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The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability. Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output. The International Society for Animal Genetics (ISAG) administered animal forensic comparison tests (AFCTs) in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification, parentage and species determination services. The AFCTs revealed that analyses of low DNA template concentrations (≤300 pg/µL) constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results. Moreover, a lack of familiarity with species testing protocols, interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results. Several laboratories showed improvement in their genotyping accuracy over time. However, the use of forensically validated standards, such as a standard forensic short tandem repeat (STR) kit, preferably with an allelic ladder, and stricter guidelines for STR typing, may have prevented some common issues from occurring, such as genotyping inaccuracies, missing data, elevated stutter products and loading errors. The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other. Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel’s proficiency in critical techniques such as low copy number (LCN) analysis and species testing. Although this is the first time that the ISAG has conducted comparison tests for forensic testing, findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing. Key PointsComparison tests allow laboratories to evaluate their analyses for accuracy and reliability.Two forensic identification, parentage and species determination comparison tests were performed.The study showed that the LCN DNA analysis represented a significant challenge to most laboratories.Lacking familiarity with species tests curbed most laboratories from reporting accurately.A reliance on forensically validated testing standards may have prevented some of the common errors.

Background: In this study, we perform a search for variants (SNVs and InDels) in the genome of a Brazilian Naturalized horse breed, using FreeBayes and GATK variant calling tools. This breed presents exclusive adaptive traits of extreme importance to semi-arid conditions, such as those that allow survival under excessive sunlight, rainfall, low forage availability and stony ground. Moreover, these traits are expressed without any detriment to the performance and perpetuation of the breed. Results: A total of 305,588,364 reads were mapped in the horse reference genome, 1,598,210 single nucleotide variations and 138,139 insertions/deletions were detected by FreeBayes, 88,838 (SNVs) and 25,232 (InDels) by GATK. Both have been used in order to increase the safety of variant calls, identify in which regions of the genome they are present and check for variants in genes possibly associated with the peculiar traits exhibited by the breed. Conclusions: The variants annotation identified numerous non-synonymous SNVs and frameshift InDels, which could affect phenotypic variation. We found 28 and 392 Emsembl gene IDs containing high and moderate impact SNVs, including GTPase family members, olfactory receptors, mitochondrial complex and defense genes. Functional enrichment analysis was performed and revealed that variants in the olfactory transduction pathway were overrepresented.

Species substitution in meat products is a common problem reported worldwide. This type of food fraud is, typically, an intentional act for economic gain, using sources of low‐priced meats in high‐value meat products. Consequences include economic, health, and religious concerns. Highly sensitive and efficient techniques are thus required to detect meat species. This paper describes a method based on real‐time PCR to detect 10 animal species (Bos taurus, Sus scrofa, Ovis aries, Capra hircus, Gallus gallus, Meleagris gallopavo, Bubalus bubalis, Equus caballus, Felis catus, and Canis familiaris) in meat product. The method combines species‐specific and universal (used here as internal positive control) primers, and applies melt curve analysis for amplicon checking. Method accuracy was evaluated on 46 experimental meat mixtures and all species were correctly identified in all cases, at 1% test sensitivity. Analysis of 14 commercial meat products revealed that 6 of 14 samples had nondeclared bovine and/or chicken material. We performed an interlaboratory comparison using the reference meat mixtures and commercial samples, achieving 100% of reproducibility. The developed test proved to be effective and reliable for routine analysis of meat products. This paper describes a fast and reliable method for species detection in meat products based on real‐time PCR. It can be applied for analysis of in natura or processed meat. The method proposed here can play an important role in controlling the origin of meat products, ensuring their quality and safety for the entire food industry—producers to consumers.

Haemonchus placei (Nematoda:Trichostrongyloidea:Haemonchidae) is a parasite of cattle and other ruminants that reduces the weight gain and increases mortality causing significant economic losses to livestock worldwide. Here we sequenced and annotated its complete mitochondrion (mt) genome, obtaining a circular molecule of 13,664 bp long and 78.8% A + T content (KT955899). Besides, all 10 mt genomes publicly available for the Trichostrongyloidea superfamily were downloaded from GenBank database. In the comparison between the complete mt genome sequences of the 3 species from the Haemonchidae family, an AT-rich region of variable size was observed. A primer pair that amplifies this region was proposed to allow the molecular diagnostic and differentiation between H . placei and H . contortus infections. Furthermore, the evolutionary relationship between H . placei and other members of Trichostrongyloidea was inferred based on a phylogenomics approach. All mitochondrial gene sequences were aligned and then concatenated into a supermatrix dataset. A maximum likelihood tree was produced and confirmed the monophyly of Haemonchidae family. The superfamily Trichostrongyloidae was also recovered as a monophyletic clade. The complete mt genome sequence reported here may be used to develop new genetic markers and investigate phylogenetic and geographic relationships between isolates as well as to assess population diversity and allow health control programs monitoring.

The trade in live animals between India and Brazil dates from the late nineteenth century when European travellers traded animals of Indian origin for display in zoos. Considering the origin of coffee and sugar cane, as well as the expertise related to mineral evaluation, we need to consider that India was involved in important economic cycles of Brazil, even indirectly. This virtuous flow of trade has been maintained and intensified throughout modern history, especially after these two nations gained political independence from their colonisers, thereby becoming independent in mercantile affairs. This paper addresses the main points related to the use of animals of Indian origin in Brazil. We revisit some of the historical aspects of the process of colonisation of Brazil, as well as the importation of animals from India. The restrictions imposed on this process due to the occurrence of diseases in cattle and buffalo in India will be examined. At the end of the text, emphasis will be given to the risks of introducing exotic diseases into Brazil.

Standard diagnostic methods currently in use for the identification of helminth infections in ruminants are based on the morphological analysis of immature and adult stages of parasites. This paper describes a method for the semiquantitative identification of nematodes, mainly Trichostrongyloidea, at species-level resolution. The method is based on amplification and fragment analysis followed by minisequencing of the ITS-2 region (internal transcribed spacer 2) of the ribosomal DNA of parasite eggs or larvae. This method allows for the identification of seven genera (Chabertia, Cooperia, Haemonchus, Oesophagostomum, Ostertagia, Teladorsagia, and Trichostrongylus) and 12 species (Chabertia ovina, Cooperia curticei, Cooperia punctata, Cooperia oncophora/Cooperia surnabada, Haemonchus contortus, Haemonchus placei, Haemonchus longistipes, Oesophagostomum asperum, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei, and Trichostrongylus colubriformis) of infectious nematodes of domestic ruminants. The concordance between the morphological and molecular analyses in the detection of genera ranged from 0.84 to 0.99, suggesting the proposed detection method is specific, semiquantitative, less laborious, and highly cost-efficient.

Para avaliar a viabilidade da metodologia da Reação em Cadeia da Polimerase associada com o Polimorfismo de Fragmentos de DNA (PCR-RFLP) na identificação de fraude intencional e contaminação acidental em produtos cárneos de origem bubalina, in natura e processados, foram testadas amostras puras e amostras de carnes com misturas controladas, produzidas em laboratório, com adição de 1%, 5%, 10% e 50% de carne bovina em carne de búfalo, homogeneizada crua e em amostras autoclavada. Foram comparados, ainda, diferentes métodos de extração, usando um kit comercial e a técnica clássica, utilizando fenol/clorofórmio. O resultado estatístico foi obtido por tabela de contingência, analisada pelo teste do qui-quadrado (χ2) e do exato de Fisher. A especificidade encontrada foi altamente significativa (P

Species substitution in meat products is a common problem reported worldwide. This type of food fraud is, typically, an intentional act for economic gain, using sources of low-priced meats in high-value meat products. Consequences include economic, health, and religious concerns. Highly sensitive and efficient techniques are thus required to detect meat species. This paper describes a method based on real-time PCR to detect 10 animal species (Bos taurus, Sus scrofa, Ovis aries, Capra hircus, Gallus gallus, Meleagris gallopavo, Bubalus bubalis, Equus caballus, Felis catus, and Canis familiaris) in meat product. The method combines species-specific and universal (used here as internal positive control) primers, and applies melt curve analysis for amplicon checking. Method accuracy was evaluated on 46 experimental meat mixtures and all species were correctly identified in all cases, at 1% test sensitivity. Analysis of 14 commercial meat products revealed that 6 of 14 samples had nondeclared bovine and/or chicken material. We performed an interlaboratory comparison using the reference meat mixtures and commercial samples, achieving 100% of reproducibility. The developed test proved to be effective and reliable for routine analysis of meat products.This paper describes a fast and reliable method for species detection in meat products based on real-time PCR. It can be applied for analysis of in natura or processed meat. The method proposed here can play an important role in controlling the origin of meat products, ensuring their quality and safety for the entire food industry-producers to consumers.

