Isolation, fractionation and anticoagulant activity of a sulfated galactan extracted from the green algae Penicillus capitatus / Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

Marine algae are natural sources of macromolecules known as sulfated polysaccharides. This class of compounds has attracted the interest of Pharmaceutical Sciences due to its pharmacological anticoagulant, antiplatelet and antithrombotic properties. Therefore, this study evaluated the anticoagulant potential of sulfated polysaccharides extracted from the algae Penicillus capitatus. The extracted sulfated polysaccharides were purified, partially characterized and their anticoagulant activity was evaluated. The extraction process followed by ethanol precipitation resulted in five fractions. Among the analyzed fractions, F44 contained highest concentration of sulfated polysaccharides. After the purified fraction F23, F44 displayed in vitro anticoagulant activity in a time testing for activated partial thromboplastin time and pro-thrombin time. The preferential mechanism effect was based on interactions between thrombin and factor Xa. Additional studies on structure pharmacological are required to test the viability of the use of sulfated polysaccharides as therapeutic agents.

As algas marinhas são fontes naturais de macromoléculas conhecidas como polissacarídeos sulfatados. Esta classe de compostos atraiu o interesse das Ciências Farmacêuticas devido às suas propriedades farmacológicas como anticoagulante, antiplaquetária e antitrombótica. Portanto, este estudo tem como objetivo avaliar o potencial anticoagulante de polissacarídeos sulfatados extraídos de algas de Penicillus capitatus. Os polissacarídeos sulfatados extraídos foram purificados, parcialmente caracterizados e sua atividade anticoagulante foi avaliada. O processo de extração seguido pela precipitação com etanol resultou em cinco frações. Entre as frações analisadas, F44 foi a maior concentração de polissacarídeos sulfatados. Após a purificação, as frações F23 e F44 mostraram atividade anticoagulante in vitro em um teste de tempo de tromboplastina parcialmente ativada e tempo de protrombina. Seu mecanismo preferencial é baseado nas interações entre trombina e fator Xa. Estudos adicionais sobre a estrutura farmacológica são necessários para testar a viabilidade do uso como agente terapêutico.

Isolation, fractionation and anticoagulant activity of a sulfated galactan extracted from the green algae Penicillus capitatus / Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

Marine algae are natural sources of macromolecules known as sulfated polysaccharides. This class of compounds has attracted the interest of Pharmaceutical Sciences due to its pharmacological anticoagulant, antiplatelet and antithrombotic properties. Therefore, this study evaluated the anticoagulant potential of sulfated polysaccharides extracted from the algae Penicillus capitatus. The extracted sulfated polysaccharides were purified, partially characterized and their anticoagulant activity was evaluated. The extraction process followed by ethanol precipitation resulted in five fractions. Among the analyzed fractions, F44 contained highest concentration of sulfated polysaccharides. After the purified fraction F23, F44 displayed in vitro anticoagulant activity in a time testing for activated partial thromboplastin time and pro-thrombin time. The preferential mechanism effect was based on interactions between thrombin and factor Xa. Additional studies on structure pharmacological are required to test the viability of the use of sulfated polysaccharides as therapeutic agents.(AU)

As algas marinhas são fontes naturais de macromoléculas conhecidas como polissacarídeos sulfatados. Esta classe de compostos atraiu o interesse das Ciências Farmacêuticas devido às suas propriedades farmacológicas como anticoagulante, antiplaquetária e antitrombótica. Portanto, este estudo tem como objetivo avaliar o potencial anticoagulante de polissacarídeos sulfatados extraídos de algas de Penicillus capitatus. Os polissacarídeos sulfatados extraídos foram purificados, parcialmente caracterizados e sua atividade anticoagulante foi avaliada. O processo de extração seguido pela precipitação com etanol resultou em cinco frações. Entre as frações analisadas, F44 foi a maior concentração de polissacarídeos sulfatados. Após a purificação, as frações F23 e F44 mostraram atividade anticoagulante in vitro em um teste de tempo de tromboplastina parcialmente ativada e tempo de protrombina. Seu mecanismo preferencial é baseado nas interações entre trombina e fator Xa. Estudos adicionais sobre a estrutura farmacológica são necessários para testar a viabilidade do uso como agente terapêutico.(AU)

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)

Soroprevalência e fatores de risco da infecção por Chlamydophila abortus em caprinos do estado do Rio Grande do Norte, Brasil / Seroprevalence and risk factors of Chlamydophila abortus infection in goats of the state of Rio Grande do Norte, Brazil

Background: Chlamydophilosis is a disease caused by the bacterium Chlamydophila abortus, responsible for reproductive damage in small ruminants, and economic losses to animal production. In addition, it is classified as a zoonosis once it presents symptoms similar to numerous bacterial and viral infections of the reproductive system, making clinical diagnosis difficult. The objective of this work was to estimate the seroprevalence of anti-C. abortus antibodies in goats and to identify the risk factors for the disease in two mesoregions of Rio Grande do Norte, Brazil.Material, Methods & Results: This research was carried out in two mesoregions of the State of Rio Grande do Norte (Central Potiguar - CP and West Potiguar - WP) located in the Northeast of Brazil. These mesoregions together represent 83% of the regional herd. A total of 54 properties were studied. Those are distributed in seven municipalities: Afonso Bezerra, Angicos, Lages and Pedro Avelino, in the CP mesoregion; and Apodi, Caraúbas, and Mossoró, in the OP mesoregion. We used 540 animals from herds characterized as non-defined breed (NDB) and native breed, regardless of their productive functions, being frequent the consortium of caprine and ovine species. Blood samples were collected through jugular venipuncture by vacuum system with 5 mL tubes without anticoagulant. After collection, the tubes were centrifuged at 1500 g for 15 min. Detection of anti-Chlamydophila sp. antibodies was performed by the Complement Fixation Test. In parallel with the collection, a questionnaire was applied addressing general property and producer characteristics. Seroprevalence of chlamydophilosis in the CP mesoregion was 3.04% (7/230) for the animals and 26.1% (6/23) for the properties, whereas, in the WP mesoregion, the values were 4.2% (13/310) and 35.5% (11/31), respectively.[...]

