Comparative expression profiles of genes related to cellular stress and development in oocyte of goats fed with distinct concentrations of lipids

Background: Lipotoxicity is characterized by an excess of saturated fatty acids in the blood stream, in which other nonadipose cells begin to store them, thereby altering the expression of genes related to endoplasmic reticulum stress, whoseeffects have been associated with decreased oocyte quality in several species, decreased mitochondrial activity, and increasedapoptosis. The present study was conducted to investigate the lipotoxicity effect of diets with increasing fat levels on geneexpression in goats oocyte and granulosa cells.Materials, Methods & Results: Thirty does were divided into three groups of 10 animals each, which received forage andconcentrate to provide respectively 2.7% of lipids (LL group), 3.9% lipids (LI group) and 5.1% lipids (LH group) for 28days. Three days before oocyte harvest, follicular wave was synchronized by 1 mL PGF2α intramuscularly, followed bythe insertion of an intravaginal progesterone release device. Viable oocytes and granulosa cells were subjected to qRT-PCRto determine the expression of PLIN2, ATF4, CHOP10, BAX, BCL2, HSP70 genes, were checked, evaluated using thedissociation curve analysis, which obtained the following values: 77.8, 80.2, 83.6, 78.0, 81.0 and 82.0ºC, respectively. Thesteps of qPCR thermal cycle was: denaturation and polymerase activation, annealing and final extension.Throughout theexperimental period, blood samples were taken for cholesterol, triglycerides and total lipids measurements. Total plasmalipids determination was calculated with the following equation: 2 x (cholesterol + triglycerides) × 1.1 with sensitivity ofthe assay for cholesterol was 1.472 mg/dL and for triglycerides, 2.845 mg/dL. In LH group, it was recorded a more pronounced increase in total lipids (+ 60.1%) (P < 0.05), cholesterol (+ 18.8%) and triglycerides (+ 8.5%). These increaseswere three times higher than that in LL and LI groups. All genes were expressed in oocytes...(AU)

Comparative expression profiles of genes related to cellular stress and development in oocyte of goats fed with distinct concentrations of lipids

Background: Lipotoxicity is characterized by an excess of saturated fatty acids in the blood stream, in which other nonadipose cells begin to store them, thereby altering the expression of genes related to endoplasmic reticulum stress, whoseeffects have been associated with decreased oocyte quality in several species, decreased mitochondrial activity, and increasedapoptosis. The present study was conducted to investigate the lipotoxicity effect of diets with increasing fat levels on geneexpression in goat’s oocyte and granulosa cells.Materials, Methods & Results: Thirty does were divided into three groups of 10 animals each, which received forage andconcentrate to provide respectively 2.7% of lipids (LL group), 3.9% lipids (LI group) and 5.1% lipids (LH group) for 28days. Three days before oocyte harvest, follicular wave was synchronized by 1 mL PGF2α intramuscularly, followed bythe insertion of an intravaginal progesterone release device. Viable oocytes and granulosa cells were subjected to qRT-PCRto determine the expression of PLIN2, ATF4, CHOP10, BAX, BCL2, HSP70 genes, were checked, evaluated using thedissociation curve analysis, which obtained the following values: 77.8, 80.2, 83.6, 78.0, 81.0 and 82.0ºC, respectively. Thesteps of qPCR thermal cycle was: denaturation and polymerase activation, annealing and final extension.Throughout theexperimental period, blood samples were taken for cholesterol, triglycerides and total lipids measurements. Total plasmalipids determination was calculated with the following equation: 2 x (cholesterol + triglycerides) × 1.1 with sensitivity ofthe assay for cholesterol was 1.472 mg/dL and for triglycerides, 2.845 mg/dL. In LH group, it was recorded a more pronounced increase in total lipids (+ 60.1%) (P < 0.05), cholesterol (+ 18.8%) and triglycerides (+ 8.5%). These increaseswere three times higher than that in LL and LI groups. All genes were expressed in oocytes...

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...](AU)

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...]

