Toxinology in the proteomics era: a review on arachnid venom proteomics

Abstract The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000s. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms (scorpion venom) AND (proteome) for scorpion venomics, and (spider venom) AND (proteome) for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.

Toxinology in the proteomics era: a review on arachnid venom proteomics

The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000's. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms "(scorpion venom) AND (proteome)" for scorpion venomics, and "(spider venom) AND (proteome)" for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.(AU)

New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Neonatal diarrhea and rotavirus A infection in beef and dairy calves, Brazil, 2006-2015 / Diarreia neonatal e infecção por rotavírus A em bezerros de corte e leite, Brasil, 2006-2015

Calf diarrhea causes substantial economic losses in the cattle industry worldwide. Bovine rotavirus A (RVA) is the main viral agent that leads to enteric infection and diarrhea outbreaks in calves throughout the world. The aim of this retrospective (2006-2015) study was to determine the frequency of RVA detection in diarrheic fecal samples from beef and dairy calves from the three main cattle-producing regions of Brazil. Diarrheic fecal samples (n=1,498) of 124 beef and 56 dairy cattle herds from the Midwest, South, and Southeast geographical regions of Brazil were evaluated using the silver-stained polyacrylamide gel electrophoresis (ss-PAGE) technique. RVA double stranded-RNA was identified by the ss-PAGE technique in 410 (27.4%) fecal samples. The frequency of positive samples found in beef calves (31.9%; 328/1,027) was higher than the frequency found in diarrheic fecal samples from dairy calves (17.4%; 82/471). RVA infection was identified in calves from the three Brazilian geographical regions analyzed. However, the frequency of positive diarrheic calves in the Midwest region (39.4%), predominantly beef calves, was higher than in the South (19.4%) and Southeast (17.6%) regions. The temporal distribution of RVA-infected calves evaluated by two five-year periods (2006-2010, 24.5%; 2011-2015, 28.8%) demonstrated a very similar frequency of RVA in both periods. Considering the wide regional and temporal scope of this study, it can be concluded that RVA remains an important etiology of neonatal diarrhea in calves of Brazilian cattle herds.(AU)

A diarreia neonatal ocasiona perdas econômicas importantes na pecuária bovina em todo o mundo. Rotavírus A (RVA) é o principal agente etiológico viral de infecções entéricas e surtos de diarreia em bezerros de rebanhos de corte e leite. O objetivo deste estudo retrospectivo (2006-2015) foi determinar a frequência de detecção de RVA em amostras de fezes diarreicas de bezerros de corte e leite das três principais regiões produtoras de bovinos do Brasil. Amostras de fezes diarreicas (n=1.498) de 124 rebanhos bovinos de corte e 56 rebanhos bovinos de leite das regiões Centro-Oeste, Sul e Sudeste do Brasil foram avaliadas utilizando a técnica de eletroforese em gel de poliacrilamida (EGPA). O genoma segmentado de RVA foi identificado pela técnica de EGPA em 410 (27,4%) amostras de fezes. A frequência de amostras positivas encontrada em bezerros de rebanhos de corte (31,9%; 328/1.027) foi maior que a frequência identificada em amostras de fezes diarreicas de bezerros de rebanhos leiteiros (17,4%; 82/471). A infecção por RVA foi identificada em bezerros das três regiões geográficas brasileiras analisadas. No entanto, a frequência de bezerros com diarreia positivos para RVA na região Centro-Oeste (39,4%), predominantemente de bezerros de rebanhos de corte, foi maior que nas regiões Sul (19,4%) e Sudeste (17,6%). A distribuição temporal dos bezerros infectados com RVA avaliados por dois períodos de cinco anos (2006-2010, 24,5%; 2011-2015, 28,8%) demonstrou uma frequência muito semelhante em ambos os períodos. Considerando a amplitude regional e temporal deste estudo, pode-se concluir que RVA continua sendo uma importante etiologia de diarreia neonatal em bezerros de rebanhos bovinos brasileiros.(AU)

Neonatal diarrhea and rotavirus A infection in beef and dairy calves, Brazil, 2006-2015 / Diarreia neonatal e infecção por rotavírus A em bezerros de corte e leite, Brasil, 2006-2015

Calf diarrhea causes substantial economic losses in the cattle industry worldwide. Bovine rotavirus A (RVA) is the main viral agent that leads to enteric infection and diarrhea outbreaks in calves throughout the world. The aim of this retrospective (2006-2015) study was to determine the frequency of RVA detection in diarrheic fecal samples from beef and dairy calves from the three main cattle-producing regions of Brazil. Diarrheic fecal samples (n=1,498) of 124 beef and 56 dairy cattle herds from the Midwest, South, and Southeast geographical regions of Brazil were evaluated using the silver-stained polyacrylamide gel electrophoresis (ss-PAGE) technique. RVA double stranded-RNA was identified by the ss-PAGE technique in 410 (27.4%) fecal samples. The frequency of positive samples found in beef calves (31.9%; 328/1,027) was higher than the frequency found in diarrheic fecal samples from dairy calves (17.4%; 82/471). RVA infection was identified in calves from the three Brazilian geographical regions analyzed. However, the frequency of positive diarrheic calves in the Midwest region (39.4%), predominantly beef calves, was higher than in the South (19.4%) and Southeast (17.6%) regions. The temporal distribution of RVA-infected calves evaluated by two five-year periods (2006-2010, 24.5%; 2011-2015, 28.8%) demonstrated a very similar frequency of RVA in both periods. Considering the wide regional and temporal scope of this study, it can be concluded that RVA remains an important etiology of neonatal diarrhea in calves of Brazilian cattle herds.(AU)

A diarreia neonatal ocasiona perdas econômicas importantes na pecuária bovina em todo o mundo. Rotavírus A (RVA) é o principal agente etiológico viral de infecções entéricas e surtos de diarreia em bezerros de rebanhos de corte e leite. O objetivo deste estudo retrospectivo (2006-2015) foi determinar a frequência de detecção de RVA em amostras de fezes diarreicas de bezerros de corte e leite das três principais regiões produtoras de bovinos do Brasil. Amostras de fezes diarreicas (n=1.498) de 124 rebanhos bovinos de corte e 56 rebanhos bovinos de leite das regiões Centro-Oeste, Sul e Sudeste do Brasil foram avaliadas utilizando a técnica de eletroforese em gel de poliacrilamida (EGPA). O genoma segmentado de RVA foi identificado pela técnica de EGPA em 410 (27,4%) amostras de fezes. A frequência de amostras positivas encontrada em bezerros de rebanhos de corte (31,9%; 328/1.027) foi maior que a frequência identificada em amostras de fezes diarreicas de bezerros de rebanhos leiteiros (17,4%; 82/471). A infecção por RVA foi identificada em bezerros das três regiões geográficas brasileiras analisadas. No entanto, a frequência de bezerros com diarreia positivos para RVA na região Centro-Oeste (39,4%), predominantemente de bezerros de rebanhos de corte, foi maior que nas regiões Sul (19,4%) e Sudeste (17,6%). A distribuição temporal dos bezerros infectados com RVA avaliados por dois períodos de cinco anos (2006-2010, 24,5%; 2011-2015, 28,8%) demonstrou uma frequência muito semelhante em ambos os períodos. Considerando a amplitude regional e temporal deste estudo, pode-se concluir que RVA continua sendo uma importante etiologia de diarreia neonatal em bezerros de rebanhos bovinos brasileiros.(AU)

Uterine infusion of conceptus fragments changes the protein profile from cyclic mares

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.