Soroprevalência e fatores de risco da infecção por Chlamydophila abortus em caprinos do estado do Rio Grande do Norte, Brasil / Seroprevalence and risk factors of Chlamydophila abortus infection in goats of the state of Rio Grande do Norte, Brazil

Background: Chlamydophilosis is a disease caused by the bacterium Chlamydophila abortus, responsible for reproductive damage in small ruminants, and economic losses to animal production. In addition, it is classified as a zoonosis once it presents symptoms similar to numerous bacterial and viral infections of the reproductive system, making clinical diagnosis difficult. The objective of this work was to estimate the seroprevalence of anti-C. abortus antibodies in goats and to identify the risk factors for the disease in two mesoregions of Rio Grande do Norte, Brazil.Material, Methods & Results: This research was carried out in two mesoregions of the State of Rio Grande do Norte (Central Potiguar - CP and West Potiguar - WP) located in the Northeast of Brazil. These mesoregions together represent 83% of the regional herd. A total of 54 properties were studied. Those are distributed in seven municipalities: Afonso Bezerra, Angicos, Lages and Pedro Avelino, in the CP mesoregion; and Apodi, Caraúbas, and Mossoró, in the OP mesoregion. We used 540 animals from herds characterized as non-defined breed (NDB) and native breed, regardless of their productive functions, being frequent the consortium of caprine and ovine species. Blood samples were collected through jugular venipuncture by vacuum system with 5 mL tubes without anticoagulant. After collection, the tubes were centrifuged at 1500 g for 15 min. Detection of anti-Chlamydophila sp. antibodies was performed by the Complement Fixation Test. In parallel with the collection, a questionnaire was applied addressing general property and producer characteristics. Seroprevalence of chlamydophilosis in the CP mesoregion was 3.04% (7/230) for the animals and 26.1% (6/23) for the properties, whereas, in the WP mesoregion, the values were 4.2% (13/310) and 35.5% (11/31), respectively.[...](AU)

In vitro inactivation of thrombin generation by polysulfated fractions isolated from the tropical coenocytic green seaweed Caulerpa racemosa (Caulerpaceae, Bryopsidales) / Inativação in vitro da geração de trombina por frações polissulfatadas isoladas damacroalga verde cenocítica tropical Caulerpa racemosa (Caulerpaceae; Bryopsidales)

Pharmacological efficacy of Caulerpa racemosa (Chlorophyta) sulfated polysaccharidic (SPs) fractions (F IIII) on models of coagulation and inflammation has been demonstrated, but not their effects on thrombin generation (TG). This study examined fractions for composition and physical-chemical characteristics and in vitro inactivation of TG by F I and F II in 60-fold diluted human plasma using continuous method. Papain-extraction yield of 0.7% revealed F IIII by DEAE-cellulose chromatography, with differences among the relative proportions of sulfate (17.37-24.00%), total sugars (30.03-48.34%) and absence of proteins. Charge density patterns and molecular sizes > 100 kDa of the fractions were verified by both agarose/polyacrylamide analyses, respectively. These electrophoreses combined with toluidine blue/Stains-All also indicated nonSPs. Anticoagulant effects of 4.76 (F I), 12.00 (F II) and 2.32 (F II) IU mg-1 by activated partial thromboplastin time test were recorded against heparin (193 IU mg-1), without changes in prothrombin time. Diluted plasma treated with F I and F II reduced concentration-dependent and sulfation pattern TG by both intrinsic and extrinsic pathways, with 50% inactivation by intrinsic pathway of F II even at 4.1 µg. Heparin abolished TG at least 4-fold lower. Therefore, C. racemosa produces SPs with TG inhibition.(AU)

Eficácia farmacológica de frações (F I→III) polissacarídicas sulfatadas (PSs) da Chlorophyta Caulerpa racemosa sobre modelos de coagulação e inflamação tem sido demonstrada, exceto seus efeitos sobre geração de trombina (GT). Examinaram-se frações quanto à composição, características físico-químicas e inativação in vitro de GT por F I e F II, em plasma humano diluído 60 vezes usando método contínuo. Rendimento de extração-papaína (0,7%) revelou, por cromatografia de DEAE-celulose, F I→III com diferenças entre as proporções relativas de sulfato (17,37-24,00%), açúcares totais (30,03-48,34%) e ausência de proteínas. Foram verificados, por ambas as análises agarose/poliacrilamida, graus de densidade de carga e tamanhos moleculares > 100 kDa das frações, respectivamente. Também essas eletroforeses, combinadas com azul de toluidina/Stains-All, indicaram polissacarídeos não sulfatados. Foram registrados, pelo teste do tempo de tromboplastina parcial ativada, efeitos anticoagulantes de 4,76 (F I), 12,00 (F II) e 2,32 (F II) UI mg-1 contra heparina (193 UI mg-1), porém não modificando tempo de protrombina. Plasma diluído tratado com F I e F II reduziu GT por ambas as vias intrinsíca/extrínsica, dependente de concentração e grau de sulfatação, com F II em 4,1 μg apresentando eficácia de 50% pela via intrínsica. Heparina, quatro vezes menos, aboliu GT. Portanto, C. racemosa produz PSs com inibição de GT.(AU)

DIAGNÓSTICO MOLECULAR DE Ehrlichia canis E Babesia vogeli, E DIAGNÓSTICO SOROLÓGICO DE Neospora caninum E Toxoplasma gondii EM CÃES PROVENIENTES DO MUNICÍPIO DE CÂNDIDO SALES-BA E MICRORREGIÃO

Cães domésticos (Canis lupus familiaris) estão sujeitos a uma variedade de agentes infecciosos. A toxoplasmose, neosporose, erliquiose monocítica e a babesiose são doenças que causam importantes alterações orgânicas em animais dessa espécie. Potencial zoonótico é também descrito para alguns desses agentes, como o T. gondii e E. canis. O conhecimento do comportamento e da prevalência desses patógenos favorece a adoção de medidas estratégicas eficazes para o controle e prevenção das doenças causadas por eles. Até o momento existem poucos estudos na Bahia sobre a prevalência de infecção por T. gondii, N. caninum, E. canis e B. canis vogeli em cães, principalmente na mesorregião centro-sul do estado, onde não há nenhum estudo. Dessa forma, objetivou-se avaliar a frequência da infecção por esses agentes na região de Cândido Sales BA. Foram incluídos no estudo 131 cães domésticos domiciliados e semi-domiciliados provenientes da zona urbana e rural da região. Inicialmente, foram obtidas informações junto aos tutores sobre o histórico e manejo dos animais, bem como sobre queixas clínicas eventuais a fim de verificar os fatores de risco/queixas clínicas associados às infecções. Em seguinda, foram coletados de cada animal aproximadamente 5ml de sangue por meio de punção venosa cefálica ou jugular, o qual foi dispensado em dois tubos, um com anticoagulante Ácido Etilenodiamino Tetra-Acético (EDTA) e outro com ativador de coágulo. O sangue acondicionado nos tubos com anticoagulante foi destinado à extração de DNA para posterior realização de diagnóstico molecular e os tubos com ativador de coágulo foram submetidos à centrifugação para obtenção do soro, que foi armazenado em microtubos de 2ml a -20ºC para posterior realização de diagnóstico sorológico. A técnica de RIFI foi utilizada para o diagnóstico da infecção por T. gondii e N. caninum, a técnica de nested-PCR foi realizada para detecção do DNA de E. canis e a PCR convencional foi utilizada para verificação da infecção po B. canis vogeli. Adicionalmente foram confeccionados esfregaços sanguíneos de sangue ponta de orelha e de sangue total para realização de dignóstico parasitológico de E. canis e B. canis vogeli. A prevalência de infecção por N. caninum foi de 70,2%, de 67,9% para T. gondii e 37,4% para E. canis. Nenhum cão avaliado foi positivo para B. canis vogeli. A idade (acima 3 anos) foi considerada como fator de risco para as infecções por E. canis (p=0,0079) e N. caninum (p=0,0042). Além disso, o habitat (rural) foi considerado fator de risco para infecção por N. caninum (p=0,0046). As coinfecções foram frequentes, principalmente por N. caninum e T. gondii, que foi diagnosticada em 45,03% dos cães. A ocorrência de infecção por E. canis se mostrou significativa para o favorecimento da infecção por N. caninum (p=0,0014) e T. gondii (p=0,025). Queixas clínicas foram descritas em apenas 27,48% dos animais. Sugere-se que sejam realizados estudos com um maior número de animais para que sejam elucidados os fatores de risco especialmente para infecções por T. gondii, e para confirmar a ausência da infecção por B. vogeli nos cães da região de Cândido Sales-BA.

