Production and Evaluation of Immunoglobulin Y Anti-Brucella abortus (Vaccinal Strain B19)

Background: The immunoglobulin Y is a principal antibody current in the blood of hens, which are transferred from the maternal blood serum to the egg yolk. The extraction of IgY from the egg yolk apply animal welfare when compared to the extraction of IgG, reducing the number of animals and prevent a bleeding of hens through the extraction of the IgY from eggs, besides that IgY presenting high specificity for antigenic binding. The objective of this study was to produce specific polyclonal IgY antibodies anti-Brucella abortus by immunizing hens with B19 vaccine and evaluate their reactivity through Buffered Acidified Plate Antigen (BAPA), 2-Mercaptoethanol (2-ME) and indirect ELISA diagnostic tests.Materials, Methods & Results: Four 25-week-old White Leghorn hens were immunized, two of them comprising the control group (Group 1) with phosphate-buffered saline (PBS) with adjuvant, and the others two immunized with B19 vaccine (Brucella abortus vaccine strain B19), representing the Group 2. The immunizations occurred six times with a 15-day interval between each. Blood samples were taken biweekly (seven times); and daily, the eggs were collected for 13 weeks, the first collection of blood and eggs, performed one week before the first immunization of each group. The IgY was purified from egg yolk, using the method of dilution in acid water and precipitation with ammonium sulfate for delipidation. BAPA, 2-ME and ELISA tests performed to verify the specificity of IgY confirmed the reactivity of polyclonal antibodies specific to the antigen used both in blood serum samples and in the purified egg yolks. The hens from the control group did not present reactivity in the diagnostic tests used, which was already expected, since no antigen was used in any of their immunizations. Hens immunized with the Brucella abortus B19 vaccine produced detectable reactive antibodies in the three tests used on blood serum and IgY samples extracted from the egg yolk. In Group 2 (vaccine B19), blood serum samples started to react one week after the first inoculation, and the IgY samples extracted from the egg yolk were reagent two weeks after serum IgY appear reactivity, showing the transfer of specific antibodies to the egg yolk, was late.Discussion: Although the transfer of serum Igy to egg yolk was late when compared to others authors which found that the transovarian passage of immunoglobulin Y occurred in approximately three to six days after IgY being detected in blood serum, the results of this study showed the occurrence of the transfer of blood serum IgY anti-Brucella abortus to egg yolk of hens immunized with B19 vaccine, the same found by others researches found the same results with others antigens. Thus, it can be concluded that immunoglobulins Y produced in this study can be used as specific antibodies in diagnostic tests for the detection of the Brucella abortus antigen, in addition, this process guarantees the welfare of the animal, since it avoids bleeding and it is possible to obtain high concentrations of antibodies directly from the hen egg, which is a great advantage, because IgY can be easily isolated from the egg yolk by the precipitation technique discarding the need of invasive and painful procedures that involve bloody interventions to obtain the serum antibodies like occur in mammals for extraction of IgG