Made available in DSpace on 2018-12-11T16:49:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Ministero dell’Istruzione, dell’Università e della Ricerca Background: The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. Methods: The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. Results: This 90K “SNP-Chip” was tested in several river buffalo populations and found to have *70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. Conclusion: The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised. AIA-LGS Associazione Italiana Allevatori–Laboratorio Genetica e Servizi Fondazione Parco Tecnologico Padano Bioinformatics core Parco Tecnologico Padano Animal Genomics and Improvement Laboratory BARC USDA-ARS Department of Animal Science Iowa State University Institute of Zootechnics Università Cattolica del S. Cuore ANASB-Associazione Nazionale Allevatori Specie Bufalina Universidade Estadual Paulista (UNESP) Câmpus de Araçatuba COMSATS Institute of Information Technology Dipartimento di Scienze Zootecniche ed Ispezione degli Alimenti Facoltà di Agraria Università degli Studi di Napoli Federico II Universidade Federal De Minas Gerais (UFMG) Affymetrix UK Ltd Davies Research Centre School of Animal and Veterinary Sciences University of Adelaide Department of Animal Science University of Arkansas Recombinetics Inc. Council on Dairy Cattle Breeding, 4201 Northview Dr. One Town Centre Universidade Estadual Paulista (UNESP) Câmpus de Araçatuba

Fraudulent species substitution in food products is a reality in many markets around the world. The consequences are serious and impact governments, consumers and producers. Molecular methods, including PCR-based techniques, are available to prevent species substitutions and to authenticate food content. Here, we describe a method for calculating the bovine and buffalo content in milk- and meat-derived food products. The method applies real-time PCR using primers designed to specifically amplify bovine or buffalo DNA. The amplification efficiencies of both primer sets were assessed using the TaqMan and SYBR Green systems. Both sets of primers showed satisfactory results. A quantification procedure is proposed and involves amplifying a sample with both primer sets and then normalizing the total DNA using the total non-normalized bovine and buffalo DNA. To correct for the potential deviations between the real and measured DNA quantity caused by biological differences between species, we propose the use of calibration curves generated from each analyzed matrix. These curves include a set of controlled admixtures of bovine and buffalo material. Using this method for dairy samples containing known bovine and buffalo content, the use of the calibration curve always approximated the measured to the expected quantities. This technique was then tested on commercial samples. Our method was efficacious and reliable and can be applied to the routine analysis of these products. Although we only tested the methodology on dairy products, it can also be applied to other food matrices, such as meat-derived products. A patent document for the technique has been submitted to the Brazilian Patent Office (INPI – Instituto Nacional da Propriedade Industrial – www.inpi.gov.br ) under the number 014110002844.

There is little published information on genetic covariance estimates among meat yield traits in Nile tilapia. Longitudinal genetic analyses to evaluate patterns of environmental and genetic covariances among such traits during growth would help to establish selection criteria and predict direct and correlated response to selection. Accordingly, longitudinal genetic studies from 106 to 245 days of age were performed by random regression models for carcass and fillet weights and yields in Nile tilapia. For each analysis, the statistical model included random family common environmental and genetic effects and assuming heterogeneity of residual variances. For carcass and fillet yield, genetic correlations between closely paired ages (from 140 to 200 days) were higher (> 0.80) than those separated by moderate (> 0.40) or longer (approaching zero) intervals. For carcass and fillet weight, genetic correlations when both ages were greater than 125 days post-hatching were larger than 0.60, and those when both ages were greater than 149 days post-hatching were larger than 0.80. Heritability estimates for fillet and carcass yields ranged from moderate (0.12) to high (0.52), suggesting the likelihood that selection programs could improve these traits. The manner in which fillets were removed seems to influence the results. Heritability estimates for carcass and fillet weight ranged from high (0.52) to low (0.01). The family common environmental effect was important for reducing the heritability estimates of these two traits, probably due to high correlation of each trait to body weight at slaughter, which is substantially affected by the common family environmental effect. Accordingly, to increase the accuracy of predicted breeding values for these traits, it is important to reduce family effects by minimizing the duration during which families are reared separately, then tagging individual fish and initiating communal stocking at as early a life stage as possible. Based upon estimated genetic parameters among the traits, direct selection for body weight, carcass weight and fillet weight is not recommended if the breeding goal is to increase fillet yield as a correlated response. Contrarily, the high genetic correlations between body weight and fillet and carcass weights (> 0.98) suggest that body weight could be used as a selection criterion to avoid slaughtering potential breeders to acquire data and to achieve higher selection intensity.

The accuracy of predicted breeding values used for selection in animal breeding programs is dependent upon the amount and quality of information, the adequacy of statistical models and the analytical methodology employed. Random regression models more adequately describe growth than repeatability and multitrait models; because they account for continuous changes in phenotype and for related genetic and environment effects while using all available information without pre-adjustment of the data. In this experiment, longitudinal genetic studies of body weight of Nile tilapia Oreochromis niloticus recorded between 106 and 245 days of age were conducted by fitting random regression models. Four models, inclusion or exclusion of a family effect and the assumption of homogeneity vs. heterogeneity of residual variance, were compared by Bayesian Information Criteria (BIC). The lowest BIC (best fit) was observed for the model considering heterogeneous residual variance and including a family effect. Heritability estimates for live body weight ranged from .02 to .52 during the age interval examined, and all estimates beyond 190 days of age were greater than .30. Part of the variance among families was attributable to direct additive genetic, common environmental, maternal and other heritable genetic effects. Thus, the inclusion of a family effect in the model would result in lower estimates of additive genetic variance, particularly at the beginning of the growth period, close to when fish were individually identified. Until that point, fish within family groups were kept in a common environment, which may explain the low heritability estimates obtained at the beginning of the experiment. Genetic correlations between body weights at different ages all were greater than .60, suggesting correlated response in body weight at older ages (245 days) could be achieved by selecting fish at earlier ages (106 days).

Background and Aims. Molecular markers have contributed to species authentication by flagging mislabeling and the misidentification of commercial landings. Such tools are of great value since the market substitution of fish of lower value for highly commercialized species is expected to become more pronounced due to a shortage of natural stocks.Materials and Methods. Here we report on the molecular identification 4results from processed fish products (i.e. fillets) and whole fishes sold in Brazilian markets under the common name surubim (Pseudoplatystoma spp.).Results. DNA barcoding revealed the incorrect labeling of around 80% of all samples analyzed, with mislabeling being more pronounced within fillets rather than whole fish.Conclusion. To our knowledge, this is the first report correlating the rate of fraud with processed fish products. The establishment of an official list of acceptable common names for freshwater fish and seafood is urgently needed in Brazil for further trade regulations to take place.

Background and aims. The application of DNA barcoding as a global standard for fish identification is probing diverse worldwide realms (Nearctic, Australian and the Neotropics) and environments (e.g. marine and freshwater). Comparing the patterns of sequence divergence among conspecific and congeneric taxa between realms can provide valuable information on recent evolutionary histories of lineages as barcode data accumulates. Materials and methods. Herein, we have analyzed over 100 species (around 50%) of the Neotropical fish fauna from the Sao Francisco River, in southeast Brazil. Our aims were to test the performance of DNA barcoding in this biodiversity-rich region, and to compare patterns of genetic divergence with previous studies. Results. The mean Kimura two-parameter distances within species, genera, families, orders, and classes were 0.5, 10.6, 21.0, 22.7, and 24.4%, respectively, with 100% of the species examined successfully differentiated by barcoding. With the exception of Astyanax bimaculatus lacustris, Piabina argentea, and Bryconamericus stramineus, all other species yield a single, cohesive cluster of barcode sequences. The average 'nearest-neighbor distance' was 11.12%, 21-fold higher than the mean within species distance of around 0.54%. In a few instances, deep lineage divergences among conspecifics (up to 10%) and congenerics (up to 22.9%) taxa were revealed. Conclusions. Reflecting possible cases of cryptic speciation and the deeper phylogeographic history of Sao Francisco fish fauna, with some higher clades extending back into the late Cretaceous and Cenozoic (90 mya), when much of the diversification of the Neotropical region apparently took place. In addition, barcodes also highlighted misidentifications and helped to document range extensions for known species.