Domestic dogs (Canis lupus familiaris) are subject to a variety of infectious agents. Toxoplasmosis, neosporosis, monocytic ehrlichiosis and babesiosis are diseases that cause important organic changes in animals of this species. Zoonotic potential is also described for some of these agents, such as T. gondii and E. canis. Knowledge of the behavior and prevalence of these pathogens favors the adoption of effective strategic measures for the control and prevention of diseases caused by them. To date, there are few studies in Bahia on the prevalence of infection by T. gondii, N. caninum, E. canis and B. canis vogeli in dogs, mainly in the central-southern mesoregion of the state, where there is no study. Thus, the objective was to evaluate the frequency of infection by these agents in the region of Cândido Sales - BA. The study included 131 domesticated and semi-domesticated domestic dogs from the urban and rural areas of the region. Initially, information was obtained from the tutors about the history and management of the animals, as well as about eventual clinical complaints in order to verify the risk factors/clinical complaints associated with the infections. Then, approximately 5 ml of blood was collected from each animal by means of cephalic or jugular venipuncture, which was dispensed in two tubes, one with anticoagulant Ethylenediamine Tetra-Acetic Acid (EDTA) and the other with clot activator. The blood stored in the anticoagulant tubes was destined for DNA extraction for subsequent molecular diagnosis and the tubes with clot activator were subjected to centrifugation to obtain the serum, which was stored in 2ml microtubes at -20ºC for further diagnosis serological.The RIFI technique was used for the diagnosis of infection by T. gondii and N. caninum, the nested-PCR technique was performed to detect the DNA of E. canis and the conventional PCR was used to check for infection by B. canis vogeli. Additionally, blood smears of ear tip blood and whole blood were made to carry out parasitological diagnosis of E. canis and B. canis vogeli. Infection with N. caninum infection was 70.2%, 67.9% for T. gondii and 37.4% for E. canis. No dogs evaluated were positive for B. canis vogeli. Age (over 3 years) was considered a risk factor for infections with E. canis (p = 0.0079) and N. caninum (p= 0.0042). In addition, the habitat (rural) was considered a risk factor for infection by N. caninum (p = 0.0046). Co-infections were frequent, mainly by N. caninum and T. gondii, that was diagnosed in 45.03% of the dogs. The presence of infection by E. canis is related to the promotion of infection by N. caninum (p = 0.0014) and T. gondii (p = 0.025). Clinical complaints were described in only 27.48% of the animals. It is suggested that further studies be carried out with a larger number of animals in order to elucidate the risk factors especially for those infected with T. gondii, and to confirm the absence of infection by B. vogeli in dogs in the region of Cândido Sales-BA.

Prevalência e fatores de risco da neosporose bovina na microrregião de Teresina-PI / Prevalence and bovine neosporosis risk factors in the micro region of Teresina - PI

A biggest problem in breeding cattle are fetal and newborn losses in the herd, which cause manyeconomic losses to producers. These losses may be related to the protozoan Neospora caninum, which causesthe disease known as neosporosis. The present study was carried in 14 production units of cattle in themunicipalities near from Teresina. Blood collection was performed by jugular venipuncture, using a 15 mlcollection tubes without anticoagulant. For the identification of anti-N antibodies. caninum used the technique ofindirect immunofluorescence (IIF) for detection of antibodies of the IgG class. The prevalence of infection by theprotozoan parasite for the samples examined was 15,23% (64/420) of seropositive animals and 84,76%(356/420) seronegative, distributed in 100% (14/14) of the herds studied. Also demonstrated in this study, theoccurrence of at least one animal tested positive for N. caninum, in each flock evaluated.(AU)

Prevalência e fatores de risco da toxoplasmose bovina na microrregião de Teresina PI / Prevalence and bovine toxoplasmosis risk factors in the micro region of Teresina - PI

Toxoplasmosis is caused by the protozoan Toxoplasma gondii, a cosmopolitan zoonosis that affectsseveral warm-blooded animal species. The main transmission routes of T. gondii are fecal-oral, carnivorous andcongenital.Congenital transmission, when the parasite reaches the concept via the transplanted route, is oftenmore severe, which transmits tachyzoites to the fetus.. The present study was carried in 13 production units ofcattle in the municipalities near from Teresina. Blood collection was performed by jugular venipuncture, using a15 ml collection tubes without anticoagulant. For the identification of. anti-T. gondii used the technique ofindirect immunofluorescence (IIF) for detection of antibodies of the IgG class. The prevalence of infection by theprotozoan parasite for the samples examined was 17,44% (68/390) of seropositive animals and 82,56%(322/390) seronegative, distributed in 100% (13/13) of the herds studied. Also demonstrated in this study, theoccurrence of at least one animal tested positive for T. gondii, in each flock evaluated.(AU)

Prevalência e fatores de risco da neosporose bovina na microrregião de Teresina-PI / Prevalence and bovine neosporosis risk factors in the micro region of Teresina - PI

A biggest problem in breeding cattle are fetal and newborn losses in the herd, which cause manyeconomic losses to producers. These losses may be related to the protozoan Neospora caninum, which causesthe disease known as neosporosis. The present study was carried in 14 production units of cattle in themunicipalities near from Teresina. Blood collection was performed by jugular venipuncture, using a 15 mlcollection tubes without anticoagulant. For the identification of anti-N antibodies. caninum used the technique ofindirect immunofluorescence (IIF) for detection of antibodies of the IgG class. The prevalence of infection by theprotozoan parasite for the samples examined was 15,23% (64/420) of seropositive animals and 84,76%(356/420) seronegative, distributed in 100% (14/14) of the herds studied. Also demonstrated in this study, theoccurrence of at least one animal tested positive for N. caninum, in each flock evaluated.