FATORES QUE INFLUENCIAM A TAXA DE CONCEPÇÃO E PERDA GESTACIONAL DE EMBRIÕES PRODUZIDOS IN VITRO NA RAÇA GIROLANDO

The in vitro production of embryos is fundamental importance for livestock, although it is still a biotechnology with varieds results than expected, being therefore necessary study ways to improve its rates. The objective of this study was to evaluate if the recipient category, season and stage of embryo development affect conception rates and gestational losses in recipients of girolando’s embryos produced in vitro and transferred at fixed time (ETFT). The experiment was carried out in a commercial dairy farm, located in the Uberlândia - MG region. A total of 1,017 TETF were evaluated. The recipients were divided into lactating cows (n=618), dry cows (n=44) and heifers (n=355). The seasons were grouped in summer (spring and summer) and winter (autumn and winter). The embryos were grouped in Group 1 (n=216) - initial blastocyst (BI) and blastocyst (BL) and Group 2 (n=801) - expanded blastocyst (BX), hatching blastocyst (BH) and hatched (BE). Receptor category effect was detected at conception rate (p<0.001) and loss of gestation (p<0.003). The summer affected negatively (p<0.044) the conception rate. Group 2 was more successful in maintaining gestation (p<0.025). It is concluded that the three evaluated factors interfere in conception rates and / or gestational loss and should be considered in the genetic improvement programs embryo transfer of the Girolando breed.A produção in vitro de embriões é de fundamental importância para a pecuária, entretanto ainda é uma biotecnologia com resultados variados sendo, portanto, necessário estudar formas de melhorar. Objetivou-se com este trabalho avaliar se a categoria das receptoras, estação do ano e estágio de desenvolvimento embrionário afetam as taxas de concepção e as perdas gestacionais em receptoras de embriões da raça Girolando produzidos in vitro e transferidos em tempo fixo (TETF). O experimento foi realizado em uma fazenda leiteira comercial, localizada na região de Uberlândia – MG. Foram avaliadas 1.017 TETF. As receptoras foram divididas em vacas em lactação (n=618), vacas secas (n=44) e novilhas (n=355). As estações do ano foram agrupadas em verão (primavera e verão) e inverno (outono e inverno). Os embriões, todos com qualidade morfológica grau 1, foram agrupados de acordo com seu estágio de desenvolvimento em: Grupo 1 (n=216) - blastocisto inicial (BI) e blastocisto (BL) e Grupo 2 (n=801) - blastocisto expandido (BX), blastocisto em eclosão (BN) e blastocisto eclodido (BE). Foi detectado efeito da categoria da receptora na taxa de concepção (p<0,001) e na perda de gestação (p<0,003). O verão afetou negativamente (p<0,044) a taxa de concepção. O Grupo 2 teve mais sucesso na manutenção da gestação (p<0,025). Conclui-se que os três fatores avaliados interferem nas taxas de concepção e/ou perda gestacional e devem ser considerados nos programas de transferência de embriões da raça Girolando

INFLUÊNCIA DO ESCORE DE COMPORTAMENTO NA TAXA DE PRENHEZ DE VACAS NELORES SUBMETIDAS À IATF

Temperament is an individual characteristic that animals have in relation to a given stimulus and is conditioned on fear. It is believed to have great importance in productive efficiency, and therefore has been seen with attention in animal production. The objective of this study was to evaluate the effect of temperament at time of artificial insemination on the pregnancy rate of Nelore animals. Nellore cow temperament (n = 168) was evaluated by trunk scores and trunk exit velocity at the time of TAI. The trunk score was evaluated with grades from 1 to 5, and the exit speed test measured the time each animal spent to travel 2 meters away. The cows underwent TAI and the pregnancy diagnosis was made 30 days after TAI. For the analyzes, the trunk score was categorized as 1.2 and ≥3; at trunk exit speeds of ≤0.67, 0.68 to 0.99 and 1.00 to 2.00 m/s. No effect of temperament on pregnancy rate (p>0.05) was detected, but the effect of trunk score on exit velocity (p<0.05) was detected. Cows with lower trunk scores came out at lower trunk speeds. It was concluded that trunk score or exit speed in animals conditioned to trunk management does not affect the pregnancy rate.O temperamento é uma característica individual que os animais têm em relação a determinado estímulo e estão condicionados ao medo. Acredita- se que possui grande importância na eficiência produtiva, e, portanto, vem sendo visto com atenção na produção animal. Objetivou-se avaliar o efeito do temperamento no momento da inseminação artificial em tempo fixo na taxa de prenhez de animais da raça Nelore. Foi avaliado do temperamento de vacas Nelore (n = 168) por meio do escores de tronco e velocidade de saída do tronco, no momento da IATF. O escore de tronco foi avaliado com notas de 1 a 5, e o teste de velocidade de saída aferiu o tempo em que cada animal gastou para percorrer 2 metros de distância. As vacas foram submetidas a IATF e o diagnóstico de gestação foi realizado 30 dias após a IATF. Para as análises o escore de tronco foi categorizado em 1,2 e ≥3; a velocidades de saída de tronco em ≤0,67, 0,68 a 0,99 e 1,00 a 2,00 m/s. Não foi detectado efeito do temperamento na taxa de prenhez (p>0,05), porem foi detectado efeito do escore de tronco na velocidade de saída (p<0,05). As vacas com menor escore de tronco saíram em menor velocidade do tronco. Concluiu-se que a taxa de prenhez não é afetada pelo escore de tronco e nem pela velocidade de saída em animais condicionados ao manejo de tronco

