Potent inhibition of Western Equine Encephalitis virus by a fraction rich in flavonoids and phenolic acids obtained from Achyrocline satureioides

ABSTRACT Achyrocline satureioides (Lam.) DC. Asteraceae, ‘marcela del campo', possess several pharmacological properties. Previously we reported antiviral activity of an aqueous extract of A. satureioides against an alphavirus, Western Equine Encephalitis virus. Alphaviruses are highly virulent pathogens which cause encephalitis in humans and equines. There are no effective antiviral to treat its infections. The aim of this study was to evaluate in vitro cytotoxic and antiviral activities against Western Equine Encephalitis virus of five water extract chromatographic fractions from A. satureioides and identify the main compounds of the bioactive fraction. Also, it was to assess in vivo cytogenotoxic ability of the active fraction. Cytotoxicity studies revealed low toxicity of the most of fractions in Vero and in equine peripheral blood mononuclear cells. Antiviral studies showed that the water crude extract – Sephadex LH 20 – fraction 3 MeOH–H2O (Fraction 3) was active against Western Equine Encephalitis virus with Effective Concentration 50% = 5 µg/ml. Selectivity Indices were 126.0 on Vero and 133.6 on peripheral blood mononuclear cells, four times higher than aqueous extract selectivity index. Regarding the mechanism of action we demonstrated that F3 exerted its action in intracellular replication stages. Further, fraction 3 showed important virucidal action. Fraction 3 contains, in order of highest to lowest: chlorogenic acid, luteolin, 5,7,8-trimethoxyflavone, 3-O-methylquercetin and caffeic acid. Fraction 3 did not induce in vivo toxic nor mutagenic effect. Therefore, it is safe its application as antiviral potential. Further studies of antiviral activity in vivo will be developed using a murine model.

Nascimento de bezerros normais após inseminação artificial utilizando espermatozóides criopreservados obtidos de epidídimos refrigerados de bovinos após a morte / Birth of normal calves after artificial insemination using cryopreserved spermatozoa obtained from refrigerated epididymides of death bovine

O objetivo deste estudo foi avaliar as características morfológicas e funcionais dos espermatozóides bovinos recuperados de epidídimos resfriados por longos períodos e posteriormente criopreservados. Testículos bovinos foram coletados no abatedouro, transportados ao laboratório e armazenados a 5°C por 0, 24, 48 e 72 horas (n=10 para cada tratamento). Os espermatozóides foram extraídos de cada epidídimo, avaliados e diluídos em meio tris-gema-glicerol a 7 por cento e criopreservados em nitrogênio líquido. As características morfológicas e funcionais dos espermatozóides foram avaliadas in vitro por análise microscópica e in vivo, por meio de inseminação artificial. Foram observadas alterações morfológicas características da imaturidade dos espermatozóides e redução da motilidade após 72 horas de refrigeração dos epidídimos. Esses parâmetros também foram alterados após o descongelamento, em todos os tratamentos. A manutenção dos espermatozoides a 5°C por 72h reduziu a motilidade espermática. Em todos os tratamentos foram observadas alterações morfológicas características da imaturidade dos espermatozoides e redução da motilidade após o descongelamento. A integridade de membrana plasmática e acrossoma somente foram afetadas pós criopreservação nos grupos mantidos a 5°C durante 48 ou 72h antes da criopreservação. Contudo, a capacidade de fecundação dos espermatozóides mantidos a 5°C durante 24 ou 72h antes da criopreservação foi suficiente para promover duas gestações e nascimento de bezerros saudáveis. Esses resultados indicam que a recuperação e a criopreservação de espermatozóides obtidos de epidídimos mantidos a 5°C, até 72h, provenientes de animais mortos é uma opção viável para preservar gametas masculinos para compor um banco de germoplasma.

