María Verónica Ripoli

DNA Typing in a Cattle Stealing Case

DNA profiling was used as probative evidence in a cattle stealing case. The carcasses of the dead animals were found from a report and a farmer recognized the remains as those corresponding to the stolen animals by the farm mark on the coat. Those remains were collected as reference samples. Meat pieces were sequestered from a butchery and then sent to our Laboratory by the Justice Department of Buenos Aires (Argentine) to perform a DNA comparative analysis with the reference. Matches were found between the evidences and the references, supporting the hypothesis that the meat pieces had been obtained from the stolen animals. The butcher was suspected of stealing animals but no direct incrimination had been made yet.

Characterization of bovine MHC DRB3 diversity in Latin American Creole cattle breeds

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for diseases and immunological traits. However, none of the highly adapted Latin American Creole breeds have been characterized for BoLA gene polymorphism by high resolution typing methods. In this work, we sequenced exon 2 of the BoLA class II DRB3 gene from 179 cattle (113 Bolivian Yacumeño cattle and 66 Colombian Hartón del Valle cattle breeds) using a polymerase chain reaction sequence-based typing (PCR-SBT) method. We identified 36 previously reported alleles and three novel alleles. Thirty-five (32 reported and three new) and 24 alleles (22 reported and two new) were detected in Yacumeño and Hartón del Valle breeds, respectively. Interestingly, Latin American Creole cattle showed a high degree of gene diversity despite their small population sizes, and 10 alleles including three new alleles were found only in these two Creole breeds. We next compared the degree of genetic variability at the population and sequence levels and the genetic distance in the two breeds with those previously reported in five other breeds: Holstein, Japanese Shorthorn, Japanese Black, Jersey, and Hanwoo. Both Creole breeds presented gene diversity higher than 0.90, a nucleotide diversity higher than 0.07, and mean number of pairwise differences higher than 19, indicating that Creole cattle had similar genetic diversity at BoLA-DRB3 to the other breeds. A neutrality test showed that the high degree of genetic variability may be maintained by balancing selection. The FST index and the exact G test showed significant differences across all cattle populations (FST=0.0478; p<0.001). Results from the principal components analysis and the phylogenetic tree showed that Yacumeño and Hartón del Valle breeds were closely related to each other. Collectively, our results suggest that the high level of genetic diversity could be explained by the multiple origins of the Creole germplasm (European, African and Indicus), and this diversity might be maintained by balancing selection.

Recent patents for detecting the species of origin in animal feedstuff, and raw and processed meat products.

The value of the traceability and labeling of food is attributable to two main aspects: health safety and/or product or process certification. The identification of the species related to meat production is still a major concern for economic, religious and health reasons. Many approaches and technologies have been used for species identification in animal feedstuff and food. The early methods for meat products identification include physical, anatomical, histological and chemical. Since 1970, a variety of methods were developed, these include electrophoresis (i.e. isoelectrofocusing), chromatography (i.e. HPLC), immunological techniques (i.e. ELISA), Nuclear Magnetic Resonance, Mass Spectrometry and PCR (DNA and RNA based methods). The recent patents on species detection in animal feedstuffs, raw meat and meat processed products, listed in this work, are mainly based on monoclonal antibodies and PCR, especially RT-PCR. The new developments under research are looking for more sensible, specific, less time consuming and quantitatively detection methods, which can be used in highly processed or heated treated meat food.

Assignment of paternity in a judicial dispute between two neighbor Holstein dairy farmers.

DNA profiling was used as evidence to assign paternity in a dispute between two neighbors in a judicial case of undue appropriation of cattle offspring from five alleged Holstein sires. Five offspring were genotyped using ten genetic markers (nine microsatellites and the BOLA-DRB3 locus). The computer program CERVUS was used to estimate the LOD score values and the confidence of paternity assignments. The results presented here show that three out of five paternity cases were assigned at 95% of confidence to a single sire with a LOD score ranging from 2.53 to 3.55. A fourth male was assigned using its delta value. Finally, all alleged sires were excluded from the paternity of the fifth offspring, probably due to the existence of an non-sampled male in the studied population. We concluded that the likelihood-based approach, included into CERVUS program, was a powerful tool in cattle kinship analysis when dealing with judicial dispute particularly when the dams genotype was absent, allowing the assignments of paternity at 95% level of confidence in situations usually used by dairy and beef cattle producers in Argentine (e.g., multi-sire pasture mating).

