Testicular alterations and semen quality in a selected group of breeding buffaloes.

Testicular ultrasound enables the evaluation of changes in the testicular parenchyma. This study aimed to report the occurrence of hypoechogenic testicular alterations and their relationship with semen quality in five breeding buffaloes. Two buffaloes presented with hyperechoic points characteristic of fibrosis and anechoic density content between the parietal and visceral tunica. The two bulls without ultrasonographic changes showed higher average trajectory speed, linear velocity, curvilinear velocity, amplitude of lateral displacement of the spermatic head, total motility, progressive motility, fast speed, and acrosomal membrane values within the normal range. The number of spermatozoa with major and total defects was higher in the group of animals without alterations. The three buffaloes that presented with testicular alterations produced semen within established freezing standards.

Key points for the thermal comfort of water buffaloes in Eastern Amazon / Pontos-chave do conforto térmico de bubalinos na Amazônia Oriental

This study presented relevant aspects about the Amazonian environment and how it impacts the thermal comfort of domestic buffaloes (Bubalus bubalis) raised in the Eastern Amazon. Furthermore, strategies for monitoring and mitigating animal heat stress are presented, based on research results with the species. Although domestic buffaloes are considered adaptable animals, exposure to intense solar radiation causes thermal discomfort. This condition is expressed in biophysical indicators, in metabolic, endocrine, behavioral responses, and in body thermographic patterns. Therefore, the biometeorological monitoring of production is crucial to support decision-making regarding environmental management strategies, genetic selection of thermotolerant individuals, and increase in animal welfare. Lastly, the use of silvopastoral systems can help to provide higher thermal comfort, which is a condition that directly impacts the productivity of milk and meat buffaloes when they are raised in tropical regions, such as in the Eastern Amazon.

Objetivou-se apresentar aspectos relevantes sobre o ambiente amazônico e como este impacta no conforto térmico de búfalos domésticos (Bubalus bubalis) criados na Amazônia Oriental. Adicionalmente, são apresentadas estratégias para monitoramento e mitigação do estresse térmico animal, a partir de resultados de pesquisa com a espécie. Apesar dos búfalos domésticos serem considerados animais adaptáveis, a exposição à intensa radiação solar provoca desconforto térmico. Essa condição é expressa em indicadores biofísicos, nas respostas metabólicas, endócrinas, comportamentais e nos padrões termográficos corporais. Assim, o monitoramento biometeorológico da produção é crucial para subsidiar tomadas de decisão em relação a estratégias de manejo ambiental, seleção genética de indivíduos termotolerantes e incremento do bem-estar animal. Por fim, o uso de sistemas silvipastoris pode auxiliar na oferta de maior conforto térmico, que é uma condição que impacta diretamente na produtividade de bubalinos de leite e carne, quando estes são criados em regiões tropicais, como na Amazônia Oriental.

Key points for the thermal comfort of water buffaloes in Eastern Amazon / Pontos-chave do conforto térmico de bubalinos na Amazônia Oriental

ABSTRACT: This study presented relevant aspects about the Amazonian environment and how it impacts the thermal comfort of domestic buffaloes (Bubalus bubalis) raised in the Eastern Amazon. Furthermore, strategies for monitoring and mitigating animal heat stress are presented, based on research results with the species. Although domestic buffaloes are considered adaptable animals, exposure to intense solar radiation causes thermal discomfort. This condition is expressed in biophysical indicators, in metabolic, endocrine, behavioral responses, and in body thermographic patterns. Therefore, the biometeorological monitoring of production is crucial to support decision-making regarding environmental management strategies, genetic selection of thermotolerant individuals, and increase in animal welfare. Lastly, the use of silvopastoral systems can help to provide higher thermal comfort, which is a condition that directly impacts the productivity of milk and meat buffaloes when they are raised in tropical regions, such as in the Eastern Amazon.

RESUMO: Objetivou-se apresentar aspectos relevantes sobre o ambiente amazônico e como este impacta no conforto térmico de búfalos domésticos (Bubalus bubalis) criados na Amazônia Oriental. Adicionalmente, são apresentadas estratégias para monitoramento e mitigação do estresse térmico animal, a partir de resultados de pesquisa com a espécie. Apesar dos búfalos domésticos serem considerados animais adaptáveis, a exposição à intensa radiação solar provoca desconforto térmico. Essa condição é expressa em indicadores biofísicos, nas respostas metabólicas, endócrinas, comportamentais e nos padrões termográficos corporais. Assim, o monitoramento biometeorológico da produção é crucial para subsidiar tomadas de decisão em relação a estratégias de manejo ambiental, seleção genética de indivíduos termotolerantes e incremento do bem-estar animal. Por fim, o uso de sistemas silvipastoris pode auxiliar na oferta de maior conforto térmico, que é uma condição que impacta diretamente na produtividade de bubalinos de leite e carne, quando estes são criados em regiões tropicais, como na Amazônia Oriental.