Foram utilizados 159 cavalos Pampa, registrados na Associação Brasileira dos Criadores de Cavalo Pampa, e um grupo-controle, de 32 cavalos da raça Paint, ambos os grupos provenientes de plantéis de diferentes regiões brasileiras, com o objetivo de comparar os testes bioquímico e molecular para detecção de marcadores genéticos para pelagem tobiana em cavalos Pampa. Houve diferença significativa (P

Avaliou-se a variação da resposta secundária ao estresse causado por hipóxia durante 18 dias, em sistema de recirculação, em uma linhagem de tilápia nilótica (Oreochromis niloticus), chitralada, refletida no perfil proteico eletroforético do peixe e avaliou-se a diferença entre sexos para essa resposta. Foram utilizados 126 peixes, sendo 60 machos e 66 fêmeas, ambos com média de peso de 800g. O estresse crônico por hipóxia alterou (P

Knowledge of genetic diversity and relationships between breeds is very important for conservation programs. Hair samples were collected from 393 individual Nordestino horses and genotyped using 14 microsatellite markers in order to investigate the genetic relationship between this breed and 66 international horse breeds. There was high allelic diversity and inbreeding coefficient within population values were not significant, which was probably due to crossbreeding. Despite the Nordestino horse population being in Hardy-Weinberg equilibrium, a global deficit of heterozygotes was observed. This may represent evidence of repeated use of the same stallions for breeding, which is consistent with the high number of castrated males found. Campolina, Mangalarga Marchador, and Mangalarga were the Brazilian horse breeds most closely related to the Nordestino horse, which is a reflection of recent introgressions. Among Iberian horse breeds, the Sorraia breed appears to have had an important influence on the genetics of the Nordestino horse. Those results provide important information that can guide future conservation programs.

The frequency of the normal myostatin gene (GDF-8) and the mutant allele in Marchigiana herds was detected. Three hundred and seventy-seven animals of Marchigiana breed raised in Sao Paulo and Parana States, Brazil, were tested. The results showed that 37.9% were homozygous normal animals, 55.2% heterozygous and 6.9% homozygous double muscling. The results suggest the interest of the breeders in having interest in the character double muscling, randomly promoting, the selection in favor of the mutation.

The effect of the bovine major histocompatibility complex (BoLA) locus on animal health may be due to a direct action of its alleles on immune functions, whereas its indirect effect on production traits might be explained by the better general health conditions of more productive animals. In the present study, the BoLA-DRB3 gene was investigated in 1058 cows belonging to seven Brazilian Gyr Dairy herds (Bos indicus, Zebu cattle). A total of 37 alleles were identified, 15 of them described for the first time in a Zebu breed. A highly significant association (p < 0.02) was observed between allele *54 and a decrease (-26.1 kg) in milk protein yield and there was a significant association (p < 0.05) between this allele and lower (-26.07 kg) milk fat yield. There was also a significant association (p < 0.05) between allele *6 and decreased (-12.47 kg) milk protein and allele *7 and increased (12.72 kg) milk protein. There were also indications of association (p < 0.10) between somatic cell score (SCS) and alleles *3 (SCS increased by 0.54 units) and *31 (SCS increased by 0.46 units). The highly significant association of allele *54 with lower protein yield suggests the possible use of this allele in marker-assisted selection programs.

DNA samples of six bovines obtained from three tissues (blood, semen and hair) were extracted using two different techniques. After the extraction procedures the samples were divided in six fractions. Three were stored at -20° C and three at 4° C. Every three months one sample of each tissue/extraction procedure was analyzed in spectrophotometer, to determine the quantity of the DNA and the extract was amplified using the primer RM 29. No differences in the DNA quantity or in the level of protein contamination among the three periods of analyses were observed. All the DNA extracted by quick extraction technique showed good amplification patterns during the nine months, meaning that this technique can be used in laboratory routine instead of the permanent extraction technique. The extract obtained from blood, using the permanent extraction technique, showed the higher quantity of DNA with the smaller index of protein contamination. The high quantity of protein contamination found in the semen samples preserved in egg yolk demanded modifications in both extraction techniques. After that the results were positive, showing good amplification patterns.

To evaluate the precision of the DNA tests using the non-automatized technique for individual identification and parentage tests, 105 Rottweiler dogs were studied using the primer CMR S. The sample was composed of 39 animals belonging to 11 complete families and their progenies, and 66 non related individuals until the second generation, derived from kennels located in the states of Minas Gerais and Sao Paulo. The CMR S primer was used for the Polimerase Chain Reaction (PCR). The results showed the inefficiency of the technique, even when analyzed through the automated gel analysis system. Also showed the impossibility of its commercial use due to the fact of does not permit the storage of data for subsequent use.

This study analyzed four remnant subpopulations of Nordestino horse breed to detect genetic structure and diversity through 14 microsatellite markers. Hair root follicles from a total of 393 horses were collected. There were 61 animals from Salitre Valley (JUAZ-BA) located at Bahia state, 89 from North and Central North ecoregions located at Piauí state (NCEN-PI), 185 animals from Sertão and Sertão do São Francisco ecoregions (SERT-PE) and 58 animals from Agrestina city (AGRE-PE) located at Pernambuco state. Genetic diversity, genetic differentiation and bottleneck effects were examined in the 4 remnant subpopulations of Nordestino horse breed. There was high allelic diversity and the Fis value did not show evidence of a significant predominance of mating among relatives, probably because of crossbreeding among populations. Recent bottleneck effects were not detected in the 4 subpopulations, but the IAM and TPM model did suggest a bottleneck effect. This may be a reflection of the decreased number of breeding animals caused by castration of males, mechanization processes and changes in life style in the rural areas. The bottleneck event was not enough to lead a genetic differentiation among the 4 remnant subpopulations of Nordestino horse. There was no evidence of genetic differentiation, so the 4 subpopulations formed one genetic group. O presente trabalho teve como objetivo avaliar a estrutura e diversidade genética de quatro subpopulações do cavalo Nordestino utilizando 14 marcadores microssatélites. Amostras de bulbo capilar de 393 cavalos foram coletadas, distribuídos em: 61 animais do vale do Salitre (JUAZ-BA) localizado no estado da Bahia, 89 das mesorregiões Norte e Centro-Norte do estado do Piauí, 58 animais da cidade de Agrestina (AGRE-PE) e 185 das mesorregiões Sertão e Sertão do São Francisco localizado no estado de Pernambuco (SERT-PE). A diversidade genética, diferenciação genética e efeito de gargalo genético foram avaliados nas 4 subpopulações do cavalo Nordestino. Observou-se elevada diversidade genética e valores de Fis que não evidenciaram níveis de consanguinidade significativa, provavelmente isso se deve à acasalamentos entre as subpopulações. Efeito de gargalo genético recente não foi detectado para as quatro subpopulações, porém os modelos IAM e TPM sugeriram um efeito de gargalo genético não recente. Isso pode ser reflexo da redução no número de reprodutores decorrente da castração de machos, processo de mecanização e mudanças no estilo de vida das populações da zona rural. Eventos mais antigos do efeito de gargalo genético não foram suficientes para evidenciar uma diferenciação genética entre as 4 subpopulações, portanto elas formam um só grupo genético.