Clonagem do cDNA e sequenciamento parcial do gene que codifica a enzima glutamato desidrogenase hepática de ovino / Cloning of the cDNA and partial sequencing of the gene that codifi es the sheep hepatic enzyme glutamate dehydrogenase

A enzima mitocondrial glutamato desidrogenase (GDH: EC 1.4.1.2) catalisa a desaminação reversível do L-glutamato para 2-oxoglutarato (α-cetoglutarato) usando o NAD+ e NADP+ como coenzimas. É uma das mais importantes enzimas hepáticas encontradas em hepatócitos de bovinos, ovinos e caprinos. Infecções por Fasciola spp., intoxicação grave aguda por toxinas de plantas, tais como Xanthium spp. e Senecio spp. e intoxicação por cobre resultam na liberação dessa enzima no sangue. O aumento da GDH indica danos ou necrose hepática em bovinos e ovinos. Esta é a enzima de escolha para avaliar a função hepática dos ruminantes. No presente trabalho o cDNA que codifica a enzima GDH do hepatócito de ovino foi sintetizado por meio de RT-PCR utilizando mRNA extraído do fígado de ovino. Parte da região de codificação do cDNA da GDH de ovino foi amplificada por PCR usando oligonucleotídeos iniciadores sintetizados a partir do alinhamento de sequências de ORFs de Ovis aries, Bos taurus, Homo sapiens, Rattus norvegicus e Mus musculus disponíveis em banco de dados. O cDNA foi clonado no vetor pGEM® -T Easy (Promega) e inserido em células cálcio-competentes de Escherichia coli DH10B através de choque térmico. O DNA plasmidial foi purificado e após o sequenciamento a presença de um inserto de 1292 pb foi confirmado. O alinhamento da sequência deduzida de aminoácidos com outras espécies revelou alta homologia entre as GDH.

The mitochondrial enzyme Glutamate Dehydrogenase (GDH: EC 1.4.1.2) catalyzes the reversible deamination of the L-glutamate for 2-oxoglutarate (α-ketoglutarate) using NAD+ and NADP+ as coenzymes. It is one of the most important liver enzymes found in hepatocytes of cattle, sheep and goats. Infections by Fasciola spp., severe acute intoxication by toxins of plants such as Xanthium spp. and Senecio spp. as well as intoxication by copper result in the release of this enzyme in blood. The increase of the GDH indicates damage or hepatic necrosis in cattle and sheep. This is an enzyme of choice to evaluate the function of the ruminants. In the present study the cDNA, that codifies the GDH enzyme of the hepatocyte of sheep, was synthesized by means of RT-PCR making use of mRNA extracted from the liver of sheep. Part of the region where the cDNA of the GDH of the ovine is codified was amplified by PCR from primers synthesized through the comparison of the aligned sequences of Ovis aries, Bos taurus, Homo sapiens, Rattus norvegicus and Mus musculus available in the database. The cDNA was cloned in the vector pGEM®-T Easy (Promega) and inserted in Escherichia coli DH10B calcium competent cells by heat shock procedure. The plasmid DNA was purified and after sequencing, the presence of 1292 pb was confirmed. The alignment of the sequence deduced of amino acid with other species revealed high homology among the GDHs.

Clonagem do cDNA e sequenciamento parcial do gene que codifica a enzima glutamato desidrogenase hepática de ovino / Cloning of the cDNA and partial sequencing of the gene that codifi es the sheep hepatic enzyme glutamate dehydrogenase

A enzima mitocondrial glutamato desidrogenase (GDH: EC 1.4.1.2) catalisa a desaminação reversível do L-glutamato para 2-oxoglutarato (α-cetoglutarato) usando o NAD+ e NADP+ como coenzimas. É uma das mais importantes enzimas hepáticas encontradas em hepatócitos de bovinos, ovinos e caprinos. Infecções por Fasciola spp., intoxicação grave aguda por toxinas de plantas, tais como Xanthium spp. e Senecio spp. e intoxicação por cobre resultam na liberação dessa enzima no sangue. O aumento da GDH indica danos ou necrose hepática em bovinos e ovinos. Esta é a enzima de escolha para avaliar a função hepática dos ruminantes. No presente trabalho o cDNA que codifica a enzima GDH do hepatócito de ovino foi sintetizado por meio de RT-PCR utilizando mRNA extraído do fígado de ovino. Parte da região de codificação do cDNA da GDH de ovino foi amplificada por PCR usando oligonucleotídeos iniciadores sintetizados a partir do alinhamento de sequências de ORFs de Ovis aries, Bos taurus, Homo sapiens, Rattus norvegicus e Mus musculus disponíveis em banco de dados. O cDNA foi clonado no vetor pGEM® -T Easy (Promega) e inserido em células cálcio-competentes de Escherichia coli DH10B através de choque térmico. O DNA plasmidial foi purificado e após o sequenciamento a presença de um inserto de 1292 pb foi confirmado. O alinhamento da sequência deduzida de aminoácidos com outras espécies revelou alta homologia entre as GDH.(AU)