Increased quality of in natura and cryopreserved semen of water buffaloes supplemented with saturated and unsaturated fatty acids from the palm oil industry

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P 0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P 0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P 0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P 0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.(AU)

Increased quality of in natura and cryopreserved semen of water buffaloes supplemented with saturated and unsaturated fatty acids from the palm oil industry

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P 0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P 0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P 0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P 0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.

Comparative evaluation between the extenders TES-TRIS and ACP-112® and the association of Sálva Marajó oil (Lippia origanoides) in the quality of cryopreserved buffalo sperm / Avaliação comparativa entre os diluidores TES-TRIS e ACP-112® e a associação com o óleo da Sálva do Marajó (Lippia origanoides) na qualidade do sêmen bubalino criopreservado

For artificial insemination, it is essential to use frozen semen, however the freezing process causes deleterious changes to the structure and integrity of sperm membranes that compromise the function of sperm. To avoid this cellular damage, extenders and suitable substrates must be used to recover the highest possible number of viable cells post-thaw. To this end, in the first experiment, we evaluated three different extenders: TES-TRIS, which is widely used for buffaloes; and an extender composed of powdered coconut water-based (ACP-112®) with or without milk (ACP-112®-milk) for buffalo semen freezing. In the second experiment, we evaluated the effect of Lippia origanoides oil extract on protecting buffalo sperm against cryoinjury arising from freezing semen. Semen was collected from ten buffalo bulls (10 ejaculates/bull) and diluted in TES-TRIS (control), ACP-112® or ACP-112®-Milk in the first experiment. In the second experiment, the samples were diluted in the diluent with the best results for sperm quality obtained in experiment I, and 2.5 μg mL-1, 5 μg mL-1 or 10 μg mL-1 of the plant extract was added to treatments; and a control group containing only the diluent was also included. The fresh semen was analyzed for conventional features such as motility, concentration, morphology and viability. After thawing, the samples were evaluated again for motility, vigor and supra-vital staining, and then, were performed the of thermal-resistance test, hypoosmotic test and evaluated sperm membrane integrity with the fluorescent probes PI, FITC-PSA and JC-1 using flow cytometry. The data were submitted to ANOVA, and the results were compared by Tukeys test at a significance of 5%.(AU)

Para a implantação da inseminação artificial é indispensável à utilização de sêmen congelado, que pode provocar mudanças deletérias na estrutura e na integridade das membranas espermáticas, comprometendo sua função. Para evitar estes danos celulares, há a necessidade de se utilizar meios diluidores e substratos adequados que recuperem o maior número possível de células viáveis pós-descongelação. Para isso, foram avaliados, no experimento I, três diferentes diluidores, o diluidor TES-TRIS, bastante utilizado para bubalinos, e um diluidor a base de água de coco em pó (ACP-112), associado ou não ao leite (ACP-112-Leite), na congelação do sêmen de bubalinos; e no experimento II, foi avaliado o efeito do óleo extraído da Lippia origanoides na proteção dos espermatozóides contra as crioinjúrias decorrentes da congelação do sêmen bubalino. Foram utilizados 10 touros bubalinos para as colheitas de sêmen (10 ejaculados/touro), sendo os ejaculados diluídos em TES-TRIS (controle), ACP-112 e ACP-112-Leite no experimento I; e no experimento II, os ejaculados foram diluídos no melhor diluidor obtido no experimento I, acrescido de 2.5 μg mL-1, 5 μg mL-1 e 10 μg mL-1 da planta e o grupo controle, constituído somente do diluidor. O sêmen recém colhido foi analisado quanto as características convencionais, tais como, motilidade, concentração, morfologia e viabilidade. Após a descongelação das amostras foram avaliados novamente, motilidade e viabilidade espermática, e posteriormente, foram realizados os testes de termo-resistência, hiposmótico e de avaliação das membranas dos espermatozóides, através das sondas fluorescentes PI, FITC-PSA e JC-1, utilizando a citometria de fluxo. Os dados obtidos foram submetidos à ANOVA e ao Teste de Tukey a 5%.(AU)