An important step in invasive biology is to assess biological variables that could be used to predict invasion success. The study of genetics, evolution, and interactions of invasive and native species in invaded ranges provides a unique opportunity to study processes in population genetics and the capability of a species' range expansion. Here, we used information from microsatellite DNA markers to test if genetic variation relates to propagule pressure in the successful invasion of an apex predator (the Amazonian cichlid Cichla) into Southeastern Brazilian River systems. Invasive populations of Cichla have negatively impacted many freshwater communities in Southeastern Brazil since the 1960s. Reduction of genetic variation was observed in all invasive populations for both Cichla kelberi (CK) and Cichla piquiti(CP). For instance, heterozygosity was lower in the invasive range when compared to native populations from the Amazon basin (CP HE= 0.179/0.44; CK HE= 0.258/0.536 respectively). Therefore, despite the successful invasion of Cichla in southeast Brazil, low genetic diversity was observed in the introduced populations. We suggest that a combination of factors, such as Cichla's reproductive and feeding strategies, the "evolutionary trap" effect and the biotic resistance hypothesis, overcome their depauperete genetic diversity, being key aspects in this apex predator invasion. Uma importante etapa na biologia da invasão é acessar variáveis biológicas que podem predizer o sucesso de invasão. O estudo da genética, evolução e interações entre invasores e espécies nativas no ambiente invadido pode prover uma oportunidade única para o estudo dos processos em genética de populações e a capacidade de uma espécie ampliar seu habitat. Nesse trabalho, nos utilizamos dados de marcadores de DNA microssatélites para testar se a variação genética é relacionada a pressão de propágulo na invasão bem sucedida do predador de topo (o ciclídeo Amazônico Cichla) nos rios do Sudeste Brasileiro. Populações invasoras de Cichla vem impactando negativamente diversas comunidades de água doce no Sudeste brasileiro deste 1960. A redução da variação genética foi observada em todas populações invasoras, tanto para Cichla kelberi (CK) como Cichla piquiti (CP). Por exemplo, a heterozigose foi menor no ambiente invadido quando comparada com as populações nativas da bacia Amazônica (CP HE = 0.179/0.44; CK HE = 0.258/0.536 respectivamente). Assim, apesar do sucesso da invasão de Cichla no sudoeste do Brasil, baixa diversidade genética foi observada nas populações introduzidas. Nós sugerimos que uma combinação de fatores, como as estratégias reprodutivas de Cichla, o efeito de "armadilha evolutiva" e a hipótese de resistências biótica superam o efeito que a diversidade genética depauperada exerce, sendo aspectos-chave na invasão desse predador de topo de cadeia.

Neste estudo foram genotipados 205 cavalos da raça Árabe, criados nos estados de Minas Gerais e São Paulo, via DNA por meio de PCR, para determinação da presença do gene mutante SCID. Os resultados mostraram 98,5% de animais normais (202/205) e 1,5% de portadores (3/205). Pela análise da genealogia dos portadores identificados pode-se, ainda, confirmar a participação de um garanhão anteriormente identificado como provável disseminador da doença. In the present study, 205 Arabian horses raised in Minas Gerais and São Paulo states, Brazil, were genotyped using the PCR technique. The results showed 98.5% of normals (202/205) and 1.5% of SCID carriers (3/205). Checking the pedigrees of the three carriers was possible to confirm the participation of one stallion first identified as the possible disease disseminator.

Foram analisados 138 resultados de tipagem sangüínea de animais da raça Gir Leiteiro, pertencentes a uma população fechada submetida a acasalamentos preferenciais por aproximadamente 30 anos. Estudou-se a associação entre os sistemas B, F, J, L e Z de grupos sangüíneos e a produção de leite à primeira lactação. Os resultados mostraram diferença significativa (P

A set of primers to amplify 10 microsatellite DNA loci was developed for the Neotropical fish Cichla piquiti, one of the largest sized cichlids in the Amazon Basin. These loci were used to genotype individuals from two populations, one native population from the Tocantins River, the other an introduced population in southeast Brazil, Upper Paraná River. Cross-amplification was also successful for another species of peacock bass, C. kelberi. An average of 4.4 alleles per locus (2-9 alleles) was detected. These markers will be useful for the characterization of genetic structure of native populations, and also for invasive biology studies since Cichla species have been introduced in many river basins outside their native ranges.

The Brazilian cattle population is mainly composed of breeds of zebuine origin and their American derivatives. Comprehensive knowledge about the genetic diversity of these populations is fundamental for animal breeding programs and the conservation of genetic resources. This study aimed to assess the phylogenetic relationships, levels of genetic diversity, and patterns of taurine/zebuine admixture among 9 commercial cattle breeds raised in Brazil. Analysis of DNA polymorphisms was performed on 2965 animals using the 11 microsatellite markers recommended by the International Society of Animal Genetics. High genetic diversity was detected in all breeds, even though significant inbreeding was observed within some. Differences among the breeds accounted for 14.72% of the total genetic variability, and genetic differentiation was higher among taurine than among zebuine cattle. Of note, Nelore cattle presented with high levels of admixture, which is consistent with the history of frequent gene flow during the establishment of this breed in Brazil. Furthermore, significant genetic variability was partitioned within the commercial cattle breeds formed in America, which, therefore, comprise important resources of genetic diversity in the tropics. The genetic characterization of these important Brazilian breeds may now facilitate the development of management and breeding programs for these populations.

Anthelmintic resistance is an increasing problem that threatens livestock production worldwide. Understanding of the genetic basis of benzimidazole resistance recently allowed the development of promising molecular diagnostic tools. In this study, isolates of Haemonchus contortus obtained from goats, sheep and buffaloes raised in Brazil were screened for presence of the polymorphism Phe200Tyr in the β-tubulin 1 gene, which confers resistance to benzimidazole. The allelic frequency of the mutation conferring resistance ranged from 7% to 43%, and indicated that resistance to benzimidazole could be found in nematodes isolated from all the ruminant species surveyed. Although significant variation in the frequency of the F200Y mutation was observed between different herds or host species, no significant variation could be found in populations isolated from animals within the same herd. These findings suggest that screening of samples from a few animals has the potential to provide information about the benzimidazole resistance status of the entire herd, which would enable a considerable reduction in the costs of diagnosis for the producer. Molecular diagnosis has practical advantages, since it can guide the choice of anthelmintic drug that will be used, before its application in the herd, thus reducing the economic losses driven by anthelmintic resistance. A resistência aos anti-helmínticos é um problema crescente que ameaça a produção pecuária em todo o mundo. A compreensão da base genética da resistência ao benzimidazol permitiu, recentemente, o desenvolvimento de métodos diagnósticos moleculares promissores. Neste estudo, isolados deHaemonchus contortus obtidos a partir de rebanhos de caprinos, ovinos e bubalinos criados no Brasil foram avaliados quanto à presença do polimorfismo F200Y no gene da β-tubulina1, o qual confere resistência ao benzimidazol. A frequência alélica da mutação variou de 7% a 43%, indicando que a resistência ao benzimidazol pode ser encontrada em nematoides isolados a partir de todas as espécies de ruminantes pesquisadas. Embora tenha sido observada variação significativa das frequências de mutação F200Y entre rebanhos/espécies hospedeiros distintos, não foi encontrada variação significativa entre populações isoladas de animais dentro de um mesmo rebanho. Estes achados sugerem que a avaliação de amostras de alguns poucos animais tem o potencial de fornecer informações sobre o nível de resistência ao benzimidazol de todo o rebanho, possibilitando uma redução considerável dos custos de diagnóstico para o produtor. O diagnóstico molecular apresenta vantagens práticas, uma vez que pode guiar a escolha da base anti-helmíntica a ser utilizada antes da sua aplicação no rebanho, reduzindo, portanto, as perdas ocasionadas pela resistência aos fármacos anti-helmínticos.