Uterine infusion of conceptus fragments changes the protein profile from cyclic mares

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.(AU)

Evaluation of the proteomic profiles of ejaculated spermatozoa from Saanen bucks (Capra hircus)

The Saanen goat breed has been widely explored in breeding programmes; however, there are few reports about the breed’s genetic and molecular composition. Thus, this study aimed to characterize the proteomic profile of spermatozoa from Saanen breeding goats. Five breeding animals with proven fertility were selected, the spermatozoa were collected, and the protein was extracted. Subsequently, the proteins were separated and analysed by two-dimensional electrophoresis and mass spectrometry; the proteins were then identified with the SwissProt database. A total of 31 proteins involved in reproduction were identified, including binding proteins on spermatozoa for fusion with the egg, acrosomal membrane proteins, metabolic enzymes, heat shock proteins, cytoskeletal proteins and spermatozoa motility proteins. The characterization of such proteins clarifies the molecular mechanisms of spermatogenesis and the modifications that ensure the success of fertilization.

Evaluation of the proteomic profiles of ejaculated spermatozoa from Saanen bucks (Capra hircus)

The Saanen goat breed has been widely explored in breeding programmes; however, there are few reports about the breeds genetic and molecular composition. Thus, this study aimed to characterize the proteomic profile of spermatozoa from Saanen breeding goats. Five breeding animals with proven fertility were selected, the spermatozoa were collected, and the protein was extracted. Subsequently, the proteins were separated and analysed by two-dimensional electrophoresis and mass spectrometry; the proteins were then identified with the SwissProt database. A total of 31 proteins involved in reproduction were identified, including binding proteins on spermatozoa for fusion with the egg, acrosomal membrane proteins, metabolic enzymes, heat shock proteins, cytoskeletal proteins and spermatozoa motility proteins. The characterization of such proteins clarifies the molecular mechanisms of spermatogenesis and the modifications that ensure the success of fertilization.(AU)

Differences in protein expression associated with ivermectin resistance in Caenorhabditis elegans / Diferenças na expressão de proteínas associadas à resistência à ivermectina em Caenorhabditis elegans

The indiscriminate administration of synthetic anthelmintics such as ivermectin contributes to the selection of subpopulations capable of resisting the drugs effects. To understand the mechanisms of ivermectin resistance in Caenorhabditis elegans, this study attempted to identify molecular targets. C. elegans lineages that were sensitive and resistant to ivermectin were used. Collected nematodes were added to an extraction buffer and macerated in liquid nitrogen for protein extraction. The extracted proteins were separated according to molecular weight by SDS-PAGE to verify their integrity. Subsequently, proteins from both lineages were separated using two-dimensional electrophoresis. The gels were analyzed and the relevant spots were excised and identified by mass spectrometry (NanoESI-Q-TOF and MASCOT®) and subsequently assessed by GO enrichment and STRING® analyses. The increased expression of proteins associated with high metabolic activity, such as ATP-2 and ENOL-1, which are responsible for ATP synthesis, was observed. Furthermore, proteins with involvement in mediating muscular function (MLC-1, ACT-1, and PDI-2), signaling (FAR-1 and FAR-2), and embryo development (VHA-2) were identified. Protein interaction analysis indicated that the majority of the identified proteins in the resistant lineages participated in the same reaction triggered by ivermectin.(AU)

A administração indiscriminada de anti-helmínticos sintéticos, como a ivermectina, contribui para a seleção de subpopulações capazes de resistir ao efeito das drogas. Para entender os mecanismos de resistência à ivermectina em Caenorhabditis elegans, este estudo visou identificar alvos moleculares. Portanto, linhagens de C. elegans sensíveis e resistentes à ivermectina foram utilizadas. Os nematóides coletados foram adicionados ao tampão de extração e macerados em nitrogênio líquido para obtenção das proteínas. As proteínas extraídas foram separadas por peso molecular em SDS-PAGE para verificar sua integridade. Posteriormente, as proteínas de ambas as linhagens foram separadas por eletroforese bidimensional. Os géis foram analisados, os spots relevantes foram excisados e identificados por espectrometria de massa (NanoESI-Q-TOF e MASCOT®), em seguida, analisados em seus termos de GO e STRING®. A expressão aumentada de proteínas associadas à alta atividade metabólica, como as proteínas ATP-2 e ENOL-1, responsáveis pela síntese de ATP, foi observada. Além disso, foram identificadas as proteínas responsáveis pelo controle da função muscular (MLC-1, ACT-1 e PDI-2), sinalização (FAR-1 e FAR-2) e desenvolvimento embrionário (VHA-2). A análise das interações proteicas indicou que a maioria das proteínas identificadas na cepa resistente participa da mesma reação desencadeada pela ivermectina.(AU)

Proteomic profile of Piper tuberculatum (Piperaceae) / Perfil proteômico e metabólico de Piper tuberculatum (Piperaceae)

Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.(AU)

Piper tuberculatum (Piperaceae) é uma espécie que acumula especialmente amidas como metabólitos secundários e diversas atividades biológicas dessa espécie foram relatadas anteriormente. No presente artigo, relatamos um estudo proteômico dessa espécie. Eletroforese bidimensional (2D SDS-PAGE) e espectrometria de massas (ESI-Q-TOF) foram utilizadas nesse estudos. Mais de cem spots e vários peptídeos foram identificados nesta espécie e as funções putativas desses peptídeos relacionadas a mecanismo de defesa como estresse biótico e abiótico foram atribuídos. As informações apresentadas ampliam a gama de informações moleculares dessa espécie.(AU)

Physical evaluation, morphological and identification of seminal proteins in Santa Ines sheep / Avaliação física, morfológica e identificação de proteínas seminais em ovinos da raça Santa Inês