Comparative evaluation between the extenders TES-TRIS and ACP-112® and the association of Sálva Marajó oil (Lippia origanoides) in the quality of cryopreserved buffalo sperm / Avaliação comparativa entre os diluidores TES-TRIS e ACP-112® e a associação com o óleo da Sálva do Marajó (Lippia origanoides) na qualidade do sêmen bubalino criopreservado

For artificial insemination, it is essential to use frozen semen, however the freezing process causes deleterious changes to the structure and integrity of sperm membranes that compromise the function of sperm. To avoid this cellular damage, extenders and suitable substrates must be used to recover the highest possible number of viable cells post-thaw. To this end, in the first experiment, we evaluated three different extenders: TES-TRIS, which is widely used for buffaloes; and an extender composed of powdered coconut water-based (ACP-112®) with or without milk (ACP-112®-milk) for buffalo semen freezing. In the second experiment, we evaluated the effect of Lippia origanoides oil extract on protecting buffalo sperm against cryoinjury arising from freezing semen. Semen was collected from ten buffalo bulls (10 ejaculates/bull) and diluted in TES-TRIS (control), ACP-112® or ACP-112®-Milk in the first experiment. In the second experiment, the samples were diluted in the diluent with the best results for sperm quality obtained in experiment I, and 2.5 μg mL-1, 5 μg mL-1 or 10 μg mL-1 of the plant extract was added to treatments; and a control group containing only the diluent was also included. The fresh semen was analyzed for conventional features such as motility, concentration, morphology and viability. After thawing, the samples were evaluated again for motility, vigor and supra-vital staining, and then, were performed the of thermal-resistance test, hypoosmotic test and evaluated sperm membrane integrity with the fluorescent probes PI, FITC-PSA and JC-1 using flow cytometry. The data were submitted to ANOVA, and the results were compared by Tukey’s test at a significance of 5%.

Para a implantação da inseminação artificial é indispensável à utilização de sêmen congelado, que pode provocar mudanças deletérias na estrutura e na integridade das membranas espermáticas, comprometendo sua função. Para evitar estes danos celulares, há a necessidade de se utilizar meios diluidores e substratos adequados que recuperem o maior número possível de células viáveis pós-descongelação. Para isso, foram avaliados, no experimento I, três diferentes diluidores, o diluidor TES-TRIS, bastante utilizado para bubalinos, e um diluidor a base de água de coco em pó (ACP-112), associado ou não ao leite (ACP-112-Leite), na congelação do sêmen de bubalinos; e no experimento II, foi avaliado o efeito do óleo extraído da Lippia origanoides na proteção dos espermatozóides contra as crioinjúrias decorrentes da congelação do sêmen bubalino. Foram utilizados 10 touros bubalinos para as colheitas de sêmen (10 ejaculados/touro), sendo os ejaculados diluídos em TES-TRIS (controle), ACP-112 e ACP-112-Leite no experimento I; e no experimento II, os ejaculados foram diluídos no melhor diluidor obtido no experimento I, acrescido de 2.5 μg mL-1, 5 μg mL-1 e 10 μg mL-1 da planta e o grupo controle, constituído somente do diluidor. O sêmen recém colhido foi analisado quanto as características convencionais, tais como, motilidade, concentração, morfologia e viabilidade. Após a descongelação das amostras foram avaliados novamente, motilidade e viabilidade espermática, e posteriormente, foram realizados os testes de termo-resistência, hiposmótico e de avaliação das membranas dos espermatozóides, através das sondas fluorescentes PI, FITC-PSA e JC-1, utilizando a citometria de fluxo. Os dados obtidos foram submetidos à ANOVA e ao Teste de Tukey a 5%.

Avaliação da viabilidade do sêmen bubalino congelado pelos métodos convencional e automatizado (resultados parciais) / Evaluation of viability the buffalo semen frozen by conventional and automated methods (partial results)

In the Artificial Insemination it is essential to the use of frozen semen, which causes damage to thestructure of sperm. To avoid these cellular damage, there is a need to assess the viability of frozen semen buffaloby conventional and automated methods, and to predict which of the methods retrieve highest number of viablecells post-thawing. The aim of this study was to evaluate the efficiency of these two methods in buffalo semenfreezing. Semen was obtained from buffalo breeding and diluted in TES-TRIS. After semen freezing, the sampleswere evaluated for motility and vigor. There was no difference between the automated and conventionalmethods, respectively, for motility (67,5%±10 and 69,37±9,28), and the vigor (3,06±0,57 and 3,06±0,68).Therefore, it is concluded that the freezing methods are effective in cryopreservation the semen buffalo, however,it is suggested that more specific tests are performed to validate the protocols. (AU)