Prevalência e fatores de risco da toxoplasmose bovina na microrregião de Teresina – PI / Prevalence and bovine toxoplasmosis risk factors in the micro region of Teresina - PI

Toxoplasmosis is caused by the protozoan Toxoplasma gondii, a cosmopolitan zoonosis that affectsseveral warm-blooded animal species. The main transmission routes of T. gondii are fecal-oral, carnivorous andcongenital.Congenital transmission, when the parasite reaches the concept via the transplanted route, is oftenmore severe, which transmits tachyzoites to the fetus.. The present study was carried in 13 production units ofcattle in the municipalities near from Teresina. Blood collection was performed by jugular venipuncture, using a15 ml collection tubes without anticoagulant. For the identification of. anti-T. gondii used the technique ofindirect immunofluorescence (IIF) for detection of antibodies of the IgG class. The prevalence of infection by theprotozoan parasite for the samples examined was 17,44% (68/390) of seropositive animals and 82,56%(322/390) seronegative, distributed in 100% (13/13) of the herds studied. Also demonstrated in this study, theoccurrence of at least one animal tested positive for T. gondii, in each flock evaluated.

Bioquimico plasmático de cães: efeitos dos diferentes anticoagulantes em comparação com o soro / Plasmatic biochemical of dogs: effects of different anticoagulants compared to serum

Na rotina clínica veterinária são utilizados diferentes tipos de anticoagulantes, escolhidos de acordo com o exame a ser solicitado. Animais agitados, agressivos, de difícil contenção e de pequeno porte podem ser fatores limitantes quando se deseja aliquotar a amostra em tubos com diferentes anticoagulantes. Assim, esse trabalho teve como objetivo avaliar os efeitos dos anticoagulantes EDTA, Citrato de Sódio e Fluoreto associado ao EDTA, nos resultados dos exames bioquímicos de rotina realizados em cães e compará-los com o soro. Verificou-se que o plasma obtido com o anticoagulante EDTA resultou em alterações nas dosagens de glicose, colesterol e cálcio. Amostras obtidas com Citrato de Sódio tiveram resultados inferiores ao do soro em todos os parâmetros avaliados, com exceção da ALT. O anticoagulante Fluoreto associado ao EDTA, apenas nas dosagens de uréia e glicose, não resultou em alterações estatísticas quando comparado ao soro.

Different types of anticoagulants are used in the routine of a veterinary clinic, which are chosen based on the exam to be performed. Agitated, aggressive, of hard contention and small size animals can be a limiting factor when it is wished to separate samples into tubes with different anticoagulants. Therefore, this study had as the objective evaluate the effects of anticoagulants: EDTA, sodium citrate and sodium fluorite associated with EDTA, in the results of routine plasma biochemistry performed in dogs and compare it with serum. Blood samples of 10 clinically healthy dogs were collected and placed in different tubes with each anticoagulant and one without to obtain serum. Albumin, alanine aminotransferase (ALT), calcium, total cholesterol, creatine, phosphorus, glucose, total protein (PT) and urea concentrations were measured by spectophotometric. It was found that plasma obtained with EDTA anticoagulant resulted in dosage alterations for glucose, cholesterol and calcium. Samples obtained with sodium citrate presented results lower than serum in all analyzed parameters, except for ALT. The anticoagulant sodium fluorite with EDTA only didn’t result in statistical differences when compared to serum in the parameters of urea and glucose. Thus, serum is the most trustworthy sample to determine the biochemical profile of dogs, however some biochemical’s can be performed with plasma with EDTA such as creatine, ALT, urea, albumin, PT and phosphorus.

Bioquimico plasmático de cães: efeitos dos diferentes anticoagulantes em comparação com o soro / Plasmatic biochemical of dogs: effects of different anticoagulants compared to serum

Na rotina clínica veterinária são utilizados diferentes tipos de anticoagulantes, escolhidos de acordo com o exame a ser solicitado. Animais agitados, agressivos, de difícil contenção e de pequeno porte podem ser fatores limitantes quando se deseja aliquotar a amostra em tubos com diferentes anticoagulantes. Assim, esse trabalho teve como objetivo avaliar os efeitos dos anticoagulantes EDTA, Citrato de Sódio e Fluoreto associado ao EDTA, nos resultados dos exames bioquímicos de rotina realizados em cães e compará-los com o soro. Verificou-se que o plasma obtido com o anticoagulante EDTA resultou em alterações nas dosagens de glicose, colesterol e cálcio. Amostras obtidas com Citrato de Sódio tiveram resultados inferiores ao do soro em todos os parâmetros avaliados, com exceção da ALT. O anticoagulante Fluoreto associado ao EDTA, apenas nas dosagens de uréia e glicose, não resultou em alterações estatísticas quando comparado ao soro.(AU)

Different types of anticoagulants are used in the routine of a veterinary clinic, which are chosen based on the exam to be performed. Agitated, aggressive, of hard contention and small size animals can be a limiting factor when it is wished to separate samples into tubes with different anticoagulants. Therefore, this study had as the objective evaluate the effects of anticoagulants: EDTA, sodium citrate and sodium fluorite associated with EDTA, in the results of routine plasma biochemistry performed in dogs and compare it with serum. Blood samples of 10 clinically healthy dogs were collected and placed in different tubes with each anticoagulant and one without to obtain serum. Albumin, alanine aminotransferase (ALT), calcium, total cholesterol, creatine, phosphorus, glucose, total protein (PT) and urea concentrations were measured by spectophotometric. It was found that plasma obtained with EDTA anticoagulant resulted in dosage alterations for glucose, cholesterol and calcium. Samples obtained with sodium citrate presented results lower than serum in all analyzed parameters, except for ALT. The anticoagulant sodium fluorite with EDTA only didnt result in statistical differences when compared to serum in the parameters of urea and glucose. Thus, serum is the most trustworthy sample to determine the biochemical profile of dogs, however some biochemicals can be performed with plasma with EDTA such as creatine, ALT, urea, albumin, PT and phosphorus.(AU)

Perfil hematológico e bioquímico de bubalinos (Bubalus bubalis) da raça Murrah criados em clima tropical

OLIVEIRA, L. F. Perfil hematológico e bioquímico de bubalinos (Bubalus bubalis) da raça Murrah, criados em clima tropical. Salvador. 2020. 109p. Dissertação (Mestre em Ciência Animal nos Trópicos) Escola de Medicina Veterinária e Zootecnia Universidade Federal da Bahia. Este trabalho teve como objetivo determinar valores dos componentes sanguíneos de bubalinos sadios criados em condições de clima tropical, avaliando-se o efeito do desenvolvimento etário e sexo. Foram avaliadas amostras de 135 bubalinos da raça Murrah com aptidão leiteira, de três propriedades localizadas no Recôncavo baiano, distribuídos em nove grupos etários: G1 (0 a 15 dias), G2 (acima de 15 a 30 dias), G3 (acima de 30 até 60 dias), G4 (acima de 60 até 120 dias), G5 (acima de 120 até 180 dias), G6 (acima de 7 meses até 12 meses), G7 (acima de 12 meses até 36 meses), G8 (acima de 36 meses até 60 meses) e G9 (acima de 60 meses), cada um com 15 animais. Do grupo G1 ao G6 foram incluídos machos e fêmeas e do G7 ao G9 apenas fêmeas. Para avaliação do fator sexo, dois subgrupos foram formados com 39 animais machos e 39 fêmeas até um ano de idade. Foram coletados amostras de sangue com anticoagulante para realização do hemograma (eritrograma e leucograma) e amostras sem anticoagulante para análises bioquímicas (proteínas totais, albumina, globulina, relação A:G, glicose, colesterol, triglicérides, AST, GGT, cálcio, fósforo, magnésio e potássio). Os dados foram submetidos à avaliação estatística inicialmente realizando-se a análise de variância e a seguir para comparação das médias entre os grupos foi aplicado Teste de Tukey com nível de significância de 5%. A idade mostrou influência com todas os componentes do hemograma, com exceção do valor absoluto de monócitos, já para os componentes bioquímicos a maioria demonstrou influência com a idade, exceto fósforo e potássio. O sexo influenciou a contagem total de leucócitos, valor absoluto de monócitos, glicose, colesterol e AST.