This study aimed to identify proteins in the seminal plasma associated with fertility in sheep of Santa Inês in Manaus, AM, using twodimensional electrophoresis techniques associated with mass spectrometry. Semen samples from eight adult sheep were collected by removing an aliquot for the physical and morphological assessments of semen and seminal plasma was subjected to SDS-PAGE profile and two-dimensional electrophoresis. Gels were stained with colloidal Coomassie, scanned and analyzed using ImageMaster 2D Platinum software, version 6.0. The selected individual spots were cut from the master gel, digested with trypsin and subjected to identification by mass spectrometry (MALDITof / Tof). Of the 108 spots detected in the gel, it selected 10 differential spots (based on the distribution thereof in the bidimensional gel and pre-analysis of the 2D ImageMaster Platinum Software) identifying 03 proteins: clusterin, a protein 14-3-3 zeta chain and Ram Seminal versicles 22kDa Protein. The identity of these proteins implies that the components of seminal plasma participate in physiological processes involved in sperm protection, motility and sperm capacitation, all associated with fertility. These proteins need to be better studied to see whether the same could be used as molecular markers of fertility as they were also found in other studies conducted with sheep Santa Ines.(AU)

Este estudo teve como objetivo identificar proteínas do plasma seminal associadas à fertilidade em ovinos da raça Santa Inês em Manaus, AM, utilizando técnicas de eletroforese bidimensional associadas à espectrometria de massa. Amostras de sêmen de oito carneiros adultos foram coletadas, retirando-se uma alíquota para as avaliações físicas e morfológicas do sêmen, e o plasma seminal foi submetido ao perfil SDS-PAGE e à eletroforese bidimensional. Os géis foram corados com Coomassie coloidal, digitalizados e analisados no aplicativo ImageMaster 2D Platinum, versão 6.0. Os spots selecionados foram cortados individualmente do gel mestre, digeridos com tripsina e submetidos à identificação por espectrometria de massa (MALDI-Tof/Tof). Dos 108 spots detectados no gel, selecionou-se 10 spots diferenciais (com base na distribuição dos mesmos no gel bidimensional e com a pré-análise destes no ImageMaster 2D Platinum Software), identificando-se 03 proteínas: a clusterina, a 14-3-3 protein zeta chain e a Ram Seminal Versicles 22kDa Protein. A identidade dessas proteínas infere que os componentes do plasma seminal participam de processos fisiológicos ligados à proteção do espermatozoide, sua motilidade e capacitação espermática, todos associados à fertilidade. Essas proteínas precisam ser melhor estudadas para verificar se as mesmas poderiam ser utilizadas como marcadores moleculares de fertilidade, já que também foram encontradas em outros trabalhos realizados com ovinos da raça Santa Inês.(AU)

Physical evaluation, morphological and identification of seminal proteins in Santa Ines sheep / Avaliação física, morfológica e identificação de proteínas seminais em ovinos da raça Santa Inês

This study aimed to identify proteins in the seminal plasma associated with fertility in sheep of Santa Inês in Manaus, AM, using twodimensional electrophoresis techniques associated with mass spectrometry. Semen samples from eight adult sheep were collected by removing an aliquot for the physical and morphological assessments of semen and seminal plasma was subjected to SDS-PAGE profile and two-dimensional electrophoresis. Gels were stained with colloidal Coomassie, scanned and analyzed using ImageMaster 2D Platinum software, version 6.0. The selected individual spots were cut from the master gel, digested with trypsin and subjected to identification by mass spectrometry (MALDITof / Tof). Of the 108 spots detected in the gel, it selected 10 differential spots (based on the distribution thereof in the bidimensional gel and pre-analysis of the 2D ImageMaster Platinum Software) identifying 03 proteins: clusterin, a protein 14-3-3 zeta chain and Ram Seminal versicles 22kDa Protein. The identity of these proteins implies that the components of seminal plasma participate in physiological processes involved in sperm protection, motility and sperm capacitation, all associated with fertility. These proteins need to be better studied to see whether the same could be used as molecular markers of fertility as they were also found in other studies conducted with sheep Santa Ines.

Este estudo teve como objetivo identificar proteínas do plasma seminal associadas à fertilidade em ovinos da raça Santa Inês em Manaus, AM, utilizando técnicas de eletroforese bidimensional associadas à espectrometria de massa. Amostras de sêmen de oito carneiros adultos foram coletadas, retirando-se uma alíquota para as avaliações físicas e morfológicas do sêmen, e o plasma seminal foi submetido ao perfil SDS-PAGE e à eletroforese bidimensional. Os géis foram corados com Coomassie coloidal, digitalizados e analisados no aplicativo ImageMaster 2D Platinum, versão 6.0. Os spots selecionados foram cortados individualmente do gel mestre, digeridos com tripsina e submetidos à identificação por espectrometria de massa (MALDI-Tof/Tof). Dos 108 spots detectados no gel, selecionou-se 10 spots diferenciais (com base na distribuição dos mesmos no gel bidimensional e com a pré-análise destes no ImageMaster 2D Platinum Software), identificando-se 03 proteínas: a clusterina, a 14-3-3 protein zeta chain e a Ram Seminal Versicles 22kDa Protein. A identidade dessas proteínas infere que os componentes do plasma seminal participam de processos fisiológicos ligados à proteção do espermatozoide, sua motilidade e capacitação espermática, todos associados à fertilidade. Essas proteínas precisam ser melhor estudadas para verificar se as mesmas poderiam ser utilizadas como marcadores moleculares de fertilidade, já que também foram encontradas em outros trabalhos realizados com ovinos da raça Santa Inês.

Proteínas do sêmen e sua relação com a resistência à congelação em ejaculados de diferentes varrões / Semen proteins and their relationship to freezing resistance in different boars

A identificação de proteínas presentes no sêmen possibilitará a busca por marcadores decongelabilidade. O presente trabalho teve como objetivo identificar proteínas espermáticas relacionadas com aproteção durante a criopreservação. O sêmen de quatorze reprodutores foi coletado uma vez por semana,descongelado, ressuspenso e analisado. Para o estudo da proteômica, estes foram submetidos à eletroforesebidimensional. A busca por marcadores de congelabilidade, foi feita pela divisão dos animais em dois grupos:congelabilidade boa (GFEs) e ruim (PFEs), através dos resultados de vigor e motilidade após a descongelação.Isto possibilitou comparar as proteínas presentes em cada grupo e correlacioná-las com a criopreservação dosêmen. O delineamento experimental utilizado foi o de blocos ao acaso. A análise estatística foi processadaatravés da avaliação das médias e desvios padrões, com os testes de Mann-Witney e o teste t de Student. Paracomparação foi utilizado o teste ANOVA com pós-teste de Tukey (5%). Os animais de GFEs = 2,2 ± 0,8 e 41,8± 22,9 e os de PFEs = 1,9 ± 0,6 e 26,8 ± 17,5. Foram encontrados 263 ± 62,2 spots por gel e um total de 234,2 ±54,6 spots de forma consistente nos géis dos animais. Foram encontrados 5 spots que divergiram entre os grupos(3 mais expressos no PFES e 2 spots no GFEs). No estudo da composição proteica da membrana espermática, oscinco spots detectados podem atuar como possíveis marcadores de congelabilidade para o sêmen suíno.(AU)