Parasitic nematodes of the genus Haemonchus infect a range of ruminant hosts and are of major veterinary and economic importance. In this study, the genetic variability of seven isolates of Haemonchus placei and Haemonchus contortus was evaluated using the mitochondrial gene cytochrome oxidase subunit I and the nuclear gene β-tubulin isotype 1. A total of 156 specimens were obtained from cattle, sheep, goat and buffalo herds raised on commercial properties from the southern and southeastern regions of Brazil and identified to the species level by sequencing of the nuclear internal transcribed spacer 2. Thirty-four percent of the specimens were identified as H. placei and 66% as H. contortus. Cattle were the preferred hosts for H. placei, whereas H. contortus was most frequent in the other three ruminant species. Analysis of genetic differentiation between isolates revealed that high rates of gene flow are operating among populations of both nematode species, including among those from different ruminant host species. Populations of H. placei were less polymorphic and presented a lower frequency of single nucleotide polymorphisms associated with benzimidazole resistance compared with H. contortus. In line with the low amount of genetic structure observed among isolates, alleles of β-tubulin1 associated with benzimidazole resistance were present at relatively high frequencies of 5–20% in isolates of H. contortus from farms that never used this class of anthelmintic. The results presented here are consistent with the hypothesis of multiple origins of alleles associated with benzimidazole resistance, with the trade of animals among properties acting as the main factor promoting the spread of anthelmintic resistance.

Foram testados três métodos de extração de DNA em amostras de queijo, com o objetivo de identificar uma técnica eficiente para extração de DNA em amostras com várias limitações, como alto teor de gordura, alto grau de degradação do DNA e grande concentração de impurezas. A técnica que faz uso do tiocianato de guanidina mostrou-se mais adequada para identificação de adição intencional não declarada de leite bovino em queijos bubalinos, podendo ser empregada para certificação de produto específico (selo de Identidade de Espécie). Three methods of DNA extraction were tested in cheese samples. The objective of this study was to identify an efficient technique for DNA extraction in different samples with several limitations, such as high fat tenor, high degree of DNA degradation and great sludge concentration. The technique using Guanidine thiocyanate was more appropriate for identification of intentional undeclared addition of bovine milk in buffalo cheeses. This technique can be used for certification of a specific product (stamp of Identity of Species).

A molecular phylogenetic analysis based on mitochondrial 16S ribosomal DNA and Control Region sequences from native and introduced populations was undertaken, in order to characterize the introduction of Cichla (peacock bass or tucunaré) species in Brazil. Mitochondrial DNA haplotypes found in introduced fish from Minas Gerais state (southeastern Brazil) clustered only with those from native species of the Tocantins River (Cichla piquiti and C. kelberi), thereby suggesting a single or, at most, few translocation acts in this area, even though with fish from the same source-population. Our study contributes to an understanding of the introduction of Cichla in regions of Brazil outside the Amazon basin, and adds phylogenetic data to the recently describe Cichla species, endemic from the Tocantins-Araguaia basin.

Utilizaram-se 195 cavalos Pampa e um grupo-controle de 41 cavalos da raça Paint, provenientes de plantéis de várias regiões brasileiras, com o objetivo de avaliar a eficiência do teste mediante uso de marcadores bioquímicos: albumina (Al) e proteína de ligação da vitamina D (Gc), para identificação dos possíveis indivíduos homozigotos dominantes para o padrão de pelagem tobiano nos cavalos Pampa. Não foram encontrados genótipos AlBB e GcSS, revelando indício de quebra de ligação gênica entre tais locos e o loco tobiano e a ineficácia do teste bioquímico na detecção dos prováveis indivíduos homozigotos dominantes para o padrão de pelagem tobiano nos cavalos Pampa. One hundred and ninety five Pampa horses and a control group of 41 Paint Horses originated from herds located in different Brazilian regions were used to objective of evaluate the efficiency of the biochemical markers albumin (Al) and vitamin D binding protein (Gc) to identify the probable homozygous dominant for the tobiano coat color pattern in Pampa horses. It was not found any genotype AlBB and GcSS, indicating a possible break of the genetic linkage between these loci and the locus Tobiano, as well as the inefficacy of the biochemical test in the detection of the probable homozygous dominant for the tobiano color pattern in Pampa horses.

Foram caracterizados geneticamente, utilizando-se cinco locos de microssatélites, 235 indivíduos de seis plantéis de tilápia (Ceará, Chitralada, Israel, Nilótica, Taiwan e Vermelha) da região Sudeste do Brasil. Verificou-se diferença genética entre os seis plantéis, obtida pelo cálculo do índice de fixação de alelos (Fst=0,3263). De modo geral, está ocorrendo perda de heterozigose nos plantéis, segundo mostrou a estimativa do coeficiente de endogamia intrapopulacional (Fis=0,0486). Os plantéis Israel e Nilótica foram os mais semelhantes geneticamente (Ig=0,6663). Os plantéis Chitralada e Taiwan foram os que menos apresentaram genes em comum (Ig=0,2463). O plantel denominado Vermelha foi o mais distinto entre todos. Two hundred and thirty five individuals from six commercial stocks of tilapias (Ceará, Chitralada, Israel, Nilótica, Taiwan and Red) from the Southeastern region of the country were genetically characterized using five microsatellite loci. The results suggest large genetic difference among the stocks, estimated through the fixation allele index (Fst = 0.3263), and a considerable loss of heterozigosity accurs in most of the stocks, according to the population inbreeding coefficient (Fis=0.0486). The Israel and Nilótica stocks were genetically similar (Ig=0.6663), while Chitralada and Taiwan showed less genes in common (Ig=0.2463). The Red stock was the most distinct stock.

Natural resistance against brucellosis in cattle is linked to the Nramp1 gene, which encodes a divalent cation transporter that localizes in the phagolysosome membrane in macrophages. Nramp1 gene in mouse plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its influence on adaptative immunity. Polymorphisms at the bovine Nramp1 3′ untranslated region (3′UTR), detectable by Single Strand Conformational Analysis (SSCA), are associated with natural resistance against brucellosis. Such polymorphisms are associated with variation in the number of GT repeats. This study compared the frequency of Nramp1 3′UTR polymorphisms between Zebu and European bovine breeds. Eighty-one Holsteins ( Bos taurus taurus ) and 167 Zebu ( Bos taurus indicus ), including the following breeds: Nelore ( n = 95), Guzera ( n = 37), and Gir ( n = 35), totaling 248 pure breed cattle studied. DNA extraction was performed using the guanidium protocol and genotyping was performed by SSCA. DNA from cattle considered genotypically resistant to brucellosis resulted in a single band (homozygous) with 175 bp, corresponding to the 3′UTR with 13 GT pairs (GT13), whereas DNA from genotypically susceptible cattle generated one single band with 177 bp (homozygous GT14) or double bands with both 175 and 177 bp, or 175 and 179 bp (heterozygous GT13/GT14 or GT13/GT15, respectively). A marked difference in the frequency of alleles was detected between the Zebu and Holstein cattle. Holsteins had an extremely homogeneous genotype, with 100% of the individuals with a GT13 genotype. In sharp contrast the Nelore breed had the most heterogeneous genotype with four allelic combinations, namely, homozygous GT13, homozygous GT14, heterozygous GT13/GT14, and heterozygous GT13/GT15. When the Zebu breeds were compared to each other, the only significant difference observed was the frequencies of the genotypes GT13 and GT14 between the Nelore and Guzera breeds. The knowledge of allelic frequencies in different breeds of cattle may prove to be very useful in the future for planning breeding strategies for selection of resistant cattle.

