PCR-based genotyping of SNP markers in sheep.

Single nucleotide polymorphisms (SNPs) are the main type of variation in genome, enabling them to be associated with traits of economic importance in livestock. Genome-wide association studies (GWAS) have led to the discovery of SNPs associated with desirable traits in sheep. However, in these studies, SNPs are genotyped by high-throughput methods in genome scale, which are expensive and require sophisticated equipment and analysis methods. Therefore, the goal of this study was to develop a reliable, rapid, and inexpensive polymerase chain reaction (PCR)-based method to genotype a medium number of animals for a few candidate SNPs previously associated with desirable phenotypes in sheep by GWAS, using markers associated with gastrointestinal nematode resistance as a model. DNA extracted from white-blood cells of 150 sheep was submitted to PCR amplification followed by agarose gel electrophoresis and determination of banding pattern. Tetra-primer ARMS-PCR was successfully optimized after changes in annealing temperature; annealing and extension times; concentration of MgCl2 and DNA; ratios of inner, outer, forward and reverse primer; and addition of adjuvants, for genotyping the OAR2_14765360, OAR6_81718546, OAR11_62887032, and OAR12_69606944 SNPs in sheep. An extensive optimization of tetra-primer ARMS-PCR resulted in a suitable, simple, cost-effective PCR-based method of genotyping four SNP markers previously detected by chip arrays.

Essential oils of Citrus aurantifolia, Anthemis nobile and Lavandula officinalis: in vitro anthelmintic activities against Haemonchus contortus.

BACKGROUND: Infections of sheep with gastrointestinal parasites, especially Haemonchus contortus, have caused serious losses in livestock production, particularly after the emergence of resistance to conventional anthelmintics. The search for new anthelmintic agents, especially those of botanical origin, has grown substantially due to the perspective of less contamination of meat and milk, as well as other advantages related to their cost and accessibility in less developed countries. The aim of this study was to evaluate the in vitro anthelmintic activity of essential oils of the plant species Citrus aurantifolia, Anthemis nobile and Lavandula officinalis against the main developmental stages of the parasite H. contortus. RESULTS: Plant species were selected based on substantial ethnopharmacological information. Analysis of the composition of each oil by gas chromatography coupled to mass spectrometry (GC-MS) demonstrated the presence of limonene (56.37%), isobutyl angelate (29.26%) and linalool acetate (35.97%) as the major constituents in C. aurantifolia, A. nobile and L. officinalis, respectively. Different concentrations of each oil were tested in vitro for their capacity to inhibit egg hatching (EHT), larval development (LDT) and adult worm motility (AWMT) using a multidrug-resistant strain of H. contortus (Embrapa 2010). The IC50 values obtained for the oils of C. aurantifolia, A. nobile and L. officinalis were 0.694, 0.842 and 0.316 mg/ml in the EHT and 0.044, 0.117 and 0.280 mg/ml in the LDT, respectively. The three oils were able to inhibit adult worm motility completely within the first 8-12 h of observation in the AWMT. CONCLUSIONS: The present results demonstrate significant anthelmintic activity of the three oils against the different developmental stages of H. contortus. Furthermore, this study is of ethnopharmacological importance by validating the anthelmintic activity of the oils studied. Although new experiments are necessary, these data contribute to the development of pharmaceutical-veterinary products for sheep farming by opening up new therapeutic possibilities against gastrointestinal infections caused by H. contortus.