The identification of proteins present in the semen wil enable the search for freezin markers. This studyaimed identify the spermatic proteins that are related to the protection during cryopreservation. The semen fromfourteen boars was collected once a week, thawed, ressuspended and analyzed. For the study of proteomic, theywere subjected to two-dimensional electrophoresis. To search for freezability markers, was made a division ofthe animals into two groups: good and bad freezability, through the results of vigour and motility after thawing.This made possible the comparison of the proteins present in each group and correlation between them andsemen cryopreservation. The experimental lineation was in randomized blocks. The statistical analysis wasmade by evaluating the means and standard deviations, in which were applied the Mann-Whitney and Student ttest. For comparison was used the ANOVA test with Tukey post-test was used (5%). The animals of GFEs = 2.2± 0.8 and 41.8 ± 22.9 and the PFEs = 1.9 ± 0.6 and 26.8 ± 17.5. Were found 263 ± 62.2 spots per gel and a totalof 234.2 ± 54.6 spots was consistently detected in the gels of the animals. The five spots that differed betweengroups were found (3 with greater intensity in PFES and 2 spots in GFEs). In the study of protein composition ofthe membrane sperm, the five spots detected can act as potential markers for boar semen freezability.(AU)

Proteínas do sêmen e sua relação com a resistência à congelação em ejaculados de diferentes varrões / Semen proteins and their relationship to freezing resistance in different boars

A identificação de proteínas presentes no sêmen possibilitará a busca por marcadores decongelabilidade. O presente trabalho teve como objetivo identificar proteínas espermáticas relacionadas com aproteção durante a criopreservação. O sêmen de quatorze reprodutores foi coletado uma vez por semana,descongelado, ressuspenso e analisado. Para o estudo da proteômica, estes foram submetidos à eletroforesebidimensional. A busca por marcadores de congelabilidade, foi feita pela divisão dos animais em dois grupos:congelabilidade boa (GFEs) e ruim (PFEs), através dos resultados de vigor e motilidade após a descongelação.Isto possibilitou comparar as proteínas presentes em cada grupo e correlacioná-las com a criopreservação dosêmen. O delineamento experimental utilizado foi o de blocos ao acaso. A análise estatística foi processadaatravés da avaliação das médias e desvios padrões, com os testes de Mann-Witney e o teste t de Student. Paracomparação foi utilizado o teste ANOVA com pós-teste de Tukey (5%). Os animais de GFEs = 2,2 ± 0,8 e 41,8± 22,9 e os de PFEs = 1,9 ± 0,6 e 26,8 ± 17,5. Foram encontrados 263 ± 62,2 spots por gel e um total de 234,2 ±54,6 spots de forma consistente nos géis dos animais. Foram encontrados 5 spots que divergiram entre os grupos(3 mais expressos no PFES e 2 spots no GFEs). No estudo da composição proteica da membrana espermática, oscinco spots detectados podem atuar como possíveis marcadores de congelabilidade para o sêmen suíno.

The identification of proteins present in the semen wil enable the search for freezin markers. This studyaimed identify the spermatic proteins that are related to the protection during cryopreservation. The semen fromfourteen boars was collected once a week, thawed, ressuspended and analyzed. For the study of proteomic, theywere subjected to two-dimensional electrophoresis. To search for freezability markers, was made a division ofthe animals into two groups: good and bad freezability, through the results of vigour and motility after thawing.This made possible the comparison of the proteins present in each group and correlation between them andsemen cryopreservation. The experimental lineation was in randomized blocks. The statistical analysis wasmade by evaluating the means and standard deviations, in which were applied the Mann-Whitney and Student ttest. For comparison was used the ANOVA test with Tukey post-test was used (5%). The animals of GFEs = 2.2± 0.8 and 41.8 ± 22.9 and the PFEs = 1.9 ± 0.6 and 26.8 ± 17.5. Were found 263 ± 62.2 spots per gel and a totalof 234.2 ± 54.6 spots was consistently detected in the gels of the animals. The five spots that differed betweengroups were found (3 with greater intensity in PFES and 2 spots in GFEs). In the study of protein composition ofthe membrane sperm, the five spots detected can act as potential markers for boar semen freezability.

SUPLEMENTAÇÃO DE FONTE INORGÂNICA DE ZINCO NO PERFIL PROTEÔMICO E METALÔMICO DA GELEIA REAL EM ABELHAS Apis mellifera L.

Este estudo teve como objetivo avaliar a qualidade nutricional do perfil proteico da geleia real produzida pelas abelhas Apis mellifera suplementadas com diferentes concentrações de zinco inorgânico (Sulfato de Zinco monoidratado - 0, 25, 50 e 75 ppm). Foi feita eletroforese bidimensional para o fracionamento das proteínas da geleia real e o nível de zinco quantificado pela técnica de espectrometria de absorção atômica com chama (FAAS). A análise de proteínas da geléia real como componente extracelular foi realizada pela plataforma Blast2Go, onde foram obtidos gráficos de funções moleculares e processos biológicos das proteínas encontradas através da análise proteômica por análise de espectrometria de massa ESI MS / MS, mostrando as proteínas Major Royal Jelly Protein 1 e Major Royal Jelly Protein 8 como as principais proteínas ativas na geléia real tanto para up como para down regulation e associada ao zinco. Todos os tratamentos independentes das concentrações de zinco mostraram número menor de Spots proteicos quando comparadas com o controle. Todas as proteínas contendo zinco foram classificadas como Proteínas Principais da Geleia Real (MRJPs). A exposição de abelhas nutrizes ao mineral zinco em sua forma inorgânica promoveu aumento na expressão de proteínas da geleia real envolvidas em sistemas de defesa (MRJP8 e MRJP9), entretanto reduziu a expressão de seis diferentes proteínas das MRJPs (sendo elas: MRJP, MRJP1, MRJP2, MRJP3, MRJP5 e MRJP7). Nossos resultados demonstram que proteínas vitais e processos metabólicos são prejudicados em abelhas nutrizes expostas ao mineral zinco em sua forma inorgânica em todas as doses utilizadas afetando a nutrição e manutenção de colônias.

