Proporção macho: fêmea de embriões bovinos cultivados na presença ou ausência de glicose após FIV com espermatozóides selecionados por Swim-up ou Gradiente de Percoll

No sistema de PIV em bovinos, tem sido obtida uma elevada porcentagem de embriões machos. Este experimento foi realizado para determinar se a presença de glicose no meio de cultivo afeta a proporção macho:fêmea (M:F) dos embriões bovinos PIV a partir da FIV com espermatozóides preparados pelos métodos do swim-up (S) ou do gradiente de Percoll (P). Após a MIV, os COCs foram divididos em dois grupos e inseminados com espermatozóides preparados por um dos métodos. Os zigotos foram cultivados em meio com ou sem 5,56mM de glicose, totalizando 4 tratamentos: S-Gli, S+Gli, P-Gli e P+Gli e 48h após a inseminação, os embriões de cada tratamento foram submetidos à sexagem por PCR (n=845). O efeito da glicose no meio de cultivo sobre a proporção M:F dos embriões PIV a partir dos dois métodos foi semelhante (teste do c²), resultando em uma porcentagem de machos menor do que 50% no estágio de 2-C (S: 30,8%; P: 23,8%: P<0,01) e maior do que 50% no estágio de 8-C (S: 79,4%; P: 68,8%: P<0,01). Estas porcentagens foram diferentes (P<0,05) das observadas quando os embriões foram cultivados sem glicose, tanto no estágio de 2-C (S: 48,5%; P: 41,5%) como no de 8-C (S: 62,5%; P: 50,8%). A presença de glicose não afetou a proporção M:F no total de embriões produzidos (S: 56,7%; P: 49,0%), que foi semelhante à observada na ausência de glicose (S: 55,7%; P: 46,2%). Portanto, a glicose exacerbou a diferença na velocidade de desenvolvimento entre os embriões machos e fêmeas.In the bovine IVP system, it has been obtained a high percentage of male embryos. This experiment was carried ou to determine if the glucose presence in the culture medium affects the sex ratio of bovine embryos IVP from the IVF with spermatozoa prepared for the swim-up (S) or Percoll gradient (P) methods. After the IVM, the COCs had been divided in two groups and inseminated with spermatozoa prepared for one of the methods. The zygotes had been cultivated in medium with or without 5.56mM of glucose, totalizing 4 treatments: S-Glu, S+Glu, P-Glu and P+Glu, and 48h post-insemination, the embryos of each treatment had been sexed for the PCR method (n=845). The effect of glucose in the culture medium on sex ratio of embryos IVP from the two methods was similar (c² test), resulting in a percentage of males lower than 50% in the 2-C stage (S: 30.8%; P: 23.8%: P<0.01) and higher than 50% in the 8-C stage (S: 79.4%; P: 68.8%: P<0.01). These percentages were different (P<0.05) of the observed when the embryos were cultivated without glucose, as much in the 2-C stage (S: 48.5%; P: 41.5%) as in the 8-C stage (S: 62.5%; P: 50.8%). The glucose presence did not affect the sex ratio in the total of produced embryos (S: 56.7%; P: 49.0%), that was similar to the observed in the glucose absence (S: 55.7%; P: 46.2%). Therefore, the glucose exacerbated the difference in the development speed between the female and male embryos

Green turtles (Chelonia mydas) foraging at Arvoredo Island in Southern Brazil: Genetic characterization and mixed stock analysis through mtDNA control region haplotypes

We analyzed mtDNA control region sequences of green turtles (Chelonia mydas) from Arvoredo Island, a foraging ground in southern Brazil, and identified eight haplotypes. Of these, CM-A8 (64%) and CM-A5 (22%) were dominant, the remainder presenting low frequencies (< 5%). Haplotype (h) and nucleotide (π) diversities were 0.5570 ± 0.0697 and 0.0021 ± 0.0016, respectively. Exact tests of differentiation and AMOVA ΦST pairwise values between the study area and eight other Atlantic foraging grounds revealed significant differences in most areas, except Ubatuba and Rocas/Noronha, in Brazil (p > 0.05). Mixed Stock Analysis, incorporating eleven Atlantic and one Mediterranean rookery as possible sources of individuals, indicated Ascension and Aves islands as the main contributing stocks to the Arvoredo aggregation (68.01% and 22.96%, respectively). These results demonstrate the extensive relationships between Arvoredo Island and other Atlantic foraging and breeding areas. Such an understanding provides a framework for establishing adequate management and conservation strategies for this endangered species

Cloning and Expression of Foreign Genes in Mycobacteria.