OLIVEIRA, L. F. Hematological and biochemical profile of healthy Murrah buffalo (Bubalus bubalis) reared in tropical climate. Salvador. 2020. 109p.Dissertation (Mestre em Ciência Animal nos Trópicos) Escola de Medicina Veterinária e Zootecnia Universidade Federal da Bahia. The objective of this study was to determine blood component values of healthy buffaloes raised under tropical climate conditions, assessing the effect of age and sex development. Samples of 135 dairy-quality Murrah buffaloes from three farms located at Recôncavo Bahia were evaluated and distributed in nine age groups: G1 (0 to 15 days), G2 (over 15 to 30 days), G3 (over 30 up to 60 days), G4 (over 60 up to 120 days), G5 (over 120 up to 180 days), G6 (over 7 months up to 12 months), G7 (over 12 months up to 36 months), G8 (up 36 months to 60 months) and G9 (over 60 months), each with 15 animals. Group G1 to G6 included males and females and from G7 to G9 only females. For sex factor evaluation, two groups were formed with 39 male and 39 female animals up to one year old. Blood samples with anticoagulant were collected for blood count (erythrogram and leukogram) and samples without anticoagulant for biochemical analyzes (total proteins, albumin, globulin, A:G ratio, glucose, cholesterol, triglycerides, AST, GGT, calcium, phosphorus, magnesium and potassium). Data were subjected to statistical evaluation initially by performing the analysis of variance and then to compare the means between groups, Tukey test with significance level of 5% was applied. Age showed influence with all blood count components, except for the absolute value of monocytes, while most biochemical components showed influence with age, except phosphorus and potassium. Gender influenced total white blood cell count, absolute monocyte value, glucose, cholesterol and AST.

PREVALÊNCIA E FATORES DE RISCO ASSOCIADOS À INFECÇÃO PELO VÍRUS DA ESTOMATITE VESICULAR EM EQUÍDEOS NO ESTADO DO RIO GRANDE DO NORTE, BRASIL

A estomatite vesicular (EV) é uma enfermidade zoonótica viral, que acomete ruminantes, equídeos, porcinos, algumas espécies silvestres e cursa com sintomatologia clínica semelhante as provocadas pelo vírus da febre aftosa. O vírus da estomatite vesicular (VEV) apresenta dois sorotipos New Jersey e Indiana sendo este último classificado em três subtipos Indiana 1, 2 e 3. Objetivou-se investigar a presença de anticorpos do VEV e potenciais fatores de risco envolvidos com a infecção pelo vírus em equídeos das mesorregiões Leste e Oeste Potiguar do estado do Rio Grande do Norte, Brasil. Foram analisadas amostras sanguíneas de 809 animais provenientes de noventa propriedades distribuídas em dezesseis municípios do estado durante os meses de julho de 2018 a fevereiro de 2019. As amostras foram obtidas por venopunção da jugular com sistema de colheita a vácuo, em tubos do tipo Vacutainer® sem anticoagulante e posteriormente centrifugadas a 3.000 rpm por 10 minutos para obtenção de soro, que foi armazenado em tubos do tipo Eppendorf® a -20ºC até o processamento para diagnóstico por soroneutralização. Os fatores de riscos foram avaliados por meio de questionário epidemiológico investigativo. Os dados foram submetidos a análise estatística no programa EpiInfo 3.5.2 com nível de confiança de 95%. A prevalência de anticorpos anti-VEV foi 24,6% (199/809), sendo 3,2% (13/402) soropositivos na mesorregião Leste e 45,7% (186/407) na região Oeste. Considerando os sorotipos observou-se uma prevalência de 3,8% (31/809) para Indiana 2, 24,5% (198/809) para Indiana 3 e 15,1% (30/198) de coinfecção para ambos. Todas as variáveis estatisticamente significantes foram submetidas a uma análise de regressão logística multivariada e concluiu-se que animais criados em áreas rurais da mesorregião Oeste, em sistemas extensivo e semi-intensivo, cujo pasto é alagado, não realiza quarentena, não desinfeta as instalações, e onde os animais enfermos são mantidos no rebanho, foram consideradas fatores de risco para infecção pelo VEV. Esses resultados demonstram que o VEV está distribuído pelo estado do Rio Grande do Norte e medidas de controle devem ser adotadas para evitar a disseminação do mesmo.

Vesicular stomatitis (VS) is a viral zoonotic disease that affects ruminants, equines, porcinis, some wild species and has clinical symptoms similar to those caused by the FMD virus. The vesicular stomatitis virus (VSV) has two serotypes New Jersey and Indiana, the latter being classified into three subtypes Indiana 1, Indiana 2 and Indiana 3. The objective of this study was to investigate the presence of VSV and its potential risk factors for the occurrence and dissemination of disease in Equidae from the eastern and Western mesoregions of the state of Rio Grande do Norte, Brazil. Blood samples were analyzed from 809 animals from 90 properties distributed in sixteen municipalities in the state from July 2018 to February 2019. The samples were obtained by venipuncture of the jugular with vacuum collection system, in tubes of the type Vacutainer® without anticoagulant. Packaged under refrigeration in a thermal box and transported to the Ruminant Laboratory of the Federal Rural University of Pernambuco for centrifugation at 3,000 rpm for 10 minutes and obtaining serum aliquots, which were stored in Eppendorf® tubes at -20ºC until diagnostic processing. by seroneutralization. Risk factors were assessed using an investigative epidemiological questionnaire. The data were submitted to statistical analysis in the EpiInfo 3.5.2 program with a 95% confidence level. The occurrence of anti-VSV antibodies was 24.6% (199/809), with 3.2% (13/402) seropositive in the eastern mesoregion and 45.7% (186/407) in the western region. Considering the serotypes, there was a prevalence of 3.8% (31/809) for Indiana 2 and 24.5% (198/809) for Indiana 3. All statistically significant variables were subjected to multivariate logistic regression analysis and it was concluded that animals raised in rural areas of the western mesoregion, in extensive and semi-intensive systems, whose pasture is flooded, does not quarantine, does not disinfect the facilities, are raised in a communal way with small ruminants and pigs, and where the animals sick are kept in the herd, risk factors for infection by VSV-IN subtypes 1 and 2 were considered. These results demonstrate that VSV is distributed across the state of Rio Grande do Norte and control measures must be adopted to prevent its spread.