This study aimed to evaluate the nutritional quality of the protein profile of royal jelly by honey bees Apis mellifera supplemented with different concentrations of inorganic zinc (Zinc Sulphate monohydrate - 0, 25, 50 and 75 ppm). Was performed two-dimensional electrophoresis for the fractionation of royal jelly proteins and the zinc level was quantified by the flame atomic absorption spectrometry (FAAS) technique. Protein analysis of royal jelly as an extracellular component was performed using the Blast2Go platform, where graphs of molecular functions and biological processes of the proteins found through proteomic analysis by mass spectrometry analysis ESI MS / MS, showing as Major Royal proteins Jelly Protein 1 and Major Royal Jelly Protein 8as the main active proteins in royal jelly for both up and down regulation and associated with zinc. All treatments independent of zinc concentrations reduced the number of protein spots when compared to the control. All zinc-containing proteins were classified as Major Royal Jelly Proteins (MRJPs). The exposure of nursing bees to the mineral zinc in its inorganic form promoted an increase in the expression of royal jelly proteins involved in defense systems (MRJP8 and MRJP9), however it reduced an expression of six different MRJPs proteins (namely: MRJP, MRJP1, MRJP2, MRJP3, MRJP5 and MRJP7). The results demonstrate that vital proteins and metabolic processes are impaired in nursing bees exposed to the mineral zinc in its inorganic form in all doses used affecting nutrition and maintenance of colonies.

EFEITO DA SUPLEMENTAÇÃO DE ZINCO NO PERFIL PROTEÔMICO E METALÔMICO DA GELEIA REAL E NO TRANSCRIPTOMA DE ABELHAS Apis mellifera L.

As abelhas A. mellifera possuem necessidades específicas de nutrientes para que possam desenvolver todo o seu potencial produtivo e reprodutivo, visando a sobrevivência e manutenção da colônia. Neste projeto avaliamos duas fontes de zinco (orgânico e inorgânico), em diferentes concentrações e seus reflexos no perfil metaloproteômico da geleia real e no transcriptoma de abelhas com 6 dias de idade. Foram utilizadas 35 colônias de abelhas A. mellifera, distribuídas aleatoriamente nos seguintes tratamentos (05 colmeias por tratamento): controle: xarope de açúcar sem suplementação de zinco; ZNI25: xarope de açúcar suplementado com 25 ppm de Zn inorgânico; ZNI50: xarope de açúcar suplementado com 50 ppm de Zn inorgânico; ZNI75: xarope de açúcar suplementado com 75 ppm de Zn inorgânico; ZNO25: xarope de açúcar suplementado com 25 ppm de Zn orgânico; ZNO50: xarope de açúcar suplementado com 50 ppm de Zn orgânico; ZNO75: xarope de açúcar suplementado com 75 ppm de Zn orgânico. A fonte de zinco inorgânico foi o sulfato de zinco monohidratado (37,4% de zinco) e a fonte orgânica de zinco metionina (16% de zinco), diluída em xarope de açúcar. Os níveis reais de zinco foram determinados por espectrometria de absorção atômica (FAAS). As abelhas A. mellifera recém-emergidas foram marcadas ao final de 30 dias de suplementação com caneta atóxica, na região pronoto e suas coletas para análise do transcriptoma foram realizadas aos 6 dias de idade em sua fase de nutriz, com exatamente 36 dias de experimento. Para a análise do metaloproteoma da geleia real, foi induzido a produção de geleia real pelo método de transferência de larvas. Assim que coletada, o teor proteico das amostras era mensurado por espectrômetro de UV visível, as proteínas eram separadas pela eletroforese bidimensional (2D page) e as metaloproteínas separadas por espectrômetro de absorção atômica. Todas as funções biológicas associadas as metaloproteínas que continham zinco foram identificados por espectrômetro de absorção atômica. Nossos resultados sugerem que as menores concentrações de zinco orgânico (ZNO 25) alteram o maior número de expressão diferencial de proteínas da geleia real e também aumentam a concentração proteica das amostras. Em relação as análises do transcriptoma, nossos resultados sugerem que os maiores níveis de zinco inorgânico (ZNI 75) alteram a maior via de genes relacionados a importantes processos fisiológicos, entretanto, somente as menores concentrações de zinco de fonte orgânica (ZNO25) apresentaram grande aumento de genes diferencialmente expressos quando relacionados ao grupo controle (ZN0).

A. mellifera bees have specific nutrient needs so that they can develop their full productive and reproductive potential, aiming at colony survival and maintenance. In this project, we evaluated two sources of zinc (organic and inorganic), at different concentrations and their effects on the metalloproteomic profile of royal jelly and the transcriptome of 6-day-old bees. Thirty-five colonies of A. mellifera bees were used, randomly distributed in the following treatments (05 hives per treatment): control: sugar syrup without zinc supplementation; ZNI25: sugar syrup supplemented with 25 ppm inorganic Zn; ZNI50: sugar syrup supplemented with 50 ppm inorganic Zn; ZNI75: sugar syrup supplemented with 75 ppm inorganic Zn; ZNO25: sugar syrup supplemented with 25 ppm organic Zn; ZNO50: sugar syrup supplemented with 50 ppm organic Zn; ZNO75: Sugar syrup supplemented with 75 ppm organic Zn. The inorganic zinc source was zinc sulfate monohydrate (37.4% zinc) and the organic zinc source methionine (16% zinc), diluted in sugar syrup. Actual zinc levels were determined by atomic absorption spectrometry (FAAS). The newly emerged A. mellifera bees were tagged at the end of 30 days of supplementation with a non-toxic pen, in the pronotum region, and their collections for transcriptome analysis were carried out at 6 days of age in their nursing phase, with exactly 36 days of the experiment. . For the analysis of the royal jelly metalloproteome, royal jelly production was induced by the larval transfer method. Once collected, the protein content of the samples was measured by a visible UV spectrometer, the proteins were separated by two-dimensional electrophoresis (2D page) and the metalloproteins were separated by an atomic absorption spectrometer. All biological functions associated with zinc-containing metalloproteins were identified by an atomic absorption spectrometer. Our results suggest that the lower concentrations of organic zinc (ZNO 25) alter the greater number of differential expressions of royal jelly proteins and also increase the protein concentration of the samples. Regarding the transcriptome analyses, our results suggest that the highest levels of inorganic zinc (ZNI 75) alter the largest pathway of genes related to important physiological processes, however, only the lowest concentrations of zinc from an organic source (ZNO25) showed a large increase of differentially expressed genes when related to the control group (ZN0)

QUALIDADE DA CARNE DE BOVINOS SUPERPRECOCES, MESTIÇOS NELORE-MONTANA DE DIFERENTES CLASSES SEXUAIS: UMA ABORDAGEM PROTEÔMICA