Associação entre o diâmetro folicular, no momento da retirada do implante de progesterona, e a taxa de ovulação de vacas após protocolo de sincronização / Association between follicular diameter at the time of removal of the progesterone and ovulation rate of cows after to the synchronization of ovulation protocol

The experiment was conducted at the Center of Biotechnology and Animal Reproduction (CEBRANUFPa),in the municipality of Castanhal, Pará State. The herd consisted of 24 crossbred cows with bodycondition score ranging between 2.5 and 3.5, divided randomly into 2 groups (G1 and G2) which were managedwith different protocols. G1 (D0-Be+P4 implant; D7-Pgf2; D9-measurement of the largest follicle + CE + eCG;D11- new measurement of FD and D15- verification of the presence or absence of CL and its measurement) G2(D0-Be + P4 implant, removal of implant D8 + FSH; D9- BE; D10-measurement of FD; D14- verification of thepresence of CL). Therefore, is possible the identification and selection of animals subjected to thesynchronization protocol for IATF.(AU)

Comparação do diâmetro do folículo dominante no momento da IATF e do corpo lúteo formado, em diferentes protocolos de sincronização da ovulação em vacas mestiças / Comparison of the diameter of the dominant follicle at the time of TAI and the corpus luteum formed in different synchronization protocols of ovulation in crossbred cows

The beef cattle industry in Brazil is characterized as an activity that goes through a period of growth.The experiment was conducted at the Center of Animal Reproduction Biotechnology (CEBRAN-UFPA), in themunicipality of Castanhal, Pará State. The herd consisted of 24 crossbred cows with body condition scoreranging between 2.5 and 3, 5, divided randomly into 2 groups (G1 and G2) which were managed with differentprotocols. G1 (D0-Be+P4 implant; D7-Pgf2; D9-measurement of the largest follicle + CE + eCG; D11- newmeasurement of FD and D15- verification of the presence or absence of CL and its measurement) G2 (D0-Be +P4 implant, removal of implant D8 + FSH; D9- BE; D10-measurement of FD; D14- verification of the presenceof CL). In the present study was not observed statistical difference for the size of follicular diameter at themoment of TAIF and CL formed in different synchronization protocols.(AU)

Associação entre o diâmetro folicular, no momento da retirada do implante de progesterona, e a taxa de ovulação de vacas após protocolo de sincronização / Association between follicular diameter at the time of removal of the progesterone and ovulation rate of cows after to the synchronization of ovulation protocol

The experiment was conducted at the Center of Biotechnology and Animal Reproduction (CEBRANUFPa),in the municipality of Castanhal, Pará State. The herd consisted of 24 crossbred cows with bodycondition score ranging between 2.5 and 3.5, divided randomly into 2 groups (G1 and G2) which were managedwith different protocols. G1 (D0-Be+P4 implant; D7-Pgf2; D9-measurement of the largest follicle + CE + eCG;D11- new measurement of FD and D15- verification of the presence or absence of CL and its measurement) G2(D0-Be + P4 implant, removal of implant D8 + FSH; D9- BE; D10-measurement of FD; D14- verification of thepresence of CL). Therefore, is possible the identification and selection of animals subjected to thesynchronization protocol for IATF.

Comparação do diâmetro do folículo dominante no momento da IATF e do corpo lúteo formado, em diferentes protocolos de sincronização da ovulação em vacas mestiças / Comparison of the diameter of the dominant follicle at the time of TAI and the corpus luteum formed in different synchronization protocols of ovulation in crossbred cows

The beef cattle industry in Brazil is characterized as an activity that goes through a period of growth.The experiment was conducted at the Center of Animal Reproduction Biotechnology (CEBRAN-UFPA), in themunicipality of Castanhal, Pará State. The herd consisted of 24 crossbred cows with body condition scoreranging between 2.5 and 3, 5, divided randomly into 2 groups (G1 and G2) which were managed with differentprotocols. G1 (D0-Be+P4 implant; D7-Pgf2; D9-measurement of the largest follicle + CE + eCG; D11- newmeasurement of FD and D15- verification of the presence or absence of CL and its measurement) G2 (D0-Be +P4 implant, removal of implant D8 + FSH; D9- BE; D10-measurement of FD; D14- verification of the presenceof CL). In the present study was not observed statistical difference for the size of follicular diameter at themoment of TAIF and CL formed in different synchronization protocols.