RELAÇÃO ENTRE TEMPERATURA RETAL E VOLUME GLOBULAR MÉDIO DE BOVINOS GIROLANDO, EM ANIMAIS NATURALMENTE INFECTADOS COM AGENTES DA TRISTEZA PARASITÁRIA BOVINA

A tristeza parasitária bovina (TPB) é uma doença causada pelos agentes Babesia bigemina, B. bovis e Anaplasma marginale, que são parasitos intraeritrocitários e têm como vetor biológico o carrapato Rhipicephalus microplus. Causam em seu hospedeiro anemia hemolítica, febre, icterícia, hemoglobinúria e anorexia, podendo levar à morte. Acometem animais na fase pós desmama, principalmente em granjas leiteiras e bovinos de raças europeias são menos resistentes à doença em relação aos zebuínos. Este trabalho foi dividido em dois experimentos, o primeiro objetivou analisar a correlação entre os valores de temperatura retal (TR) e volume globular médio (VGM) de bovinos girolando 7/8 e 15/16 naturalmente infectados pela TPB. Foram avaliados TR, VGM e parasitemia de 533 bezerras girolando de uma fazenda no estado de Goiás e realizou-se curva ROC para as variáveis VGM e TR e o critério de associação de risco para parasitemia, TR e VGM. Animais com TR 39,3°C possuíam VGM de 18,6% e riquetsemia média de 7,14%, porém quando a variável avaliada era VGM 24%, a TR média era de 39,1°C e riquetsemia de 5,04%. Concluiu-se que a anaplasmose promove queda no VGM e aumento da TR de acordo com a infecção em bezerras girolando, sendo mais acentuada em 15/16. A queda de VGM demonstrou maior sensibilidade e especificidade por apontar a anaplasmose mais precocemente, se comparado à TR, em ambos graus sanguíneos. Não foi possível inferir sobre babesiose devido à baixa ocorrência destes protozoários. O segundo experimento objetivou validar um equipamento portátil humano de mensuração de hemoglobina (Hb) e VGM para utilização em bovinos. Foram mensurados valores obtidos pelo aparelho (MP) utilizando sangue coletado com e sem EDTA de 136 bovinos em repetições de dez e de três, os resultados obtidos foram comparados com os do padrão ouro (GS). Os valores de Hb (g/dL) obtidos pelo método GS, MP sem EDTA e MP com EDTA foram 8,38±0,19, 8,42±0,21 e 7,40±0,16, respectivamente. Enquanto os valores de VGM (%) foram de 25,64±0.23, 24,78±0,61 e 21,77±0,50, respectivamente. Foi detectada correlação positiva entre os valores de Hb (P<0,0001) e VGM (P<0,0001) com as técnicas do equipamento MP e GS usando sangue com e sem EDTA. Entretanto, os valores de R² encontrados na correlação positiva para Hb (0,4882 ou 0,5053) e VGM (0,7550 ou 0,7545) foram considerados baixo/moderado e alto, respectivamente. Uma correção do fator de multiplicação foi estimada para melhorar a acurácia do equipamento MP quando o sangue bovino com EDTA é usado para medir Hb (x 1,133) e VGM (x 1,178). O equipamento MP pode ser usado para analisar principalmente o VGM do sangue bovino para auxiliar no diagnóstico à campo de hemoparasitoses, visto que o aparelho demonstrou alta correlação, precisão e acurácia similares ao método de padrão ouro.

Bovine sadness (TPB) is a disease caused by the agents Babesia bigemina, B. bovis and Anaplasma marginale, which are intra-erythrocyte parasites and have the tick Rhipicephalus microplus as their biological vector. These parasites cause in their hostos hemolytic anemia, fever, jaundice, hemoglobinuria and anorexia, which can lead to death. They affect mainly animals in the post-weaning phase in dairy farms and cattle of European breeds are less resistant to the disease compared to zebu cattle. This work was divided into two experiments, the first one aimed to analyze the correlation between the values of rectal temperature (RT) and packed cell volume (PCV) of 7/8 and 15/16 gyrolando cattle naturally infected with TPB. RT, PCV and parasitemia of 533 gyrolando calves from a farm in the state of Goiás were evaluated and a ROC curve was performed for the variables PCV and TR and the risk association criteria for parasitemia, RT and PCV. Animals with RT 39.3°C presentend 18.6% of PCV and an average rickettsemia of 7.14%, but when the variable evaluated was VGM 24%, the average RT was 39.1 ° C and rickettsemia of 5.04%. It was concluded that anaplasmosis causes a decrease in the PCV and an increase in RT according to the infection in gyrolando calves, being more accentuated in 15/16. The drop in VGM demonstrated greater sensitivity and specificity for pointing out anaplasmosis earlier, when compared to RT, in both blood grades. It was not possible to infer babesiosis due to its low occurrence. The second experiment aimed to validate a portable human aparatus to measure hemoglobin (Hb) and PCV for use in cattle, comparing the results with gold standard (GS). Bovine blood (n=136) were collected in addition or not of anticoagulant (EDTA) to perform the tests. Hb values (g/dL) obtained by gold GS method, MP without EDTA and MP with EDTA were 8.38±0.19, 8.42±0.21 and 7.40±0.16, respectively. While PCV values (%) were 25.64±0.23, 24.78±0.61 and 21.77±0.50, respectively. A positive correlation between Hb (P<0.0001) and PCV (P<0.0001) values detected by the MP equipment and the GS techniques using blood with and without addition of EDTA was observed. However, the values of R2 founded in the positive correlation for Hb (0.4882 or 0.5053) and PCV (0.7550 or 0.7545) were considered low/moderate and high, respectively. A multiplication correction factor was estimated to improve the accuracy of the MP equipment when cattle blood with EDTA is used for measure Hb (x 1.133) and PCV (x 1.178). The MP equipment can be used to analyze mainly PCV in bovine blood to assist in hemoparasitosis diagnosis in the field since the apparatus showed high correlation, precision and accuracy similar of the gold standard methods.