The mitochondrial enzyme Glutamate Dehydrogenase (GDH: EC 1.4.1.2) catalyzes the reversible deamination of the L-glutamate for 2-oxoglutarate (α-ketoglutarate) using NAD+ and NADP+ as coenzymes. It is one of the most important liver enzymes found in hepatocytes of cattle, sheep and goats. Infections by Fasciola spp., severe acute intoxication by toxins of plants such as Xanthium spp. and Senecio spp. as well as intoxication by copper result in the release of this enzyme in blood. The increase of the GDH indicates damage or hepatic necrosis in cattle and sheep. This is an enzyme of choice to evaluate the function of the ruminants. In the present study the cDNA, that codifies the GDH enzyme of the hepatocyte of sheep, was synthesized by means of RT-PCR making use of mRNA extracted from the liver of sheep. Part of the region where the cDNA of the GDH of the ovine is codified was amplified by PCR from primers synthesized through the comparison of the aligned sequences of Ovis aries, Bos taurus, Homo sapiens, Rattus norvegicus and Mus musculus available in the database. The cDNA was cloned in the vector pGEM®-T Easy (Promega) and inserted in Escherichia coli DH10B calcium competent cells by heat shock procedure. The plasmid DNA was purified and after sequencing, the presence of 1292 pb was confirmed. The alignment of the sequence deduced of amino acid with other species revealed high homology among the GDHs.(AU)

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNaiTM). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80ºC. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAiTM was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Clonagem animal: a sobrevivência dos mais aptos / Animal cloning: survival of the fittest

A clonagem animal por transferência nuclear de célula somática (TNCS) apresenta inúmeras aplicações científicas e comerciais, incluindo a produção de animais transgênicos, a preservação de animais de genética desejável, rara ou em extinção, ou mesmo a aplicação para o estudo de aspectos básicos em biologia molecular, celular e do desenvolvimento. Não obstante, a clonagem por TNCS ainda é ineficiente, com menos de 5% dos embriões clones produzidos resultando em animais nascidos vivos. O sucesso na clonagem exige o exímio domínio técnico e científico de várias disciplinas e áreas de conhecimento, havendo pelo menos cinco etapas críticas no processo associadas a falhas de desenvolvimento, desde a produção in vitro dos embriões até o nascimento de um animal viável. A identificação de fatores associados às falhas em cada etapa, em especial aqueles relacionados ao oócito receptor (citoplasto), à célula doadora (carioplasto) e aos procedimentos técnicos per se de produção de embriões clones, além da observação cuidadosa dos sinais de anormalidades subsequentes à transferência dos embriões para fêmeas receptoras, é essencial para a optimização de todos os procedimentos para a obtenção, em seu final, de um animal clonado viável e que sobreviva até a vida adulta. Esta revisão visa descrever alguns eventos técnicos e biológicos associados ao sucesso e/ou insucesso da clonagem animal.

Animal cloning by somatic cell nuclear transfer (SCNT) has numerous scientific and commercial applications, including the production of transgenic animals, preservation of animals from desirable or rare gene pools, and animals in risk of extinction, or even for the study of basic aspects in molecular, cell and developmental biology. Nevertheless, cloning by SCNT is still inefficient, with less than 5% of cloned embryos resulting in liveborn animals. The cloning success depends on a proficient technical and scientific know-how of a number of disciplines and areas of knowledge, with at least five critical steps in the process associated with developmental failures, from the in vitro production of cloned embryos through the birth of a viable animal. The identification of factors associated with failures in each step, in special to those related to the recipient oocyte (cytoplast), to the nucleus donor cell (karyoplast), and to the technical procedures for the production of cloned embryos per se, along with the careful observation of signs of abnormalities following the transfer of embryos to recipient females, is essential for the optimization of procedures that, ultimately, may result in a cloned animal that survives to adulthood. This review aims to discuss some technical and biological events associated with success and/or failure in animal cloning.

