clusterina, proteína semelhante à ribonuclease inativa 9, inibidor da anidrase carbônica isoforma X2 e albumina. O fluido da cauda do epidídimo contribui para o plasma seminal com proteínas específicas como a lipocalina-5-específica do epidídimo, proteína secretora epididimal E1, prostaglandina-H2 D-isomerase e a espermadesina-1. Comparando os mapas 2-D das proteínas de membrana do espermatozoide ejaculado e da cauda do epidídimo, há um aumento na intensidade e número de spots nos mapas da membrana do espermatozoide ejaculado. Estas proteínas são oriundas do fluido das glândulas acessórias. Futuras pesquisas sobre as proteínas identificadas em nosso estudo podem contribuir para investigações de marcadores para a qualidade espermática e fertilidade. No estudo 2 cinco carneiros adultos da raça Morada Nova foram vasectomizados e o fluido das glândulas acessórias (FGA) foi coletado por vagina artificial. Metabólitos foram extraídos e identificados por GC/MS e LC/MS. Os metabólitos obtidos foram analisados pelos bancos de dados Human Metabolome Database (HMDB), PubChem e LIPID Metabolites and Pathways Strategy. A análise das vias foi realizada utilizando o MetaboAnalyst 3.0. O uso combinado de GC e LC/MS permitiu a identificação de 371 compostos do FGA de carneiros. Estas técnicas apresentaram uma sobreposição de um único metabólito, confirmando a complementaridade das abordagens utilizadas. Lipídios e moléculas semelhantes a lipídios foi a classe mais abundantes no FGA de carneiros, seguida por aminoácidos ácidos, peptídeos e análogos. Os metabólitos mais abundantes foram frutose, glicerol, ácido cítrico, D-manitol, D-glicose e ácido L-(+)-lático. Metabólitos identificados no FGA de carneiros estavam envolvidos em 52 diferentes vias. Os metabólitos do FGA atuam como moduladores da função espermática, portanto, a análise química do fluido das glândulas sexuais acessórias é valiosa para melhor entender a reprodução animal bem como para a investigação de biomarcadores da fertilidade.

The objective of the present study was to identify and characterize, through omics technologies (proteomics and metabolomics), the components present in the secretions of the genital tract and sperm cells of Morada Nova rams. In study 1, Fresh semen samples were collected from six adult rams (using an artificial vagina) and then obtained whole seminal plasma and ejaculated sperm. After, rams were vasectomized and fluid of accessory sex glands was collected (also using an artificial vagina). Next, animals were slaughtered and the epididymides, seminal vesicles and bulbourethral glands were properly collected for recovery of reproductive fluids and epididymal sperm. Two-dimensional electrophoresis, mass spectrometry and bioinformatics tools were used to map the major proteome of secretions from reproductive tract and sperm membranes. The most abundant proteins in the seminal plasma gels appeared as binder of sperm 1 (BSP1), binder of sperm 5 (BSP5), bodhesin-2 and spermadhesin Z13-like. Proteins from composite accessory sex gland fluid that contribute to seminal plasma composition include BSPs, bodhesins, 72kDa type IV collagenase, BPI fold-containing family A member 1, clusterin, inactive ribonuclease-like protein 9, inhibitor of carbonic anhydrase-like isoform X2 and albumin. Cauda epididymal fluid contributes to seminal plasma with specific proteins, such as epididymal-specific lipocalin-5-like isoform X1 and epididymal secretory protein E1, prostaglandin-H2 D-isomerase and spermadhesin-1-like. Comparing 2-D maps of membrane proteins from ejaculated and cauda epididymal sperm membranes, there is an increase in the intensity and number of spots in the ejaculated sperm maps. Those proteins are originated from accessory sex gland fluids. Further research into the proteins identified in our study can contribute to investigations of markers for sperm quality and fertility. In the study 2, five adult Morada Nova rams were vasectomized and accessory sex gland fluid (AGF) was collected by artificial vagina. Metabolites were extracted and identified by GC/MS and LC/MS. Metabolites obtained were analyzed by Human Metabolome Database (HMDB), PubChem, and LIPID Metabolites and Pathways Strategy databases. Pathway analysis was performed using MetaboAnalyst 3.0. Combined use of GC and LC/MS allowed the identification of 371 compounds from AGF of rams. These techniques presented an overlap of one single metabolite, thus confirming the complementarity of the approaches used. Lipids and lipid-like molecules were the most abundant classes in the ram AGF, followed by amino acids, peptides, and analogues. The most abundant metabolites were fructose, glycerol, citric acid, D-mannitol, D-glucose and L-(+)-lactic acid. Metabolites identified in the ram AGF were involved in 52 different pathways. AGF metabolites are modulators of sperm function, thus chemical analysis of the accessory sex gland fluid is valuable to better understand animal reproduction and to search for biomarkers of fertility.