Objetivou-se com esta pesquisa identificar proteínas diferencialmente expressas no músculo Longissimus thoracis et lumborum (LT) e sua associação com características físico-químicas encontradas da carne maturada de novilhas versus novilhos F1 Nelore-Montana, superprecoces e terminados em confinamento. Foram avaliadas características da carcaça (peso de carcaça quente, rendimento e espessura de gordura subcutânea), composição química (teor de umidade, gordura intramuscular, proteína, colágeno total) e características de qualidade da carne como força de cisalhamento (FC), perdas por cocção, coloração instrumental (L*; a*; b*) e pH, considerando-se três tempos de maturação (3, 10, 17 dias postmortem). Amostras individuais do músculo LT foram coletadas pré rigor mortis para estudo do proteoma utilizando eletroforese bidimensional (2D-PAGE) e espectrometria de massa por ionização por eletrospray (ESI-MS). Interações entre a classe sexual e o tempo de maturação foram analisadas sobre as características de qualidade da carne. Adicionalmente, estudos de imagem dos géis 2D-PAGE foram avaliados, sendo selecionados spots proteicos diferencialmente expressos (P<0,05) para caracterização de proteínas por ESI-MS. Observou-se que o teor de gordura intramuscular (GIM) da carne foi 63,6% maior (P<0,01) nas novilhas comparados aos novilhos. Já as perdas por evaporação (PPE) foram 10,22% menor para as novilhas. No entanto, nenhuma diferença foi observada (P>0,05) sobre as demais características da carne (FC, L*, a*, b* e pH) entre as classes sexuais. No estudo do proteoma muscular, vinte e três proteínas foram identificadas entre os grupos biológicos (P<0,05). Para as novilhas, houve maior expressão de proteínas de ligação (ALVA, GLNG, TF, MYBPC1), metabolismo de carboidratos (ALDOA, GAPDH, PKM), defesa/proteção celular (HSPA8, CA3), metabolismo da creatina (CKM) e contração muscular/regulação (TNNT1, TNNI2). Esses resultados possivelmente refletem maior capacidade oxidativa em relação aos novilhos. Nestes, houve maior expressão de proteínas relacionadas à contração muscular/regulação (ACTA1, TPM2, TNNT3), ao metabolismo de carboidratos (ENO1, ENO3, PYGM, PGM1, TPI1) e de energia (ATP5F1B, PEBP1, AK1). Os resultados indicam antecipação do rigor mortis no músculo dos novilhos. A identificação de diferenças de expressão proteica por abordagem proteômica (2D-PAGE + ESI-MS) foram associadas ao metabolismo energético e melhor resposta ao estresse oxidativo dos animais, auxiliando na compreensão das diferenças fenotípicas observadas na qualidade da carne. Os resultados do proteoma foram associados ao maior teor de GIM e, possivelmente, a capacidade de retenção de água do tecido muscular das novilhas em comparação a machos castrados.

The objective of this research was to identify differentially expressed proteins in the Longissimus thoracis et lumborum (LT) muscle and its association with physico-chemical characteristics of aged meat for young F1 Nelore Montana heifers and steers finished in feedlot. Carcass characteristics (hot carcass weight, yield, and backfat thickness), chemical composition (moisture content, intramuscular fat, protein, total collagen) and meat quality characteristics such as shear force (SF), cooking loss (CL), instrumental color (L *; a *; b *) and pH, considering three aging time (3, 10, 17 days post-mortem) were evaluated. Individual samples of LT muscle were collected pre rigor mortis to study the proteome using two-dimensional electrophoresis (2D-PAGE) and electrospray ionization mass spectrometry (ESI-MS). Interactions between sexual class and aging time were analyzed on the meat quality characteristics. Additionally, image studies of 2D-PAGE gels were evaluated with differentially expressed protein spots (P<0.05) being selected for protein characterization by ESI-MS. Intramuscular fat content (IMF) was 63.6% higher (P <0.01) in the meat of heifers compared to steers. Evaporative losses (EL) were 10.22% lower for heifers. However, no difference was observed (P>0.05) regarding to other characteristics of the meat (SF, L *, a *, b * and pH) between sexual classes. In the study of muscle proteome, twenty-three proteins were identified between biological groups (P<0.05). In heifers, there was a higher expression of binding proteins (ALVA, GLNG, TF, MYBPC1), carbohydrate metabolism (ALDOA, GAPDH, PKM), defense/protection cellular (HSPA8, CA3), creatine metabolism (CKM) and muscle contraction/regulation (TNNT1, TNNI2). These results possibly reflect a greater oxidative capacity compared to steers. In these, there was a higher expression of proteins related to muscle contraction/regulation (ACTA1, TPM2, TNNT3), carbohydrate metabolism (ENO1, ENO3, PYGM, PGM1, TPI1) and energy (ATP5F1B, PEBP1, AK1). These results indicate anticipation of rigor mortis in steers. The identification of differences in protein expression by a proteomic approach (2D-PAGE + ESI-MS) were associated with energy metabolism and better response to oxidative stress leading to understanding the phenotypic differences observed in meat quality. Proteome results were associated with a higher IMF content and, possibly water holding capacity in the muscle tissue of heifers compared to steers.