Foram utilizados 46 animais da raça Gir, registrados na Associação Brasileira de Criadores de Zebu, provenientes de cinco fazendas situadas no Estado de Minas Gerais, com o objetivo de avaliar a eficiência dos microssatélites BM2113, ILSTS005, ILSTS008, ETH131 e RM88 em testes de verificação de parentesco. Os locos BM2113, ILSTS005, ETH131 e RM88 mostraram-se eficientes, apresentando valores de PE2 (probabilidade de exclusão quando os dois progenitores são genotipados) entre 0,62 e 0,69 e PIC2 (conteúdo de informação polimórfica quando os dois progenitores são genotipados) entre 0,78 e 0,83. O mesmo não ocorreu para o loco ILSTS008, o qual apresentou baixos valores de PE2 (0,24) e PIC2 (0,41). Forty six animals of the Gir breed, registered at the Brazilian Association of Zebu Breeders, coming from five farms located in Minas Gerais State, were used to analyze the efficiency of the microsatellites BM2113, ILSTS005, ILSTS008, ETH131 and RM88 in parentage tests. The loci BM2113, ILSTS005, ETH131 and RM88 showed to be efficient, presenting values of PE2 (exclusion probability when both parents are genotiped) between 0.62 and 0.69 and PIC2 (polymorphic information contents when both parents are genotiped) between 0.78 and 0.83. The same was not observed for the locus ILSTS008 that showed low values of PE2 (0.24) and PIC2 (0.41)

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A chamada de variantes genômicas, a fim de identificação de novas variantes de nucleotídeo único (SNVs) e inserções/deleções (InDELs), potencialmente associadas às características de rusticidade e resistência a ambientes áridos em equinos da raça Nordestina, a partir de dados de sequenciamento de genoma completo foi a estratégia utilizada no presente estudo para compreensão dos mecanismos adaptativos da raça para sobrevivência no semiárido do nordeste brasileiro. Essas variantes podem estar associadas à resistência exibida pelo cavalo Nordestino, por meio do padrão racial, que é altamente especializado para o desempenho de atividades que exigem força e resistência, sem quaisquer prejuízos à sua perpetuação. O genoma de um representante típico da raça foi sequenciado, sendo este o primeiro trabalho de sequenciamento de genoma completo de uma raça equina naturalizada brasileira, que utilizou a estratégia de genômica comparativa para chamada de variantes, usando-se como referência a mais recente atualização do genoma equino disponível (Ensemble, EquCab 3.0). A chamada de variantes genômicas por ferramentas baseadas em algoritmos identificou 1.598.210 SNVs e 138.139 InDels pelo software Freebayes e 88.838 SNVs e 25.232 InDELs pelo software GATK. As variantes foram classificadas quanto ao tipo, localização e efeito de impacto e foi feita a anotação genômica das regiões onde estão localizadas. Aquelas de impacto alto e moderado foram selecionadas para análise de enriquecimento funcional e para validação populacional em 60 animais provenientes dos estados da Bahia, Pernambuco e Piauí. SNVs de impacto alto ocorreram exclusivamente em genes de GTPases. Genes da família de Receptores Olfativos (OR), que continham SNVs de impacto moderado tiveram maior representatividade na análise funcional. Todos os dez loci testados, contendo SNVs características da raça apresentaram alelos polimórficos nas três populações, com frequências genotípicas elevadas de indivíduos heterozigotos e homozigotos contendo polimorfismo alélico. As frequências genotípicas de oito loci analisados foram significativamente diferentes entre as três populações, sugerindo a fixação populacional dos alelos polimórficos e diversidade genética. A presença de SNVs em OR genes, que estão diretamente relacionados aos mecanismos adaptativos, representa importantes informações genéticas para compreender a adaptação da raça ao semiárido do nordeste brasileiro e para associá-las ao fenótipo peculiar exibido por essa raça. Genomic variant calling, in order to identify new single nucleotide variants (SNVs) and insertions/deletions (InDELs), potentially associated with the characteristics of rusticity and resistance to arid climate in Nordestino horse breed, based in whole genome sequencing was the strategy used in the present study to understand the adaptive mechanisms of the breed to semiarid climate of Brazilian northeastern. These variations are possibly associated with the resistance exhibited by the breed, through the highly specialized racial pattern for the performance of activities that require strength and endurance, without any damage to its perpetuation. Complete genome sequencing of a typical representative of the breed was carried out, this being the first whole genome sequencing of a Brazilian naturalized equine breed, as a comparative genomics strategy for variants calling with the most recent reference genome equine update (Ensemble EquCab 3.0). Tools based on algorithms for variants calling identified 1,598,210 SNVs and 138,139 InDels (Freebayes software) and 88,838 SNVs and 25,232 InDELs (GATK software). Variants were classified as to the type, location and impact effect and the genomic regions were anoted. High and moderate impact variants were selected to functional enrichment analysis and for population validation in 60 animals from the states of Bahia, Pernambuco and Piaui. High-impact SNVs occurred exclusively in GTPase genes. Olfactory Receptor (OR) genes family containing moderate-impact SNVs had high representativeness in the functional analysis. All 10 loci tested, containing breed-specific SNVs, presented polymorphic alleles in the three populations, with high genotypic frequencies of heterozygous and homozygous with allelic polymorphism. The genotypic frequencies for 8 loci were significantly different between the three populations, suggesting allelic fixation and genetic diversity. The presence of SNVs in OR genes, which are related to adaptive mechanisms, represents important genetic information to understand the adaptation of the breed to the semiarid region of northeastern Brazil and to associate them with the peculiar phenotype exhibited by that breed.

A identificação de espécies em alimentos é assunto de grande interesse para toda a cadeia de produção, para a fiscalização e nos meios acadêmicos. As fraudes pela adição ou substituição de espécies infringem as legislações vigentes no Brasil e podem trazer graves consequências, incluindo perdas econômicas e riscos à saúde pública. Diversas técnicas estão disponíveis para a identificação da origem dos alimentos. Dentre essas, os métodos baseados na análise de DNA provaram ser práticos, confiáveis, específicos e extremamente sensíveis para a detecção de fraude. O presente trabalho teve como objetivo o desenvolvimento de teste de detecção de DNA das espécies Bos taurus, Sus scrofa, Ovis aries, Capra hircus, Gallus gallus, Meleagris gallopavo, Bubalus bubalis, Equus caballus, Felis catus e Canis familiaris em produtos cárneos. O método baseia-se na técnica de PCR em Tempo Real e combina o uso de iniciadores universais (utilizados como controle interno) e iniciadores específicos para a detecção do DNA alvo. Após cada reação de PCR, os produtos são submetidos à curva de dissociação, para confirmar se o DNA amplificado corresponde ao fragmento esperado de cada espécie. A precisão do método foi avaliada em 45 misturas experimentais de carnes nas proporções de 50-50% ou 99-1% e em uma mistura com 10% de cada espécie do teste. O método detectou corretamente as espécies em cada mistura, inclusive aquelas presentes em 1% do total. Em seguida, o método foi aplicado na análise de 14 produtos comerciais e os resultados revelaram que seis desses continham material bovino e/ou de frango, não declarado no rótulo. Os testes das misturas experimentais e dos produtos comerciais foram realizados nos dois laboratórios participantes (LGEV e Myleus), obtendo-se 100% de reprodutibilidade. O teste desenvolvido provou ser eficaz e confiável para uma rotina de detecção de espécies em produtos cárneos Species identification in food is a subject of significant interest for the entire production chain, for government inspection and in academic circles. Fraud by addition or substitution of species is against the law in Brazil and can lead to serious consequences, including economic losses and public health risks. Several methods are available for identifying the origin of food and, among these, the DNA techniques have proven to be practical, reliable, specific and highly sensitive for fraud detection. The present work is aimed at the development of a test to detect DNA from Bos taurus, Sus scrofa, Ovis aries, Capra hircus, Gallus gallus, Meleagris gallopavo, Bubalus bubalis, Equus caballus, Felis catus and Canis familiaris in meat products. The method is based on real-time PCR and combines the use of universal primers (for internal positive control) and species-specific primers for the detection of target DNA. After each reaction, the PCR products are subject to melt curve analysis to check if the amplified DNA corresponds to the expected fragment of each species. Method precision was evaluated on 45 experimental meat mixtures in proportions of 50-50% or 99-1% plus one mixture with 10% of each species. The test correctly detected the species in each mixture, including those present in 1% of the total content. The method was then applied to the analysis of 14 commercial meat products and the results revealed that six of these had non-declared bovine and/or chicken material. The experimental mixtures and commercial products analyses were carried out in the two participating laboratories (LGEV and Myleus), obtaining 100% reproducibility. The developed test proved to be effective and reliable for a routine of species detection in meat products