In this thesis, the construction and use of integrative vectors to express foreign genes in mycobacteria is described. These vectors are based on the transposition properties of the IS900 insertion sequence. Transformation by stable integration of multiple copies of the vector into the chromosome of Mycobacterium smegmatis, M. bovis BCG and M. vaccae was achieved. The M. leprae gene encoding the 18-kDa antigen was used to drive the expression of foreign antigens in mycobacteria. The major immunogenic site of Foot-and-Mouth disease virus (FMDV) comprising amino acids 140-160 of the virus protein 1 (VP1) was inserted in the 18kD gene and expressed as a fusion protein. Guinea pigs were vaccinated with the recombinant BCG expressing the FMDV epitope and immune responses specific for the FMDV were detected. A system for testing mycobacterial promoter strength in both extracellularly and intracellularly growing mycobacteria, as well as in E. coli was developed. It consists of a shuttle vector containing a promoterless reporter gene (lacZ). A promoter library was constructed using M. bovis BCG chromosomal DNA and screened in M. smegmatis and E. coli and a number of clones containing DNA inserts with promoter activity were isolated. The activity of each individual clone was measured in both, E. coli and M. smegmatis. Several previously characterized mycobacterial promoters including those of the Mycobacterium bovis BCG hsp60, the M. leprae 18kD and the putative iron regulated M. leprae 28kD gene were also cloned in front of the promoterless lacZ gene and the promoter strength was determined. To study intracellular expression, rBCG was used to infect murine macrophages and the activity of the reporter gene was measured using a fluorescent substrate and a FACS can system. Relative differences in the expression of the reporter gene during intracellular and extracellular growth were detected for some promoters. The BCG hsp60 promoter was shown to be the strongest of the characterized promoters also during intracellular growth. To obtain a higher level of expression of the 18kD/FMDV fusion, the 18kD promoter was replaced by the hsp60 promoter. A level of expression of the fused gene that in vitro was at least 10 fold higher was obtained with the hsp60 promoter in BCG

Efeito do gene do estresse suíno sobre características de quantidade e qualidade de carcaça

O gene do estresse suíno (gene hal), em homozigose recessiva (nn), está associado com a ocorrência da Síndrome do Estresse Porcino (PSS) e com a ocorrência da carne pálida, mole e exudativa (PSE). Em heterozigose (Nn), está relacionado com diminuição na qualidade da carne porém, com maior peso de carcaça. Neste estudo, foi caracterizado o genótipo do gene hal de 160 suínos por meio de análise do DNA extraído de um único folículo piloso. Estes animais foram abatidos e, após, mensuradas características de carcaça de cada um. Entre os 160 animais analisados, 82 (52,58%) foram caracterizados como NN, 67 (41,80%) como Nn e nove (5,62%) como nn. A variação das características de carcaça entre os genótipos foi avaliada por análise de variância, usando o SAS. Os animais dos três genótipos não diferiram quanto ao peso da carcaça quente, à espessura de toucinho, profundidade de músculo, porcentagem de carne magra e cor do músculo longissimus dorsi. Entretanto, a variação da cor ao longo da carcaça foi menor em animais NN (40,82%) do que em Nn (49,77%) e nn (53,83%). Estes resultados indicam que a presença do gene hal tanto em heterozigose como em homozigose recessiva não está associada ao maior peso de carcaça e a utilização destes animais pode acarretar em perda na qualidade da carne. Devido a isto, o uso intencional de animais homozigotos recessivos ou heterozigotos em programas de cruzamento deve ser desestimulado

Seropositivity to Leptospira interrogans Canicola local isolate associated to tongue necrosis in dog without significant hematological alterations

Leptospirosis is a global zoonosis caused by infection with the spriochetal bacterium of the genus, Leptospira. Dogs may be exposed to leptospires in the environment by contact with urine of an infected host, contaminated water or moist soil, where the bacteria may survive for several months. In this work, we report the clinical case of a dog suffering tip of the tongue necrosis caused by pahogenic L. interrogans serovar Canicola strain Tande. Laboratorial examinations demonstrated urinalysis and blood chemistry changes without significant hematological alterations. The 3.200 antibodies titers against Tande strain were detected on Microscopic Agglutination Test (MAT), confirming the diagnosis of leptopirosis. Aggressive fluid therapy and β-lactâmic antibiotics were used for treatment and to prevent and treat acute kidney damage. The dog had a complete clinical recovery after treatment.A leptospirose é uma zoonose de distribuição mundial causa por bactérias espiroquetas do gênero Leptospira spp. Cães são expostos ao agente por meio de urina de hospedeiros infectados, água ou solo contaminados, onde  bactéria pode sobreviver por muitos meses. Neste trabalho, relatamos o caso clínico de um cão apresentando como sinal clínico necrose da ponta da língua associada a uma infeção aguda pela patogênica L. iterrogans Canicola cepa Tande. A infecção aguda por esta cepa patogênica causou mudança nos parâmetros da bioquímica sérica e na urinálise, sem causar alterações hematológicas. Título de anticorpos de 3.200 contra cepa Tande foram detectados pelo teste de soroaglutinação microscópica (MAT), confirmando o diagnóstico de leptospirose. A associação de antibióticos β-lactâmicos foram utilizados para combater a infecção aguda e fluidoterapia agressiva foi adotada para previnir  insuficiência renal. O cão obteve recuperação  clínica após tratamento

Production of leptospiral LipL32 antigen in Pichia pastoris and its use in an enzyme-linked immunosorbent assay

The production of recombinant LipL32 protein using Escherichia coli has been used extensively for the development of vaccines and diagnostic tests for leptospirosis. However, E. coli has demonstrated limitations, including low yield and lack of post-translational modifications. In this study, rLipL32 was produced in eukaryotic expression system (Pichia pastoris) and evaluated the antigen by enzyme-linked immunosorbent assay (ELISA). The yield obtained from the culture supernatant reached 270 mg/L and ELISA showed an accuracy of 95.34%. In summary, the production of rLipL32 using P. pastoris did not impair the antigenic characteristics of this antigen and ensured its use for detecting the leptospiral antibodies in swine sera