The role of Kisspeptin in bovine in vitro embryo production

The aim of this study was to investigate the effect of Kisspeptin (Kp) on the medium used in different stages of in vitro production of bovine embryo (IVEP), evaluating cleavage (CR) and blastocyst (BR) rates. The study was divided into three experiments that analyzed, respectively, the action of Kp on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of bovine embryos. In experiment 1, the oocytes were matured in IVM medium and distributed into the following treatments: maturation (IVM Control, n = 102), maturation with addition of 10-7 M Kp (Kp 10-7 IVM, n = 90), and hormone-free maturation luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with the addition of 10-7M Kp (No hormones + Kp 10-7, n = 84), following maturation to normal stages of IVEP. In experiment 2, the oocytes were fertilized in IVF medium, in the following treatments: TALP-FERT without Kp (Control IVF, n = 103) and TALP-FERT with the addition of 10-7M Kp (Kp 10-7 IVF, n = 119), usually following the other steps. Finally, in the third experiment, the oocytes passed through all phases and were divided into IVC in two treatments: SOF medium without Kp (Control IVC, n = 109) and SOF medium with the addition of 10-7M Kp (Kp 10-7, N = 106). The data were analyzed by PROC GLIMMIX of the SAS program. In experiment 1, the means of CR and BR were similar (P > 0.05) between treatments (IVM Control76.47% and 37.25%, Kp 10-7 MIV80% and 33.33%, and No hormones + Kp 10-770.24% and 30.95%, respectively). In experiment 2, the means of CR were similar for the IVF Control and Kp 10-7 IVF groups (P > 0.05), 76.70% and 86.55% respectively. But, the mean of the BR of the group Kp 10-7 IVF was 38.66%, which was higher (P 0.05) were similar between the IVC Control and Kp 10-7 IVC groups (CR 83.50% and 78.30%, and BR 26.60% and 23.60%, respectively). Although at this concentration of 10-7M during IVC no change in embryo production is seen, Kp presents the same performance as both gonadotrophins in oocyte maturation and modulates the fertilization process, providing more blastocysts. With these findings, it can be seen that Kp presents a regulatory action on bovine reproduction, and can be an excellent tool to maximize IVEP indexes

ATLANTIC-PRIMATES: a dataset of communities and occurrences of primates in the Atlantic Forests of South America

Primates play an important role in ecosystem functioning and offer critical insights into human evolution, biology, behavior, and emerging infectious diseases. There are 26 primate species in the Atlantic Forests of South America, 19 of them endemic. We compiled a dataset of 5,472 georeferenced locations of 26 native and 1 introduced primate species, as hybrids in the genera Callithrix and Alouatta. The dataset includes 700 primate communities, 8,121 single species occurrences and 714 estimates of primate population sizes, covering most natural forest types of the tropical and subtropical Atlantic Forest of Brazil, Paraguay and Argentina and some other biomes. On average, primate communities of the Atlantic Forest harbor 2 ± 1 species (range = 1–6). However, about 40% of primate communities contain only one species. Alouatta guariba (N = 2,188 records) and Sapajus nigritus (N = 1,127) were the species with the most records. Callicebus barbarabrownae (N = 35), Leontopithecus caissara (N = 38), and Sapajus libidinosus (N = 41) were the species with the least records. Recorded primate densities varied from 0.004 individuals/km 2 (Alouatta guariba at Fragmento do Bugre, Paraná, Brazil) to 400 individuals/km 2 (Alouatta caraya in Santiago, Rio Grande do Sul, Brazil). Our dataset reflects disparity between the numerous primate census conducted in the Atlantic Forest, in contrast to the scarcity of estimates of population sizes and densities. With these data, researchers can develop different macroecological and regional level studies, focusing on communities, populations, species co-occurrence and distribution patterns. Moreover, the data can also be used to assess the consequences of fragmentation, defaunation, and disease outbreaks on different ecological processes, such as trophic cascades, species invasion or extinction, and community dynamics. There are no copyright restrictions. Please cite this Data Paper when the data are used in publications. We also request that researchers and teachers inform us of how they are using the data. © 2018 by the The Authors. Ecology © 2018 The Ecological Society of Americ