PROTEÔMICA DE TECIDO RUMINAL E CECAL DE BOVINOS NELORE CONFINADOS COM DIFERENTES ESTRATÉGIAS NUTRICIONAIS

A literatura apresenta poucos estudos que investigaram o proteoma de ceco e rúmen de bovinos submetidos a estratégias alimentares com uso de aditivos. Essa proposta é pioneira na identificação, expressão e compreensão do perfil proteico ruminal e cecal de bovinos nelore confinados. Artigo 1: Objetivou-se avaliar a expressão proteica, a fim identificar possíveis moléculas candidatas a biomarcadores e suas relações com funções metabólicas e nutricionais aplicados a animais confinados com diferentes níveis de amido (25, 35 e 45%) e aditivos (Monensina × blend de óleos essências + amilase exógena), as dosagens de monensina sódica, blend de óleos essenciais e amilase exógena foram 26, 90 e 560 mg/kg MS, respectivamente. Foram utilizados machos Nelore (n = 210) (Bos taurus indicus), não castrados (Peso Inicial = ± 380 kg). Após o abate foram coletadas amostras de ceco e acondicionadas imediatamente em nitrogênio líquido. Foi feito um pool de amostras utilizando 90 animais (15 animais/tratamento) no processo de precipitação do pellet proteico. A separação e caracterização do proteoma das amostras biológicas foram feitas por eletroforese bidimensional (2D-PAGE) e identificadas por cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS). Os géis de poliacrilamida foram analisados utilizando ImageMaster 2D Platinum 7.0 para verificar as diferenças na expressão de proteínas, para as comparações do % volume normalizado dos spots. Após a identificação das proteínas foram analisadas as vias metabólicas usando a função da Enciclopédia de Genes e Genomas de Kyoto (KEGG), análise de enriquecimento da via Reactome para mapear as expressões das proteínas que codificam enzimas e suas respectivas funções nas vias afetadas. O uso de Blend de Óleo essenciais associado com -Amilase como aditivo alimentar promoveu maior expressão de enzimas na via da glicólise e gliconeogênese e ausência de proteína ligada a inflamação (Leukocyte elastase inhibitor). Por outro lado, o incremento de amido nas dietas promoveu redução de enzimas ligadas a degradação de carboidrato com aumento de respostas atribuídas à injúrias inflamatórias no rúmen de bovinos Nelore confinados. Artigo 2: Objetivou-se mapear o proteoma do epitélio ruminal de bovinos nelore confinados (n=60) com diferentes níveis de amido (Baixo: 25 e Alto: 45%) e aditivos (Monensina × blend de óleos essências + amilase exógena). As separações e fracionamento das proteínas foram através da eletroforese bidimensional em gel de poliacrilamida (2D-PAGE), e posteriormente a identificação por espectrometria de massas acoplada a cromatografia líquida (LC-MS/MS). Após a identificação das proteínas foram utilizados os acessos das proteínas para a classificação em suas funções moleculares, processos biológicos e componentes celulares utilizando Blast2GO, posteriormente a análise de enriquecimento via String para mapear as redes e interação entres proteínas caracterizadas suas respectivas funções no metabolismo de glicose e ácidos graxos. Dietas contendo blend de óleos essenciais associados à amilase exógena promoveram maior expressão de macromoléculas que participam da degradação de carboidratos pela via glicolítica e cetogênica. Foram identificadas 14 proteínas com maior expressão e presentes no tecido epitelial do rúmen envolvidas na oxidação da glicose, a proteína hidroximetilglutaril-CoA liase que catalisa parte do metabolismo metabólico intermediário, uma etapa fundamental na cetogênese. Nossos resultados sugerem que houve aumento da glicólise a partir da oxidação do gliceraldeído-3-fosfato, que participa da primeira etapa da produção de acetato e butirato e da descarboxilação oxidativa no epitélio ruminal de bovinos nelore confinados. A monensina melhora os precursores de propionato, a maior expressão de metilmalonil-CoA mutase sugere síntese de propionato via propionil-CoA que participa do ciclo do ácido cítrico através do succinil-CoA, que pode aumentar a energia metabolizável e reduzir a ingestão de alimentos.

Few studies have investigated the proteome of cecum and rumen of cattle feeding diferente estrategies with the use of additives. This proposal is a pioneer in the identification, expression. and understanding of the ruminal and cecal protein profile of feedlot Nellore cattle. Manuscript 1: The objective was to evaluate protein expression in order to identify possible candidate molecules for biomarkers and their relationships with metabolic and nutritional functions applied to animals fed with different levels of starch (25, 35 and 45%) and additives (Monensin × blend of essential oils + exogenous amylase), the dosages of sodium monensin, blend of essential oils and exogenous amylase were 26, 90 and 560 mg/kg DM, respectively. Nellore bulls (n = 210) (Bos taurus indicus) (initial weight = ) 380 kg) were used. After slaughter, cecum samples were collected and immediately placed in liquid nitrogen. Samples were pooled using 90 animals (15 animals/treatment) in the protein pellet precipitation process. The separation and characterization of the proteome of biological samples were performed by twodimensional electrophoresis (2D-PAGE) and identified by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Polyacrylamide gels were analyzed using ImageMaster 2D Platinum 7.0 to verify differences in protein expression for the % volume normalized spot comparisons. After identifying the proteins, the metabolic pathways were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) function, enrichment analysis of the Reactome pathway to map the expressions of proteins encoding enzymes and their respective functions in the affected pathways. The use of blend essential oil associated with -Amylase as a feedd additive promoted greater expression of enzymes in the glycolysis and gluconeogenesis pathway and absence of protein linked to inflammation (Leukocyte elastase inhibitor). On the other hand, the increase in starch in the diets reduced enzymes linked to carbohydrate degradation with increased responses attributed to inflammatory injuries in the rumen of feedlot Nellore cattle. Manuscript 2: The objective was to map the proteome of the ruminal epithelium of feedlot Nellore cattle (n=60) with different levels of starch (Low: 25 and High: 45%) and additives (Monensin × blend of essential oils + exogenous amylase) the dosages of sodium monensin, blend of essential oils and exogenous amylase were 26, 90 and 560 mg/kg DM, respectively. Protein separations were performed using two-dimensional polyacrylamide gel electrophores (2D-PAGE), followed by identification by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). After protein identification, protein accessions were used to classify their molecular functions, biological processes and cellular component using Blast2GO, then enrichment analysis pathways String to map the networks and interaction xii between proteins characterized and their respective functions in glucose and fatty acids metabolic. Diets containing blend of essential oils associated with exogenous amylase promoted greater expression of macromolecules that participate in carbohydrate degradation via glycolytic and ketogenic pathways. We identified 14 proteins with greater expression and present in the epithelial tissue of the rumen involved in glucose oxidation, the protein hydroxymethylglutaryl-CoA lyase that catalyzes part of intermediary metabolic metabolism, a fundamental step in ketogenesis. Our results suggest that there was an increase in glycolysis from the oxidation of glyceraldehyde-3-phosphate, which participates in the first stage of acetate and butyrate production and oxidative decarboxylation in the ruminal epithelium of feedlot Nellore cattle. Monensin improves propionate precursors, the increased expression of methylmalonyl-CoA mutase suggests propionate synthesis via propionyl-CoA that participates in the citric acid cycle through succinyl-CoA, which can increase metabolizable energy and reduce feed intake.