Clonagem animal: a sobrevivência dos mais aptos / Animal cloning: survival of the fittest

A clonagem animal por transferência nuclear de célula somática (TNCS) apresenta inúmeras aplicações científicas e comerciais, incluindo a produção de animais transgênicos, a preservação de animais de genética desejável, rara ou em extinção, ou mesmo a aplicação para o estudo de aspectos básicos em biologia molecular, celular e do desenvolvimento. Não obstante, a clonagem por TNCS ainda é ineficiente, com menos de 5% dos embriões clones produzidos resultando em animais nascidos vivos. O sucesso na clonagem exige o exímio domínio técnico e científico de várias disciplinas e áreas de conhecimento, havendo pelo menos cinco etapas críticas no processo associadas a falhas de desenvolvimento, desde a produção in vitro dos embriões até o nascimento de um animal viável. A identificação de fatores associados às falhas em cada etapa, em especial aqueles relacionados ao oócito receptor (citoplasto), à célula doadora (carioplasto) e aos procedimentos técnicos per se de produção de embriões clones, além da observação cuidadosa dos sinais de anormalidades subsequentes à transferência dos embriões para fêmeas receptoras, é essencial para a optimização de todos os procedimentos para a obtenção, em seu final, de um animal clonado viável e que sobreviva até a vida adulta. Esta revisão visa descrever alguns eventos técnicos e biológicos associados ao sucesso e/ou insucesso da clonagem animal.(AU)

Animal cloning by somatic cell nuclear transfer (SCNT) has numerous scientific and commercial applications, including the production of transgenic animals, preservation of animals from desirable or rare gene pools, and animals in risk of extinction, or even for the study of basic aspects in molecular, cell and developmental biology. Nevertheless, cloning by SCNT is still inefficient, with less than 5% of cloned embryos resulting in liveborn animals. The cloning success depends on a proficient technical and scientific know-how of a number of disciplines and areas of knowledge, with at least five critical steps in the process associated with developmental failures, from the in vitro production of cloned embryos through the birth of a viable animal. The identification of factors associated with failures in each step, in special to those related to the recipient oocyte (cytoplast), to the nucleus donor cell (karyoplast), and to the technical procedures for the production of cloned embryos per se, along with the careful observation of signs of abnormalities following the transfer of embryos to recipient females, is essential for the optimization of procedures that, ultimately, may result in a cloned animal that survives to adulthood. This review aims to discuss some technical and biological events associated with success and/or failure in animal cloning.(AU)

A new therherapeutic prrotocol f for dogs infectted with Trrypanosoma evansi

Background: Trypanosomosis is a disease caused by a flagellate protozoan known as Trypanosoma evansi, transmitted by hematophagous insects. It parasites showed a large diversity of mammalian hosts. Dogs may show clinical changes such as weight loss, progressive weakness, anorexia, anemia, intermittent fever, conjunctivitis, swelling of limbs and increased of superficial lymph nodes. Treatment of trypanosomosis relies on the use of diminazene aceturate which is effective for the treatment of disease in infected animals. However, a single dose of drug are not effective for horses, mules and dogs, since drug neither cross the blood-brain barrier or has insufficient doses to control the T. evansi infection. Therefore, the present study aimed to report the curative efficacy of a new therapeutic protocol, based on diminazene aceturate, for dogs infected with T. evansi. Case: The treatment against trypanosomosis was performed in a dog, male, two years old, from the municipality of Uruguaiana, RS, Brazil.. The animal showing clinical signs such as apathy, vomit, increase of left submandibular lymph node, edema of the left face and change of gait in the hind limbs when it was evaluated by veterinarians. The hematological and biochemical parameters revealed normocytic-hypochromic anemia, thrombocytopenia, hyperglobulinemia and hypoalbuminemia. Trypomastigotes of T. evansi were identified in blood smears stained by panoptic method. The parasitism by T. evansi was confirmed by blood inoculation in two rats (xenodiagnosis) and by PCR T. evansi-specific. The canine was treated with diminazene aceturate (intramuscular injection) using a dose of 3.5 mg kg-1 for 5 days at 24h of intervals. After treatment the dog showed clinical signs of health improvement, and clinical signs disappeared after the seventh day of treatment. The parasite was not found in blood smears after the third day of treatment and PCR was negative on days 30 and 50 post-treatment. During the treatment signs of drug intoxication were not observed, as well as hepatic and renal functions were not affected. The animal showed normal biochemical and hematological parameters after 30 days of treatment. Discussion: In this study, the treatment tested was effective, leading to the cure of the disease. Previously, the same protocol was used for cats experimentally infected with T. evansi, obtaining 85.7% (6/7) of curative efficacy. In a comparative study of doses of diminazene aceturate in rats infected with T. evansi, inefficiency and death of rats treated with a single dose of 3.5 and 7.0 mg kg-1 were observed. In contrast, the cure in rats occurred when animals received a dose of 3.5 and 7.0 mg kg-1 during five consecutive days. The return of the parasitemia after treatment may be related to the impossibility of the medicament pass through the blood-brain barrier or the doses is insufficient. Our data reveal that five-dose protocol obtained higher efficiency because it provided greater passage of drug molecules through blood-brain barrier, which could eliminate the parasite from brain. After treatment, all clinical signs disappeared, biochemical and hematological parameters returned to normal levels, allowing us to conclude that this new protocol tested was effective to cure of this disease in dogs.