O presente estudo descreve a otimização e padronização do método de identificação de espécies de peixes por DNA Barcode bem como a sua validação segundo os requisitos da ABNT NBR ISO/IEC 1705 para acreditação junto ao INMETRO e posteriormente credenciamento junto ao MAPA com o objetivo de prestar o serviço de identificação, podendo auxiliar os empresários na escolha de seus fornecedores e controle de qualidade de seus produtos, bem como os consumidores a terem maior confiança no que estão consumindo. Pode ainda, auxiliar o governo nos testes de fiscalização de produtos para evitar a fraude, além de regular com maior eficácia produtos exportados e importados. Propõe também um novo método para identificação molecular de trutas e salmão em nível de gênero e/ou de espécies. Esse método é baseado na amplificação seguida de minissequenciamento de parte da região D-loop (Região controle) do DNA mitocondrial dos peixes. Por meio desse método, é possível identificar os dois gêneros e as quatros espécies de salmão e truta mais prevalentes, Salmo salar, Oncorhynchus keta, O. gorbuscha e O. mykiss. The present study describes the optimization and standardization of the method of identification of fish species by DNA Barcode as well as its validation according to the requirements of ABNT NBR ISO / IEC 1705 for accreditation with INMETRO and later accreditation with the MAPA with the objective of providing the identification service, which can help entrepreneurs choose their suppliers and quality control of their products, as well as consumers to have greater confidence in what they are consuming. It can also assist the government in testing products to prevent fraud, as well as regulate products exported and imported more effectively. It also proposes a new method for molecular identification of trout and salmon at the level of genus and / or species. This method is based on amplification followed by minissequencing of part of the D-loop region of the mitochondrial DNA of the fish. By means of this method, it is possible to identify the two genera and the four most prevalent salmon and trout species, Salmo salar, Oncorhynchus keta, O. gorbuscha and O. mykiss.

Impactos antropogênicos são uma ameaça crescente para a diversidade de peixes, especialmente em áreas em torno de grandes centros urbanos, e muitas ações efetivas de conservação dependem de identificação precisa das espécies. Considerando a utilidade do DNA Barcode como um sistema global de identificação e descoberta de espécies, este estudo tem como objetivo caracterizar molecularmente algumas espécies de peixes ornamentais e depositar sequências parciais do gene COI em uma biblioteca de referência. Seguindo essa metodologia, um fragmento da sub-unidade I da citocromo oxidase foi amplificado e sequenciado bidirecionalmente e a partir de 298 indivíduos. Uma análise de Neighbor-Joining revelou que esta abordagem pode discriminar inequivocamente todas as espécies pesquisadas. A maioria das espécies exibiu baixa distâncias genéticas intraespecíficas (1,53%), cerca de 9 vezes menor do que a distância entre as espécies dentro de um gênero. A biblioteca elaborada nesse estudo é um primeiro passo para conhecer melhor a diversidade molecular de peixes ornamentais, fornecendo uma base para futuros estudos sobre essa fauna - estendendo a capacidade de identificá-los em todas as fases da vida e até mesmo em restos ou fragmentos, fornecendo dados para uma melhor compreensão das interações entre as espécies, gerando estimativas sobre a composição de espécies e riqueza em um ecossistema, provendo ferramentas de autenticação de bioprodutos e monitoramento exploração ilegal de espécies. Anthropogenic impacts are a rising threat to the diversity of fish, especially in areas around urban centers, and many effective conservation actions depend on accurate identification of species. Considering the usefulness of DNA Barcode as a global system of identification and discovery of species, this study aims to molecularly characterize the species of ornamental fish and provide partial COI gene sequences in a reference library. Following this methodology, a fragment of the subunit I of cytochrome oxidase gene was amplified and sequenced bidirectionally from 298. A Neighbor-Joining analysis showed that this approach can clearly discriminate all the surveyed species. Most of the species showed low intraspecific genetic distances (1.53%), about 9-fold less than the distance between species within a genus. The library developed in this study is a first step to better understand the molecular diversity of ornamental fish, providing a basis for future studies on this fauna - extending the ability to identify them in all life stages and even remnants or fragments, providing data for a better understanding of the interactions between species, generating estimates of the species composition and richness in an ecosystem, providing bioproducts authentication tools and monitoring illegal exploitation of species.

O objetivo do presente trabalho foi a padronização do painel de microssatélites para testes de identificação genética e de paternidade bubalina, bem como o uso do mesmo em estudo de caracterização genética, por meio das análises de variabilidade e distância entre as quatro raças bubalinas reconhecidas pela Associação Brasileira de Criadores de Búfalos (Carabao, Jafarabadi, Mediterrânea e Murrah) e de mestiços de búfalos criados no Brasil. Foram avaliados 590 búfalos por meio de 20 marcadores microssatélites recomendados pela ISAG/2010 (International Society Animal Genetics). O painel principal padronizado com 13 marcadores microssatélites apresentou elevado PIC (0,6). Os dados confirmam a confiabilidade e a eficiência dos marcadores analisados tanto para testes de identificação genética quanto para o de genealogias. Para a caracterização genética de bubalinos foram utilizados os 20 marcadores. Foi observado pequeno déficit de heterozigosidade (Fis=0,077), porém as populações apresentaram estrutura genética intermediária (Fst=0,163). Houve desvio de HW em 15 dos 20 marcadores microssatélites. Estes dados podem ser devido ao reduzido número de propriedades, à seleção de touros, ao elevado índice de endogamia e ao cruzamento entre búfalos de Rio e de Pântano, quando foram introduzidos no Brasil. O resultado do estudo da caracterização genética da estrutura populacional determinou a formação de dois clusters, ou seja, total separação entre as duas variedades bubalinas, búfalos de Rio (Bubalus bubalis var. bubalis) e de Pântano (B. bubalis var. kerebau). Estas informações sobre a caracterização genética são necessárias para programas de conservação genética e para um adequado programa de melhoramento genético bubalino. The aim of this study was to standardize a microsatellites panel for buffalo genetic identification test as well as the study of genetic characterization by means of analyzes of variability and distance amog the four buffalo breeds recognized by the Brazilian Association of Builders Buffaloes (Carabao, Jaffarabadi, Mediterranean and Murrah) and crossbred buffaloes raised in Brazil. Were evaluated 590 buffaloes using 20 microsatellite markers recommended by ISAG/2010 (International Society for Animal Genetics). The panel of 13 microsatellite markers developed exhibited high PIC (0.6). The data confirm the reliability and efficiency of the markers analyzed for genetic identification tests as to the genealogy. To evaluate the genetic characterization were observed a low heterozygosity deficit (Fis = 0.077), but the populations had intermediate genetic structure (Fst = 0.163) . There was deviation from HW in 15 of 20 microsatellite markers. These data may be due to the small number of properties, the selection of bulls, the high level of inbreeding , the possibility of sharing the same common ancestor and the Buffaloes cross between Rio and Marsh , when introduced in Brazil . The population structure determined k = 2 , featuring full separation between the two varieties of buffaloes, Bubalus bubalis var bubalis and B. bubalis var. kerebau, River and Wamp buffaloes, respectively. This information on the genetic characterization are necessary for the development of genetic identification and paternity testing for complementation of genetic conservation programs and an appropriate program of buffalo breeding .