The objective of this study was to evaluate the morphological and functional characteristics of bovine spermatozoa retrieved from chilled epidydimides for long periods and cryopreserved. Bovine testicles were collected in abattoir, transported to the laboratory and stored at 5°C for 0, 24, 48h e 72 hours (n=10 for each storage time treatment group). The spermatozoa were retrieved from each epidydimides, evaluated and diluted in tris-egg yolk-glycerol 7 percent medium and cryopreserved in liquid nitrogen. The morphological and functional characteristics of the spermatozoa were analyzed in vitro, by microscopic evaluation and in vivo, using artificial insemination. Morphological alterations as sperm immaturity and motility reduction decreased after 72h of epididymides refrigeration and after thaw sperm were observed. The membrane and acrosome integrity were only affected in G48 and G72 groups after cryopreservation. However, the sperm capacity of fertilization post-cryopreservation was sufficient to promote two pregnancies and birth of healthy calves from G24 h and G72h groups. These results indicated that recovery and cryopreservation of chilled epididymal sperm until 72h from dead animals is a viable option to preserve male gametes to compose a germplasm bank.

DNA testing for parentage verification in a conservation nucleus of Pantaneiro horse

We investigated the genealogy of the in situ conservation nucleus of the Pantaneiro horse using DNA microsatellites by evaluating 101 horses, the group consisting of 71 adult horses (3 stallions, 40 male and 31 mares) and 27 foals (14 colts and 13 fillies). Genomic DNA was extracted from hair roots and genotyped using 12 microsatellite markers (AHT4, AHT5, ASB2, ASB17, ASB23, HMS3 HMS6, HMS7, HTG4, HTG10, LEX33 and VHL20). The number of alleles per locus varied from 6 to 13, with a mean of 7.8 and the expected heterozygosity ranged from 0.544 to 0.734 (mean 0.644). The VLH20, ASB2, HTG10, ASB23 markers had a high (> 0.8) polymorphism information content and the total exclusion probability of the 12 microsatellite loci was 0.99. The genealogical study of the Pantaneiro horse using genetic markers was efficient in detecting mistakes during paternity and maternity designation and is an important tool which can be used together with traditional systems of animal identification. The use of genetic markers is recommended in the systematic control of the genealogical registrations and conservation plans to improve genetic aspects of the Pantaneiro horse.

Body indices for the pantaneiro horse / Índices corporais para o cavalo pantaneiro

Body index measurements on 2248 horses registered by the Brazilian Association of Pantaneiro Horse Breeders (ABCCP), in the period from 1972 to 2000 were analyzed. Data was analyzed using SAS and DFREML to calculate genetic parameters. The Dactyl Thoracic Index indicated that these animals are intermediary (not light horses but not suitable for traction), while the BARON and CREVAT index confirmed this result. The Chest Index presented animals that are good for speed as they have long legs but looking at the Body index the indication was that these horses are neither suitable for speed nor traction (intermediary). The analysis of other conformation indices indicated that, in general, these horses are medilines, show good speed and chest development, making them suitable for dealing with cattle and resistance for long treks. The environmental factor analysis verified that these horses have changed in recent years possibly due to selection by the breeder which may affect their adaptive capacity.

Foram estudados índices zootécnicos de 2.248 animais registrados na Associação Brasileira de Criadores do Cavalo Pantaneiro (ABCCP), no período compreendido entre 1972 e 2000. Os dados foram analisados usando o programa SAS e o DFREML para estimação de parâmetros genéticos. Na análise dos índices corporais, o índice dáctilo torácico (IDT) apresentou animais intermediários (nem leves e nem aptos para tração). O índice de BARON e CREVAT também confirmou este resultado. Na determinação do índice peitoral (IP), os animais foram considerados, em geral, favoráveis à velocidade por apresentarem membros longos. Já no índice corporal (IC), a maioria dos animais não foi apta à velocidade e nem à tração (mediolíneos). Através da análise dos índices de conformação verificou-se que, em geral, os Pantaneiros são mediolíneos, aptos para velocidade e possuem bom desenvolvimento torácico, proporcionando velocidade na lida do gado e resistência a longas caminhadas. Na análise dos fatores ambientais, verificou-se que nos últimos anos alguns índices têm sofrido alterações, provavelmente devido à seleção já exercida pelo criador, que pode afetar a capacidade de adaptação dos cavalos.