Influence of Kisspeptin in the spermatic selection stage for in vitro production of bovine embryos

The aim of the present study was to evaluate the addition of Kisspeptin (Kp) in the in vitro process of spermatic selection, regarding the viability of bovine spermatozoa and embryo production rates. For this, 980 oocytes were obtained from slaughterhouse ovaries and 75 commercial bovine semen straws were used. The treatments were performed on the spermatozoa during the in vitro spermatic selection stage by the centrifugation method with 90% and 45% Percoll® discontinuous gradients according to each proposed treatment: Control (n = 182), Kp 10-5 M (n = 198 ), Kp 10-6 M (n = 200), Kp 10-7 M (n = 206) and 50 μM P-234 antagonist (n = 194), with the dilutions carried out on both gradients respected concentrations. The spermatozoa were analyzed before and after spermatic selection for motility and vigor, and were submitted to staining with fluorescent probes JC-1, IP and FITC-PSA, to evaluate the mitochondrial potential, plasma membrane integrity and acrosome integrity under confocal laser scanning microscope LSM 510 meta. After the treatments, spermatozoa were normally used in IVEP procedures. The production analyzes were performed on D2 and D7, based respectively on cleavage and blastocysts rates. The data were submitted to the normality and homogeneity test by guided analysis SAS, after were used the proc GLIMMIX procedure to evaluate the experimental model and later the Tukey-Kramer test for comparison of means. Differences are considered when P 0.05) among all proposed treatments. The cleavage rate (n = 846) was higher than 82% and the percentage of blastocyst production (n = 331) higher than 29% in all treatments. The mitochondrial potential of Kp 10-6 and Kp 10-7 (92.6%) groups was higher (P 0.05) Kp 10-5 (88.9%/90.9%), Kp 10-6 (90.7%/91.8%) and Kp 10-7 (90.2%/91.4%) and the P-234 antagonist (90.5%/90.8%), respectively. The Kp 10-6 treatment presented a superior result (P 0,05) entre todos os tratamentos propostos. A taxa de clivagem (n=846) foi maior que 82% e a porcentagem de produção de blastocistos (n=331) foi maior que 29%. Quanto ao potencial mitocondrial, os grupos Kp 10-6 e Kp 10-7 (92,6%) foram superiores (P 0,05) entre si Kp 10-5 (88,9%/90,9%), Kp 10-6 (90,7%/91,8%) e Kp 10-7 (90,2%/91,4%) e ao antagonista P-234 (90,5%/90,8%), respectivamente. Apenas o tratamento Kp 10-6 apresentou resultado superior (P < 0,05) ao grupo Controle (87,9%/89,1%). Ao correlacionar as variáveis analisadas pela microscopia confocal, foi observada uma correlação significativa e positiva entre alto potencial mitocondrial e integridade de membrana plasmática (r = 0,80; P < 0,0001), integridade de acrossoma e alto potencial mitocondrial (r = 0,80; P < 0,0001), integridade de membrana plasmática e integridade de acrossoma (r = 0,84; P < 0,0001). Finalmente, pode-se concluir que a concentração equilibrada da Kp proporcionou maior viabilidade aos espermatozoides bovinos referente ao potencial mitocondrial, integridade acrossômica e de membrana plasmática. Já o antagonista P-234 apresentou efeito prejudicial referente ao potencial mitocondrial

In vitro culture of bovine ovarian fragment supplemented with zinc oxide nanocomposites doped with calcium oxide or magnesium oxide