EFEITO DA ADMINISTRAÇÃO DE ANTICOAGULANTES SOBRE A RECUPERAÇÃO EMBRIONÁRIA DE ÉGUAS SUPEROVULADAS

A superovulação é uma biotécnica utilizada nos programas de transferência de embrião e proporciona diversos benefícios devido ao maior número de ovulações por ciclo. Entretanto, os protocolos superovulatórios na espécie equina ainda não são utilizados rotineiramente, pois apresentam resultados inconsistentes relacionados a baixa taxa de recuperação embrionária frente ao número de ovulações, estando muito aquém do esperado quando comparado a espécie bovina. Atualmente, sabe-se que uma das possíveis causas responsáveis por este fato pode estar relacionada pela formação de grandes coágulos de sangue obstruindo a fossa da ovulação, dificultando assim a captação do oócito para o interior da tuba uterina. Desta forma, o objetivo desse estudo foi avaliar o efeito da utilização de dois tipos de anticoagulantes parenterais (heparina não fracionada - HNF e heparina de baixo peso molecular - HBPM) em éguas superovuladas com extrato de pituitária equina (EPE), sobre a posterior taxa de recuperação embrionária. Foram utilizados quatro ciclos estrais de 11 éguas, sendo subdivididos em quatro grupos: Grupo 1 (G1) utilizado como controle; Grupo 2 (G2) receberam 25 mg de EPE intramuscular (IM) a cada 12 horas sendo a primeira e a terceira administração associada a 5 mg de dinoprost-trometamina (IM) e solução fisiológica 0,9% única aplicação 35 horas após indução da ovulação; Grupo 3 (G3) foi utilizado mesmo protocolo superovulatório, sendo substituído a solução fisiológica por um dos anticoagulantes, Heparina Sódica 450 UI/Kg/IV, e Grupo 4 (G4) onde as éguas foram superovuladas pelo mesmo protocolo mencionada acima e receberam 1 mg/Kg/IV de Enoxaparina Sódica. Os animais foram aleatoriamente designados aos quatros grupos em cross-over design, nos quais todos foram submetidos a todos os tratamentos. O intervalo de um ciclo estral para cada égua foi utilizado entre os tratamentos. Os tratamentos foram iniciados no sétimo dia pós-ovulação. A duração do tratamento com EPE não excedeu oito dias, sendo interrompido quando a maioria dos folículos atingissem diâmetro adequado para indução da ovulação. A indução da ovulação foi realizada utilizando 1 ml (250µg) de GnRH (Histrelina Acetato) intramuscular e 2500 UI de hCG (Gonadotrofina Coriônica Humana) intravenoso quando o(s) folículo(s) atingisse um diâmetro médio entre eles de 35 mm em ambos os grupos. A partir da indução da ovulação, 24 horas após as éguas foram inseminadas com sêmen a fresco de um garanhão de fertilidade comprovada. A colheita de embriões foi realizada no nono dia após a primeira ovulação detectada. Para cada animal submetido ao tratamento com anticoagulante foram realizadas avaliações laboratoriais (hemograma e coagulograma). Para isso foram padronizados os seguintes momentos de avaliação; para hemograma as amostras de sangue foram coletadas nos momentos prévio a administração do anticoagulante (T0 hora) servindo como controle, e três horas após a aplicação do anticoagulante (T3 horas), já para o coagulograma as amostras foram coletadas nos momentos T0, T1, T2, T3, T6, T9 e T12 horas após aplicação do anticoagulante. Os resultados demostraram maior (p<0,05) número de ovulações nos grupos de éguas superovuladas (G2 4,6±0,4; G3 5,2±0,5; G4 4,9±0,6) em relação ao G1 (1±0). Já o número de embriões recuperados por égua não diferiu (p>0,05) entre o G1 (0,6±0,2) e G2 (1,6±0,5), no entanto, foi maior para os grupos que receberam anticoagulantes G3 (2,5±0,3) e G4 (2,6±0,6) em relação ao G1. Quando a taxa de recuperação embrionária por folículo ovulado foi avaliado, os resultados demostraram que os grupos tratados com anticoagulantes apresentaram taxas similares (G3 50%; G4 52%) em comparação ao grupo controle (G1 64%), já o grupo superovulado controle (G2 36%) apresentou tendência de redução (P = 0,8) em relação ao G1. Em relação a avaliação laboratorial, os valores hematológicos dos animais em ambos os grupos que receberam anticoagulante (G3 e G4) no momento T3 horas não diferiu (p>0,05) em relação ao momento T0 hora, mantendo-se dentro dos valores de referência. Quanto ao coagulograma, para o G3 os valores de tempo de tromboplastina parcial ativado (TTPA) a partir de uma hora após aplicação (T1 hora) apresentou aumento significativo se manteve até doze horas após aplicação (T12 horas), onde todos os momentos diferiram-se (p<0,05) em relação ao momento (T0 hora), já para o tempo de protrombina (TP) houve um aumento (p<0,05) após aplicação do anticoagulante (T1-T2 horas) em relação ao tempo de avaliação pré aplicação (T0 hora), reduzindo-se gradativamente a partir de três horas até doze horas após aplicação (T3-T12 horas), não diferindo (p>0,05) em relação do T0 hora. Para o G4, uma hora após a aplicação (T1 hora) houve um aumento (p<0,05) da atividade plasmática fator anti-Xa, apresentando um constante declínio até doze horas (T12), não apresentando diferença (p>0,05) apenas neste momento em relação ao T9 horas e T0 hora. Desta forma, pode-se concluir que o protocolo proposto no presente estudo é uma alternativa para melhoria das taxas de recuperação embrionária em éguas superovuladas com EPE, mostrando-se seguro para o uso de ambos os tipos de anticoagulantes sem apresentar alterações hematológicas ou efeitos adversos que possam comprometer ou colocar em risco a condição clínica do animal.