EFEITOS DO ÓLEO DA SEMENTE DE MARACUJÁ NOS SISTEMAS IMUNE E ANTIOXIDANTE DE FRANGOS DE CORTE

O estresse térmico proporciona muitos prejuízos na avicultura industrial e com o objetivo de minimizá-los, estratégias nutricionais vêm sendo adotadas, com ênfase, principalmente aos alimentos naturais. O objetivo desse trabalho foi estudar o efeito da suplementação de óleo da semente de maracujá (OSM) na imunidade e respostas imunes, qualidade de carne e sistema antioxidante de frangos de corte. Para isso foram utilizados 450 pintainhos de 1 dia de idade, machos, da linhagem Cobb® 500, alojados em gaiolas de arame galvanizado, em duas câmaras com 45 gaiolas cada. O delineamento foi inteiramente casualizado, em esquema fatorial 2 x 5, sendo duas temperaturas (termoneutra e estresse cíclico pelo calor) e cinco tratamentos (controle sem inclusão de OSM + quatro níveis de inclusão de OSM (0,30; 0,50; 0,70; 0,90%) com nove repetições de cinco aves cada. A ração foi desprovida de antibióticos como melhorador de desempenho, porém acrescida de anticoccidiano em todos os tratamentos. Os dados foram analisados utilizando-se análise de variância com auxílio do SAS 9.3 (SAS Institute, 2008) e, sendo significativas a (P<0,05) no teste de média pelo teste de Tukey. Houve efeito significativo para o peso vivo ao abate e peso de cortes comerciais, para frangos criados em ambiente termoneutro, que apresentaram maiores valores em relação ao estresse por calor, e a gordura abdominal apresentou menor valor para aves desafiadas por calor. O OSM influenciou no maior peso (em gramas) das pernas. Frangos de corte criados em estresse por calor apresentaram menores valores de a* na musculatura do peito do que os frangos criados em ambiente termoneutro. Em relação a análise proteômica eletroforese bidimensional em gel de poliacrilamida (2D-PAGE) do plasma sanguíneo para resultados voltados a qualidade de carne, a proteína Actin alpha skeletal muscle, apresentou diferença significativa nos tratamentos com inclusão de 0,9% de OSM em ambas as temperaturas (estresse por calor e termoneutra) e, Neuropilin-1, no tratamento termoneutro com 0% de inclusão de OSM. Com 21 dias de idade foi observado uma interação significativa para titulação de anticorpos, sendo que frangos criados em ambiente de estresse que receberam o nível de 0,90% de OSM, apresentaram maior valor de titulação vacinal. Aos 28 dias de idade, novamente os animais que receberam 0,90% de inclusão de OSM e, independente do ambiente de criação, apresentaram maiores valores de títulos de anticorpos. Na análise proteômica do plasma sanguíneo (2D-PAGE) foram encontradas diversas proteínas que 11 estão relacionadas com o sistema imunológico dos frangos de corte: Complement factor B-like protease (Fragment); Glyceraldehyde-3-phosphate dehydrogenase; Mannose-binding protein; Zinc finger protein ubi-d4; Ig lambda chain C region; Ig lambda chain V-1 region; Apolipoprotein A-I. A análise proteômica também apresentou resultados referentes a manutenção do epitélio gastrointestinal, com a expressão diferencial de Retinol-binding protein 4 e, Homeodomain-only protein. Houve maior % de redução de DPPH com a maior inclusão do OSM, assim como maior expressão de Alcohol dehydrogenase 1, nos tratamentos com maior inclusão de OSM. Também houve diferença significativa para o efeito temperatura sobre a atividade da enzima catalase que se apresentou maior em ambiente termoneutro. Assim como, Nuclear receptor coactivator 7 que foi diferencialmente expresso no tratamento 0% de frangos de corte criados em ambiente termoneutro. Hemoglobin subunit epsilon teve sua expressão diferenciada no tratamento 0,9% de frangos de corte criados em ambiente de estresse. Além disso, vários spots proteicos relacionados as chaperonas moleculares foram diferencialmente expressas nos tratamentos 0,5% e 0,9% de inclusão de OSM em ambas as temperaturas de criação. Membrane-associated progesterone receptor component 1 foi diferencialmente expresso nos tratamentos de 0,9% de inclusão de OSM em ambos os ambientes de criação.

Heat stress causes many damages in industrial poultry farming and in order to minimize them, nutritional strategies have been adopted, with emphasis, mainly on natural foods. The objective of this work was to study the effect of passion fruit seed oil (OSM) supplementation on immunity and immune responses, meat quality and antioxidant system of broiler chickens. For this, 450 1-day-old male chicks of the Cobb® 500 strain were used, housed in galvanized wire cages, in two chambers with 45 cages each. The design was completely randomized, in a 2 x 5 factorial scheme, with two temperatures (thermoneutral and cyclic heat stress) and five treatments (control without inclusion of OSM + four levels of inclusion of OSM (0.30; 0.50; 0) .70; 0.90%) with nine replicates of five birds each.The ration was devoid of antibiotics as a performance enhancer, but added anticoccidial in all treatments. Data were analyzed using analysis of variance with the aid of SAS 9.3 (SAS Institute, 2008) and, being significant (P<0.05) in the mean test by the Tukey test. which had higher values in relation to heat stress, and abdominal fat had lower value for heat challenged birds. OSM influenced the higher weight (in grams) of the legs. Broilers reared under heat stress had lower value a* in the breast musculature than broilers raised in a thermoneutral environment. Regarding proteomic two-dimensional electrophoresis analysis in polyacrylamide gel (2D-PAGE) of blood plasma for meat quality results, the protein Actin alpha skeletal muscle showed a significant difference in treatments with inclusion of 0.9% OSM in both temperatures (heat stress and thermoneutral) and, Neuropilin-1, in thermoneutral treatment with 0% inclusion of OSM. At 21 days of age, a significant interaction for antibody titration was observed, and broilers reared in a stress environment that received the level of 0.90% of OSM had a higher value of vaccine titration. At 28 days of age, again the animals that received 0.90% inclusion of OSM and, regardless of the rearing environment, had higher antibody titers. In the proteomic analysis of blood plasma (2D-PAGE) several proteins that are related to the immune system of broiler chickens were found: Complement factor B-like protease (Fragment); Glyceraldehyde-3-phosphate dehydrogenase; Mannose-binding protein; Zinc finger protein ubi-d4; Ig lambda chain C region; Ig lambda chain V-1 region; Apolipoprotein A-I. Proteomic analysis also showed results regarding the maintenance of the gastrointestinal epithelium, with the differential expression of Retinol-binding protein 4 and Homeodomain-only protein. There was a greater % reduction of DPPH with the 13 greater inclusion of OSM, as well as greater expression of Alcohol dehydrogenase 1, in treatments with greater inclusion of OSM. There was also a significant difference for the temperature effect on the catalase enzyme activity, which was higher in a thermoneutral environment. As well as, Nuclear receptor coactivator 7 which was differentially expressed in the 0% treatment of broilers reared in thermoneutral environment. Hemoglobin subunit epsilon had its expression differentiated in the treatment of 0.9% of broilers reared in stress environment. Furthermore, several protein spots related to molecular chaperones were differentially expressed in the 0.5% and 0.9% OSM inclusion treatments at both rearing temperatures. Membrane-associated progesterone receptor component 1 was differentially expressed in treatments of 0.9% inclusion of OSM in both rearing environments.