A identificação ou certificação de peixes tornou-se uma ferramenta importante para a detecção de fraude ou substituições intencionais frequentemente encontradas na forma de substituições por produtos de maior disponibilidade e menor valor comercial. A ocorrência de substituições em peixes tem sido relatada em vários países, como o Canadá, Estados Unidos, Japão, Coréia e Brasil, principalmente devido à ausência de caracteres morfológicos em espécies processadas. Além disso, a técnica de identificação de espécies por eletroforese de proteínas é laboriosa, requerendo trabalho especializado e não pode ser aplicada a produtos quimicamente ou termicamente processados. Neste trabalho, utilizouse a técnica de identificação molecular de DNA barcoding, para identificação da composição de espécies de peixes e frutos do mar, in natura e processados. Vinte e dois por cento das amostras foram consideradas substituições, 60% das amostra fidedignas e 18% não puderam ser analisadas por falta de informações nos rótulos. O pescado onde mais se observou substituição foi a Merluza (70%), sendo que a espécie mais comercializada com esse nome popular foi a Gadus chalcogrammus (n= 17). Em segundo lugar em número de fraudes está o Bacalhau, sendo identificadas as espécies Molva molva e Pollachius virens, não consideradas como bacalhau segundo portaria do MAPA, respondendo então, por 63% da amostragem considerada como "substituição". Em outros tipos de pescado como o Panga, Salmão e Traíra, também foram observadas substituições. Sendo assim, pode-se afirmar que a técnica de DNA Barcoding é uma ferramenta importante e útil na detecção de substituições em peixes e frutos do mar, processados e in natura. Portanto, recomenda-se o estabelecimento de uma lista válida de nomes comerciais e científicos para os peixes e frutos do mar comercializados no Brasil. Tal lista de referência tornaria possível aos órgãos reguladores do Brasil, a detecção de fraudes e substituições na comercialização de produtos. Além disso, os serviços aduaneiros teriam respaldo para regular e fiscalizar itens importados / exportados, para fins de tributação e também para a proteção do consumidor. The certification or identification of fish has become an important tool for the detection of fraud or replacement intentional often found as replacements for products of higher availability and low commercial value. The occurrence of substitutions in fish has been reported in several countries, such as Canada, USA, Japan, Korea and Brazil, mainly due to the absence of morphological characters in species processed. Furthermore, the protein electrophoresis technique for species identification is laborious, requiring skilled labor and cant be applied to chemically or thermally processed. In this work, we used the technique of molecular identification of DNA barcoding to identify the species composition of fish and seafood, fresh and processed. Twenty-two percent of the samples were considered replacements, 60% of the sample reliable and 18% could not be analyzed due to lack of information on labels. The fish where more substitution observed was Merluza (70%), and the kind marketed under the name most popular has been the Gadus chalcogrammus (n = 17). Second in number of frauds is the Cod, identified species Molva molva and Pollachius virens, that were not considered codas second order of the MAPA, accounting then for 63% of the sample considered "replacement". In other types of fish such as Panga, Salmon and Traíra, substitutions were also observed. Thus, one can say that the technique of DNA Barcoding is an important tool and useful in detecting substitutions in fish and seafood, processed and fresh. Therefore, we recommend the establishment of a valid list of commercial and scientific names for fish and seafood sold in Brazil. This reference list would make it possible for regulators of Brazil, fraud detection and replacement market. In addition, customs services would support regulating and supervising items imported / exported, for tax purposes, and also to protect the consumer.

A conservação dos cães Fila Brasileiro depende de projetos e ações que perpassem a caracterização genética das principais populações preservadas in vivo e sua análise comparativa, a criação e manutenção de bancos de sêmen para a preservação in vitro, além de um trabalho de conscientização para a asociação de criadores conservacionistas. Objetivou-se com este trabalho caracterizar geneticamente uma população representativa da raça e testar diferentes diluídores para congelar o sêmen de seus reprodutores. Foram realizados dois experimentos. Primeiro, 40 cães Fila Brasileiro, enquadrados no padrão Paulo Santos Cruz, foram genotipados por meio de 21 marcadores microsatélites de DNA. No segundo experimento, realizaram-se análises clínicas, andrológicas e zootécnicas, com posterior coleta de sêmen de seis reprodutores Fila Brasileiro. Foram avaliadas variáveis do sêmen fresco imediatamente após a coleta e sua congelabilidade em quatro diluídores sob um protocolo de congelação. Foram também avaliadas a motilididade dos espermatozóides bem como variáveis pós-congelação por meio do sistema CASA e por testes de morfologia e funcionais de integridade das membranas espermáticas. Houve sucesso na criopreservação possibilitando o uso da inseminação artificial (IA) com sêmen congelado na preservação e na crisção. Conclui-se que genotipar cães Fila Brasileiro- PSC pode cotribuir com a preservação e criação desse grupamento, especialmente se associadas ao uso da IA com sêmen congelado. The conservation of FIla Brasileiro dogs depends on projects and actions related to genetic characterization of the main in vivo preserved bloodlines and its comparative analyses, the creation and maintenance of banks for the in vitro preservation and an educational work directed to the association of conservationist breeders. The aim of the present study was to genetically characterize a pure Fila population and to test different extenders for the semen freezing process for the breed. Two experiments were conducted. In the first experiment 40 Fila Brasileiro. phenotipically cassified according to Paulo Santos Cruz standart, were genotyped using 21 DNA microsatellites marker (ISAG, 2008). In the second, clinical andrological examinations and semen collections of six Fila were perfomed. Characteristics of semen were evaluated immediately after sampling and also its freezability in four different extenders. Motility, as well as posthawing characteristics, were obtained by CASA and by Functional tests for the integrity of spermatic membranes (HOST, FCDA and PI) during a thermo-resistance test. Successful semen cryopreservation were obtained and allows the artificial insemination (AI) with frozen stored semen usefull for preservation programs and breeding. It can be concluded tha genotyping Fila Brasileira-PSC dogs using genetic analyses from DNA microstallite markers can help Fila preservation programs and breeding, specially associated with the use of frozen semen AI.

O objetivo deste trabalho é caracterizar geneticamente a resistência ao anti-helmíntico benzimidazol (BZ) em helmintos Haemonchus sp isolados de ruminantes. Para tanto trinta animais pertencentes a um rebanho de bovinos, um rebanho de Caprinos, um rebanho de Ovinos e um rebanho de Bubalinos, tiveram amostras de fezes coletadas e analisadas para a presença de ovos de Tricostrongilídeos. As propriedades foram avaliadas quanto ao histórico de uso de BZ para controle de nematódeos. Nos rebanhos bovinos e ovinos é utilizado BZ a mais de dez anos enquanto que nos rebanhos de bubalinos e caprinos não se utiliza este fármaco a pelo menos cinco anos. Dentre os 120 animais testados, dezoito (~15%) apresentaram ovos de Tricostrongilídeos nas fezes, com contagens de 200 a 33000 ovos por grama de fezes. A presença da mutação Phe200Tyr no gene da -tubulina, que confere a resistência ao BZ foi avaliada em todas as amostras e suas frequências variaram de 0,074 a 0,476 (N= ~20 larvas/hospedeiro). Para a identificação do helminto que infectava os rebanhos foi realizado o sequenciamento do DNA nuclear na região ITS-2 de ovos e larvas. No rebanho de bovinos o principal nematódeo presente foi identificado como Haemonchus placei e nos demais rebanhos foi identificado a espécie Haemonchus contortus. O estudo revelou a presença de alelos resistentes em alta frequência entre espécimes de Haemonchus sp, principalmente nas propriedades que mantêm contínua pressão seletiva por meio do uso de BZ. A identificação da resistência nas subpopulações dos rebanhos de ruminantes foi realizada por genotipagem, os testes estatísticos 2 realizados revelaram não haver diferença significativa entre os isolados de Haemonchus presentes em diferentes animais de um mesmo rebanho. Todavia, foram encontradas diferenças significativas entre rebanhos. Este estudo reforça a necessidade do uso de métodos moleculares para o monitoramento da ocorrência da resistência anti-helmíntica, visando o uso racional de antiparasitários. The purpose of this study is to genetically characterize the resistance to benzimidazole (BZ) anthelmintic in Haemonchus sp helminths isolated from ruminants. With this purpose, thirty animals from herds of cattle, goats, sheep and buffalos, had stool samples collected and analyzed for the presence of trichostrongylus eggs. Properties were evaluated as for the history of using BZ to control nematodes. In herds of cattle and sheep, BZ has been used for over ten years whereas in herds of buffalos and goats, it has not been used for at least five years. Among the 120 animals tested, eighteen (~15%) had trichostrongylus eggs in their feces, with counts from 200 to 33000 eggs per gram of feces. The presence of Phe200Tyr mutation in the -tubulin gene, which confers a resistance to BZ was evaluated in all samples and its frequencies ranged from 0.074 to 0.476 (N= ~20 larvae/host). In order to identify the helminth which infected herds, nuclear DNA was sequenced at the ITS-2 region of eggs and larvae. In the herd of cattle, the main nematode was identified as Haemonchus placei and in the other herds, it was the Haemonchus contortus species. The study has shown the presence of resistant alleles in a high frequency among specimens of Haemonchus sp, particularly in properties which maintain a continuous selective pressure by using BZ. Resistance in isolates of ruminant livestock was identified through genotyping; 2 statistical tests were carried out and have not shown a significant difference among isolates of Haemonchus present in different animals from the same herd. However, significant differences were observed among herds. This study emphasizes the need to use molecular methods for monitoring the occurrence of anthelmintic resistance, encouraging the rational use of anti-parasitic drugs.