The aim of this study was to test the influence of supplementing the in vitro culture medium with Nanocomposites (NCs) of zinc oxide doped with magnesium chloride (ZnO: 0.5 Mg-MgO) and NCs of zinc oxide doped with calcium chloride (ZnO: 0.5 Ca-CaO), compared to the pure supplementation of these minerals MgCl2 and CaCl2, in three concentrations 10, 20 and 30 µg/ml on the viability of bovine ovarian tissue fragments cultured in vitro. For this purpose, 2 experiments were carried out with the following treatments: 1) Fresh Control (CF), Control (CONT), 10 µg/mL of calcium chloride (CaCl2) (CA10), 20 µg/mL of CaCl2 (CA20), 30 µg/mL of CaCl2 (CA30), 10 µg/mL of ZnO NP:0.5Ca (NPCA10), 20 µg/mL of ZnO NP:0.5Ca (NPCA20), 30 µg/mL of ZnO NP:0.5Ca (NPCA30); 2) CF, CONT, 10 µg/mL magnesium chloride (MgCl2) (MG10), 20 µg/mL MgCl2 (MG20), 30 µg/mL MgCl2 (MG30), 10 µg/mL ZnO NP:0.5Mg (NPMG10), 20 µg/mL ZnO NP:0.5Mg (NPMG20), 30 µg/mL ZnO NP:0.5Mg (NPMG30). Cell degeneration, respiratory metabolism of cells by NAD and FAD emission, redox state, ROS production and finally follicular activation and viability were analyzed. For statistical analysis, SigmaPlot version 11.0 software (Systat Software, Inc., USA) was used, applying analysis of variance and Fisher's LSD post hoc test to compare means, with significance P < 0.05. The NPCA20 treatment showed higher levels of cell degeneration, higher ROS production and lower NAD(P)H and FAD emission, compared to CF and its respective CA20 treatment. The NPMG20 treatment showed a lower rate of cell degeneration, higher NAD and FAD production, lower ROS production and good follicular viability when compared to the controls and MG20. Thus, it is concluded that ZnO:0.5Ca NP at a concentration of 20 μg/mL impaired cell and follicular development, proposing cytotoxic effects in the culture of bovine ovarian cortex fragments. In the second experiment, the doping process was efficient in the ZnO:0.5Mg NP, providing a favorable environment for cellular and follicular development, as the doping process and the correct combination of ions reverse a possible toxicity scenario.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorTese (Doutorado)Objetivou-se testar a influência da suplementação do meio de cultivo in vitro com Nanocompósitos (NCs) de óxido de zinco dopado com cloreto de magnésio (ZnO:0,5 Mg-MgO) e NCs de óxido de zinco dopado com cloreto de cálcio (ZnO:0,5 Ca-CaO), comparados a suplementação pura desses minerais MgCl2 e CaCl2, em três concentrações 10, 20 e 30 µg/ml na viabilidade de fragmentos de tecido ovariano bovino cultivados in vitro. Para isso foram realizados 2 experimentos com os seguintes tratamentos: 1) Controle Fresco (CF), Controle (CONT), 10 µg/mL de cloreto de cálcio (CaCl2) (CA10), 20 µg/mL de CaCl2 (CA20), 30 µg/mL de CaCl2 (CA30), 10 µg/mL de NP de ZnO:0,5Ca (NPCA10), 20 µg/mL de NP de ZnO:0,5Ca (NPCA20), 30 µg/mL de NP de ZnO:0,5Ca (NPCA30); 2) CF, CONT, 10 µg/mL de cloreto de magnésio (MgCl2) (MG10), 20 µg/mL de MgCl2 (MG20), 30 µg/mL de MgCl2 (MG30), 10 µg/mL de NP de ZnO:0,5Mg (NPMG10), 20 µg/mL de NP de ZnO:0,5Mg (NPMG20), 30 µg/mL de NP de ZnO:0,5Mg (NPMG30). Foi analisada a degeneração celular, metabolismo respiratório das células pela emissão de NAD, FAD, estado redox, produção de EROs e por fim ativação e viabilidade folicular. Para a análise estatística foi utilizado o software SigmaPlot versão 11.0 (Systat Software, Inc., EUA), aplicando-se análise de variância e teste post hoc de Fisher LSD para comparação de médias, com significância P < 0,05. O tratamento NPCA20 apresentou maiores níveis de degeneração celular, maior produção de EROs e menor emissão de NAD(P)H e FAD, comparado ao CF e seu respectivo tratamento de CA20. O tratamento NPMG20 apresentou menor taxa de degeneração celular, maior produção de NAD e FAD, menor produção de EROs e boa viabilidade folicular quando comparado com os controles e MG20. Com isso conclui-se que a NP de ZnO:0,5Ca na concentração de 20 μg/mL prejudicou o desenvolvimento celular e folicular propondo efeitos citotóxicos no cultivo de fragmentos do córtex ovariano bovino. Já no segundo experimento, o processo de dopagem foi eficiente na NP ZnO:0,5Mg, proporcionando um ambiente favorável ao desenvolvimento celular e folicular, pois o processo de dopagem e a combinação correta dos íons revertem um possível quadro de toxicidade.2025-09-2