A new therherapeutic prrotocol f for dogs infectted with Trrypanosoma evansi

Background: Trypanosomosis is a disease caused by a flagellate protozoan known as Trypanosoma evansi, transmitted by hematophagous insects. It parasites showed a large diversity of mammalian hosts. Dogs may show clinical changes such as weight loss, progressive weakness, anorexia, anemia, intermittent fever, conjunctivitis, swelling of limbs and increased of superficial lymph nodes. Treatment of trypanosomosis relies on the use of diminazene aceturate which is effective for the treatment of disease in infected animals. However, a single dose of drug are not effective for horses, mules and dogs, since drug neither cross the blood-brain barrier or has insufficient doses to control the T. evansi infection. Therefore, the present study aimed to report the curative efficacy of a new therapeutic protocol, based on diminazene aceturate, for dogs infected with T. evansi. Case: The treatment against trypanosomosis was performed in a dog, male, two years old, from the municipality of Uruguaiana, RS, Brazil.. The animal showing clinical signs such as apathy, vomit, increase of left submandibular lymph node, edema of the left face and change of gait in the hind limbs when it was evaluated by veterinarians. The hematological and biochemical parameters revealed normocytic-hypochromic anemia, thrombocytopenia, hyperglobulinemia and hypoalbuminemia. Trypomastigotes of T. evansi were identified in blood smears stained by panoptic method. The parasitism by T. evansi was confirmed by blood inoculation in two rats (xenodiagnosis) and by PCR T. evansi-specific. The canine was treated with diminazene aceturate (intramuscular injection) using a dose of 3.5 mg kg-1 for 5 days at 24h of intervals. After treatment the dog showed clinical signs of health improvement, and clinical signs disappeared after the seventh day of treatment. The parasite was not found in blood smears after the third day of treatment and PCR was negative on days 30 and 50 post-treatment. During the treatment signs of drug intoxication were not observed, as well as hepatic and renal functions were not affected. The animal showed normal biochemical and hematological parameters after 30 days of treatment. Discussion: In this study, the treatment tested was effective, leading to the cure of the disease. Previously, the same protocol was used for cats experimentally infected with T. evansi, obtaining 85.7% (6/7) of curative efficacy. In a comparative study of doses of diminazene aceturate in rats infected with T. evansi, inefficiency and death of rats treated with a single dose of 3.5 and 7.0 mg kg-1 were observed. In contrast, the cure in rats occurred when animals received a dose of 3.5 and 7.0 mg kg-1 during five consecutive days. The return of the parasitemia after treatment may be related to the impossibility of the medicament pass through the blood-brain barrier or the doses is insufficient. Our data reveal that five-dose protocol obtained higher efficiency because it provided greater passage of drug molecules through blood-brain barrier, which could eliminate the parasite from brain. After treatment, all clinical signs disappeared, biochemical and hematological parameters returned to normal levels, allowing us to conclude that this new protocol tested was effective to cure of this disease in dogs.(AU)