Caracterização do metaboloma sérico de bovinos Nelore e sua potencial associação à eficiência alimentar

NOVAIS, F.J. Caracterização do metaboloma sérico de bovinos Nelore e sua potencial associação à eficiência alimentar. 92 f. Dissertação (Mestrado) Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2017. A seleção de animais para consumo alimentar residual (RFI) está intrinsecamente associada com a diminuição do consumo matéria seca e é independente do ganho de peso corporal, selecionando animais de eficiência produtiva e econômica, além também de diminuir a emissão de gases de efeito estufa provinda do gado. Neste estudo, amostras de soro de 16 animais selecionados divergentemente para eficiência de alimentação foram coletadas antes do confinamento (dia -21) e avaliadas em uma abordagem metabolômica global, com o objetivo de usar análise diferencial, análise de co-expressão e enriquecimento funcional, identificando marcadores para eficiência de alimentação antes do confinamento. Um analito foi diferencialmente presente entre os animais de baixo e alto RFI. A análise WGCNA identificou 22 e 25 módulos no modo positivo e negativo, respectivamente e, 1 módulo de cada modo foi fortemente associado a RFI (r = 0,53, p-valor <0,05 e r = 0,52, p-valor <0,1 nos modos negativo e positivo, respectivamente). A análise de enriquecimento funcional predize 13 processos biológicos associados à eficiência alimentar, incluindo alterações no metabolismo de vitaminas lipossolúveis, inflamação, estresse oxidativo, metabolismo de aminoácidos e metabolismo de ácidos graxos. Esse trabalho evidencia a possibilidade de se identificar um biomarcador para eficiência alimentar e também sugerem que as diferenças nas respostas ao estresse oxidativo e nos processos inflamatórios já influenciam na variação da eficiência alimentar previamente ao confinamento.

NOVAIS, F.J. Serum metabolite characterization and their potential association with feed efficiency in Nellore cattle. 92 p. M.Sc. Dissertation Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2017. Animal selection for residual feed intake (RFI) is intrinsically associated with decreased consumption of dry matter independent of body weight gain, selecting yielding increased production and economic efficiency but also decreasing the greenhouse gas emission of livestock. In this study, serum samples of 16 animals selected for divergent feed efficiency were collected prior to feedlot (day -21) and evaluated in an untargeted metabolomics approach, with the goal of using differential analysis, co-expression analysis and functional enrichment to identifier markers for feed efficiency prior to the feedlot. One feature was differentially accumulated between low and high RFI. WGCNA analysis identified 22 and 25 modules in positive and negative mode, respectively, of 1 module of each mode was strongly associated with RFI (r= 0.53, p-value <0.05 and r=0.52, p-value < 0.1 to negative and positive mode, respectively). Pathway enrichment analysis yielded 13 biological processes associated with feed efficiency including alterations in vitamins liposoluble metabolism, inflammation, oxidative stress, amino acid metabolism and fatty acid metabolism. Our findings suggest the possibility to identify a biomarker for feed efficiency and also discuss that differences in oxidative stress responses and inflammatory processes could explain the feed efficiency variation prior to feedlot.

Reproductive technologies and epigenetics: their implications for genomic selection in cattle

Background: The development of genomic selection allowing a better selection for multiple traits (both for production and functional traits) induces dramatic changes in the way selection schemes are to be conducted. The associated needs for genomic selection which is to produce a large number of genotyped candidates as quick as possible may/will influence the way reproductive techniques are used to produce them. As the effect of the environment is no more integrated in the evaluation of performances based on genotype, there is also a need to better understand epigenetic effects and their possible implications while implementing genomic selection. Review: Information brought by reproductive physiology through access to powerful research tools in the fields of genomics, transcriptomics, proteomics and metabolomics will provide new genetic markers and will contribute to improve the precision of phenotypes. The combination of the two types of information is susceptible to increase considerably the efficiency of selection for reproductive traits. As better reproduction may facilitate the way to run selection schemes (more choice among candidates and production of those candidates at a young age), this knowledge can be profitable also to increase the efficiency of multiple trait selection. In this context, and depending on population characteristics, the interest of the reproductive techniques including assisted embryo based reproductive technologies (Multiple Ovulation Embryo Transfer (MOET) and Ovum pick up associated to in vitro Fertilization (OPU-IVF)) should be also revisited. The efficiency of systems based on scenarios involving several reproductive techniques taken in combination should be tested. The recent results obtained with embryo typing, which are compatible with the use of the last generation of chips for genotype analysis may lead to very promising applications for the breeding industry. The combined use of several embryo based reproductive technologies will probably be more important in the near future for selection purposes to satisfy the needs of genomic selection by increasing the number of candidates and to preserve at the same time genetic variability. Since several years, genotyping has been used more or less intensively by breeding companies to genotype males and females within nucleus herds As any farmer will get access to the genotype of the females present in their herd, an increased use of embryo based reproductive technologies may result also from the demand of individual farmers who may wish to valorize as well and as quick as possible the genetic potential of their best heifers following genotyping. In the near future, a better knowledge on epigenetics will allow to estimate the interactions between genotype and environment and their impact on performances of present or future generations. This represents a critical information when evaluating performances and when selecting future sires on genomic based information especially with the objective of implementing sustainable breeding schemes. Conclusion: The manuscript describes the new context and corresponding needs for genomic selection and how reproductive technologies and additional knowledge on epigenetics can be used to meet those needs.