Fracionamento de doses de sêmen bovino criopreservado para utilização na fertilização in vitro de embriões / Fractionation of bovine semen doses cryopreserved for use in vitro fertilization

The use of frozen semen for PIVE is not fully utilized, as usually using a full dose of semen being wasteda large number of sperm that could be used in other PIVE, for these reasons, the objective of this work was toevaluate the fractionation of 0.25 ml straws in frozen bovine semen doses, divided into four equal sections forlater use in IVF technique in order to avoid wasting sperm present in the straw. Motility, vigor and spermconcentration analyzes were performed. There was observed no effect of vane fractionation process on spermparameters evaluated, such as motility 58,75%±7,6, vigor 2,75±1,6, and spermatic concentration average(3,7x106) between the sectioned parts. Thus, it is concluded that the semen dose fractionation method ofcryopreserved bull into four sections is viable for in vitro fertilization techniques, provided that there is nocompromise on the number and viability of sperm cells.

Avaliação da viabilidade do sêmen bubalino congelado pelos métodos convencional e automatizado (resultados parciais) / Evaluation of viability the buffalo semen frozen by conventional and automated methods (partial results)

In the Artificial Insemination it is essential to the use of frozen semen, which causes damage to thestructure of sperm. To avoid these cellular damage, there is a need to assess the viability of frozen semen buffaloby conventional and automated methods, and to predict which of the methods retrieve highest number of viablecells post-thawing. The aim of this study was to evaluate the efficiency of these two methods in buffalo semenfreezing. Semen was obtained from buffalo breeding and diluted in TES-TRIS. After semen freezing, the sampleswere evaluated for motility and vigor. There was no difference between the automated and conventionalmethods, respectively, for motility (67,5%±10 and 69,37±9,28), and the vigor (3,06±0,57 and 3,06±0,68).Therefore, it is concluded that the freezing methods are effective in cryopreservation the semen buffalo, however,it is suggested that more specific tests are performed to validate the protocols.

Fracionamento de doses de sêmen bovino criopreservado para utilização na fertilização in vitro de embriões / Fractionation of bovine semen doses cryopreserved for use in vitro fertilization

The use of frozen semen for PIVE is not fully utilized, as usually using a full dose of semen being wasteda large number of sperm that could be used in other PIVE, for these reasons, the objective of this work was toevaluate the fractionation of 0.25 ml straws in frozen bovine semen doses, divided into four equal sections forlater use in IVF technique in order to avoid wasting sperm present in the straw. Motility, vigor and spermconcentration analyzes were performed. There was observed no effect of vane fractionation process on spermparameters evaluated, such as motility 58,75%±7,6, vigor 2,75±1,6, and spermatic concentration average(3,7x106) between the sectioned parts. Thus, it is concluded that the semen dose fractionation method ofcryopreserved bull into four sections is viable for in vitro fertilization techniques, provided that there is nocompromise on the number and viability of sperm cells.(AU)

Ocorrência da leucose enzoótica dos bovinos no Estado do Pará, Brasil / Occurrence of enzootic bovine leukosis in the State of Pará, Brazil

The occurrence of the infection with Bovine Leukosis Virus (BLV) was examined in agar-gel immunodiffusion (AGID) test and with indirect enzyme-linked immunosorbent assay (ELISA), in the State of Pará, Brazil. The blood sera were collected from different breeds including Nelore, Piemontes, Simental, Holstein Frisian, Indubrasil, Girolanda, Simbrasil and their cross-breedings. The majority of the animals were adults. The overall occurrence of infections was 49.8% (359/721) and 26.0% (174/668) for ELISA and AGID test, respectively. All animal groups examined showed infection in ELISA, however in the AGID test two groups were sera negative.(AU)

A ocorrência da infecção pelo Vírus da Leucose Enzoótica dos Bovinos (BLV) no Estado do Pará, foi estudada através do método de imunodifusão em ágar-gel (AGID) e por um ensaio imunoenzimatico (ELISA) indireto, paralelamente. Os exames foram realizados com amostras de soros sanguíneos oriundos de bovinos de diferentes raças sendo a maioria deles adultos. A prevalência observada foi de 49,8% (359/721) no ELISA e 26,0% (174/668) no AGID. Todos os 14 grupos dos animais estudados pelo ELISA indireto, mostraram a existência da infeção, enquanto que pelo método da AGID, dois grupos de animais foram negativos.(AU)