Superovulation is a biotechnic from embryo transfer programs providing several benefits due to the higher number of ovulations per cycle. However, superovulation protocols in the equine species are not yet routinely used, as they present inconsistent results related to the low embryonic recovery rate concerning the number of ovulations, being far below expectations when compared to the bovine species. Currently, one of the possible causes responsible for this fact may be related to the development of large blood clots obstructing the ovulation fossa, making it difficult to capture the oocyte into the fallopian tube. Therefore, this study aimed to evaluate the effect of two types of parenteral anticoagulants (unfractionated heparin - HNF and low molecular weight heparin - LMWH) on equine pituitary extract (EPE) superovulated mares of embryonic recovery. Four estrous cycles of 11 mares were used and subdivided into four groups: Group 1 (G1) as a control; Group 2 (G2) received 25 mg intramuscular EPE (MI) every 12 hours being the first and third administration associated with 5 mg dinoprost-tromethamine (IM) and 0.9% saline only application 35 hours after ovulation induction; Group 3 (G3) in the same superovulation protocol, replacing the physiological solution with one of the anticoagulants, Sodium Heparin 450 IU / kg / IV, and Group 4 (G4) where the mares were superovulated by the same protocol mentioned above and received 1 mg. / Kg / IV of Enoxaparin Sodium. All animals were randomly assigned to the four cross-over design groups, in which all underwent all treatments. The interval of one estrous cycle for each mare was used between treatments. Treatments started on the seventh day after ovulation. EPE treatment did not exceed eight days being interrupted when most follicles reached the appropriate diameter for ovulation induction. Ovulation induction was performed using 1 ml (250µg) of intramuscular GnRH (Histrelin Acetate) and 2500 IU of intravenous hCG (Human Chorionic Gonadotropin) when the follicle (s) reached a mean diameter of 35 mm in both groups. Twenty-four hours afterward ovulation induction the mares were inseminated with fresh semen from a proven fertility stallion. Embryo collection was performed on the ninth day after the first ovulation detected. Each animal at anticoagulant treatment was submitted to laboratory evaluations (blood count and coagulogram). For blood count blood samples were collected before anticoagulant administration (T0 hour) serving as a control, and three hours after applying anticoagulant (T3 hours), while for the coagulogram the samples were collected at T0, T1, T2, T3, T6, T9 and T12 hours after anticoagulant application. Groups of superovulated mares showed higher (p <0.05) number of ovulations (G2 - 4.6 ± 0.4; G3 - 5.2 ± 0.5; G4 - 4.9 ± 0.6) in relation to G1 (1 ± 0). The number of embryos recovered by mare did not differ (p> 0.05) between G1 (0.6 ± 0.2) and G2 (1.6 ± 0.5), however, it was higher for the groups that received anticoagulants G3 (2.5 ± 0.3) and G4 (2.6 ± 0.6) concerning G1. Evaluating the embryo recovery rate by ovulated follicle, anticoagulant-treated groups had similar rates (G3 - 50%; G4 - 52%) compared to the control group (G1 - 64%). Superovulated control (G2 - 36%) showed a tendency to decrease (P = 0.8) concerning G1. Hematological values in both groups receiving anticoagulant (G3 and G4) at T3 hours did not differ (p> 0.05) from T0 hour, remaining the reference values. As for the coagulogram, for G3 the activated partial thromboplastin time (TTPA) values from one hour after application (T1 hour) showed a significant increase until twelve hours after application (T12 hours), where all moments differed (p <0.05) to (T0 hour). Prothrombin time (PT) had an increase (p <0.05) after anticoagulant application (T1-T2 hours) about pre-application evaluation (T0 hour), gradually decreasing from three hours to twelve hours after application (T3-T12 hours), not differing (p> 0.05) from T0 hour. For G4, one hour after application (T1 hour) there was an increase (p <0.05) of anti-Xa factor plasma activity, showing a constant decline up to twelve hours (T12), with no difference (p> 0, 05) only at this moment in relation to T9 hours and T0 hour. The protocol proposed in this study is an alternative for improving embryo recovery rates in EPE superovulated mares, proving safe use of both types of anticoagulants without presenting hematological alterations or adverse effects compromising or endangering the clinical condition of the animal.

Alterations of primary hemostasis in azotemic dogs / Alterações da hemostasia primária em cães azotêmicos

Background: Uremia is a cause of acquired platelet dysfunction. Normal platelet counts and normal coagulation tests, associated with prolonged bleeding time, suggest a qualitative platelet defect. The objective of this study was to verify the presence of abnormalities in primary hemostasis associated with azotemia in dogs, and the correlation of the buccal mucosal bleeding time (BMBT) with urea and creatinine levels. Materials, Methods & Results: Forty azotemic dogs were evaluated for hemostasis. Dogs were included in the study if serum urea concentration was > 60 mg / dL and serum creatinine concentration was > 2.0 mg / dL. The BMBT was performed on the inner surface of the upper lip, using a commercial device. Blood was collected from de jugular vein in EDTA-K2, sodium citrate 3.2% tubes and tubes without anticoagulant. EDTA samples were used to perform red blood cell (RBC) and platelets count. Serum was used for determination of urea, creatinine, alanine aminotransferase (ALT), alkaline phosfatase (ALP), albumin and total protein. Citrated plasma was used to determine activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen levels, von Willebrand factor antigen (vWF:Ag) and fibrin degradation products (FDPs). Statistical analysis used Mann-Whitney test, Kruskal-Wallis test and/or Spearman correlation coefficient. A multiple linear regression

A uremia é causa de disfunção plaquetária adquirida em pessoas e animais. Dentre os sinais clínicos encontrados em cães com síndrome urêmica, o sangramento pode estar presente e manifestar-se sob a forma de petéquias, equimoses, sangramento gastrintestinal, gengival e dos locais de venopunção. Sangramentos associados a quantidade normal de plaquetas e concentração de fatores da coagulação normais sugerem um defeito qualitativo plaquetário. O prolongamento no tempo de sangramento da mucosa oral (TSMO) em quatro de oito cães com insufi ciência renal induzida foi um achado consistente com o defeito de adesão plaquetária. [...]

Platelet rich plasma intralesional injection as bedside therapy for tendinitis in athletic horse

Background: Tendinitis result in a considerable economic loss to the equine industry due to decreased performance, prolonged rehabilitation, recurrent injuries and early retirement. It is well known that damaged tendons have a low healing potential and old therapies often lead to mechanical side effect of a scar tissue with reduced functionality and high risks of re-injury. Regenerative therapies that induce the restoration of the tendon's normal structure and function, rather than formation of less functional scar tissue, decrease the risk of re-injury tissue repair. In particular, platelet rich plasma (PRP) therapy reproduce those processes of development in which there are spatial and temporal interactions between scaffold, growth factors and cell populations that lead to a normal structure and function of neo-formed tendon tissue. According to this knowledge, the aim of this study was to assess autologous PRP therapy as bedside procedure to treat equine tendon injuries. Materials, Methods & Results: Following a clinical and ultrasound examination, fifteen Thoroughbred horses affected by tendinitis of the superficial or deep digital flexor tendons were treated with intralesional injection of autologous PRP on-field preparation. On each horse blood samples were aseptically collected from the jugular vein into sterile tubes. Two sequential centrifugations of whole blood, added with Anticoagulant Citrate Dextrose Solution "A", were performed at 400 g for 10 min and at 620 g for 12 min, respectively, to obtain the PRP. After the PRP injection, horses were subjected to a standardized rehabilitation program that lasted about 6 months and an ultrasound checkup on the 50th day after the administration of PRP showed echographic aspect of the treated tendons comparable to healthy tendons. At the time of ultrasound checkup, all horses showed a marked clinical improvement. A month after completing the rehabilitation program the horses return to train and compete in 1200 m gallop competitions, on average once a month. In no case re-injury occurred within 12 months from the beginning of the therapy. Discussion: PRP treatment improves the regeneration of tissues with a low healing potential like tendons through increased levels of several growth factors including transforming growth factor-b (TGF-b), platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), that are released after platelet degranulation in the damage site and enhance tissue regeneration by stimulating cell proliferation. The effects of PRP therapy in equine tendinitis include also the enhanced gene expression of tendon matrix molecules, including cartilage oligomeric matrix protein (COMP), and the increased collagen type I/collagen type III ratio, with no concomitant increase in the catabolic molecule matrix metalloproteinase. There are many methods for sampling and preparing PRP: test tubes, quadruple blood bags, apheresis, manual preparation or automatic preparation systems; but most of these methods require expensive and sophisticated technical equipment. Since treated horses showed neither local nor systemic side effect after the PRP administration, our study suggests that maintaining aseptic conditions PRP treatment is a minimally invasive and low cost therapy that is quite easy and safe to realize in the field to treat equine tendinitis. After a year follow-up all horses treated with the PRP were either performing at their previous workload and they were back in racing. None of them showed signs of re-injury.