Production and Evaluation of Immunoglobulin Y Anti-Brucella abortus (Vaccinal Strain B19)

Background: The immunoglobulin Y is a principal antibody current in the blood of hens, which are transferred from the maternal blood serum to the egg yolk. The extraction of IgY from the egg yolk apply animal welfare when compared to the extraction of IgG, reducing the number of animals and prevent a bleeding of hens through the extraction of the IgY from eggs, besides that IgY presenting high specificity for antigenic binding. The objective of this study was to produce specific polyclonal IgY antibodies anti-Brucella abortus by immunizing hens with B19 vaccine and evaluate their reactivity through Buffered Acidified Plate Antigen (BAPA), 2-Mercaptoethanol (2-ME) and indirect ELISA diagnostic tests.Materials, Methods &amp; Results: Four 25-week-old White Leghorn hens were immunized, two of them comprising the control group (Group 1) with phosphate-buffered saline (PBS) with adjuvant, and the others two immunized with B19 vaccine (Brucella abortus vaccine strain B19), representing the Group 2. The immunizations occurred six times with a 15-day interval between each. Blood samples were taken biweekly (seven times); and daily, the eggs were collected for 13 weeks, the first collection of blood and eggs, performed one week before the first immunization of each group. The IgY was purified from egg yolk, using the method of dilution in acid water and precipitation with ammonium sulfate for delipidation. BAPA, 2-ME and ELISA tests performed to verify the specificity of IgY confirmed the reactivity of polyclonal antibodies specific to the antigen used both in blood serum samples and in the purified egg yolks. The hens from the control group did not present reactivity in the diagnostic tests used, which was already expected, since no antigen was used in any of their immunizations. Hens immunized with the Brucella abortus B19 vaccine produced detectable reactive antibodies in the three tests used on blood serum and IgY samples extracted from the egg yolk. In Group 2 (vaccine B19), blood serum samples started to react one week after the first inoculation, and the IgY samples extracted from the egg yolk were reagent two weeks after serum IgY appear reactivity, showing the transfer of specific antibodies to the egg yolk, was late.Discussion: Although the transfer of serum Igy to egg yolk was late when compared to others authors which found that the transovarian passage of immunoglobulin Y occurred in approximately three to six days after IgY being detected in blood serum, the results of this study showed the occurrence of the transfer of blood serum IgY anti-Brucella abortus to egg yolk of hens immunized with B19 vaccine, the same found by others researches found the same results with others antigens. Thus, it can be concluded that immunoglobulins Y produced in this study can be used as specific antibodies in diagnostic tests for the detection of the Brucella abortus antigen, in addition, this process guarantees the welfare of the animal, since it avoids bleeding and it is possible to obtain high concentrations of antibodies directly from the hen egg, which is a great advantage, because IgY can be easily isolated from the egg yolk by the precipitation technique discarding the need of invasive and painful procedures that involve bloody interventions to obtain the serum antibodies like occur in mammals for extraction of IgG