Characterization of the bovine gene LIPE and possible influence on fatty acid composition of meat

LIPE is an intracellular neutral lipase, which is capable of hydrolyzing a variety of esters and plays a key role in the mobilization of fatty acids from diacylglycerols. The objectives of this study were to characterize the genetic polymorphism of bovine LIPE gene and to evaluate the possible association between three SNPs in the coding regions of this gene with the fatty acid composition of meat in a cattle population. Forty-three unrelated animals from different cattle breeds were re-sequenced and 21 SNPs were detected over approximately 2600 bp, five of these SNPs were novel. Three SNPs were selected, on the basis of evolutionary conservation, to perform validation and association studies in a crossbred cattle population. Our results may suggest a possible association of SNP1 with contents of oleic acid and total monounsaturated fatty acids (p < 0.01), and SNP2 and SNP3 with Heneicosylic acid content (p < 0.01), may be helpful to improve the quality of meat and improve health.

Effectiveness of single-nucleotide polymorphisms to investigate cattle rustling.

Short tandem repeats (STR)s have been the eligible markers for forensic animal genetics, despite single‐nucleotide polymorphisms (SNP)s became acceptable. The technology, the type, and amount of markers could limit the investigation in degraded forensic samples. The performance of a 32‐SNP panel genotyped through OpenArraysTM (real‐time PCR based) was evaluated to resolve cattle‐specific forensic cases. DNA from different biological sources was used, including samples from an alleged instance of cattle rustling. SNPs and STRs performance and repeatability were compared. SNP call rate was variable among sample type (average = 80.18%), while forensic samples showed the lowest value (70.94%). The repeatability obtained (98.7%) supports the used technology. SNPs had better call rates than STRs in 12 of 20 casework samples, while forensic index values were similar for both panels. In conclusion, the 32‐SNPs used are as informative as the standard bovine STR battery and hence are suitable to resolve cattle rustling investigations.

Genetic Variation in FABP4 and Evaluation of Its Effects on Beef Cattle Fat Content

ABSTRACT FABP4 is a protein primarily expressed in adipocytes and macrophages that plays a key role in fatty acid trafficking and lipid hydrolysis. FABP4 gene polymorphisms have been associated with meat quality traits in cattle, mostly in Asian breeds under feedlot conditions. The objectives of this work were to characterize FABP4 genetic variation in several worldwide cattle breeds and evaluate possible genotype effects on fat content in a pasture-fed crossbred (Angus-Hereford-Limousin) population. We re-sequenced 43 unrelated animals from nine cattle breeds (Angus, Brahman, Creole, Hereford, Holstein, Limousin, Nelore, Shorthorn, and Wagyu) and obtained 22 single nucleotide polymorphisms (SNPs) over 3,164 bp, including four novel polymorphisms. Haplotypes and linkage disequilibrium analyses showed a high variability. Five SNPs were selected to perform validation and association studies in our crossbred population. Four SNPs showed well-balanced allele frequencies (minor frequency > 0.159), and three showed no significant deviations from Hardy-Weinberg proportions. SNPs showed significant effects on backfat thickness and fatty acid composition (P < 0.05). The protein structure of one of the missense SNPs was analyzed to elucidate its possible effect on fat content in our studied population. Our results revealed a possible blockage of the fatty acid binding site by the missense mutation.

Polymorphisms of BoLA-DRB3 gene and its association with resistance / susceptibility to Leucosis in Holstein cattle from La Pampa

EBL is a disease of adult cattle caused by the retrovirus, bovine leukemia. It can manifest: aleukemic an asymptomatic form, with a normal number of B lymphocytes in the blood; a form of permanent lymphocytosis, with an increase in the number of B lymphocytes and finally a lymphoproliferative tumor presentation in the form of lymphosarcoma. The alleles of the genes of the Bovine Major Histocompatibility Complex (BoLA) have been associated with resistance and susceptibility to infectious diseases. The overall objective of this study was to associate polymorphisms of the BoLA-DRB3.2 gene, defined by the technique of polymerase chain reaction technique and polymorphisms length of the restriction fragment (PCR-RFLP), with resistance / susceptibility to leucosis in Holstein cows of La Pampa. It relied on a case/control design, for which blood samples were taken to 150 animals in 3 opportunities with intervals of three months each. The case group comprised animals that were positive in the immunodiffusion agar animals test (DIDA) and whose blood lymphocyte count was ≥ 10.000 linf/μl and the control group included cows that ware negative for DIDA and present < 10.000 linf/μl of blood. Fisher’s Exact test and Odds Ratio (OR) of Woolf-Haldane were used to study the association between lymphocyte count and DIDA results with allelic variants. In 82 animals genotyped by PCR-RFLP, the DRB3.2*22 allele showed an