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Genetic Tool

Molecular markers identify tender meat in Nelore cattle

Schefer Everton / EmbrapaCanchim cattle: genes tied to thickness of fat layerSchefer Everton / Embrapa

Soon it should not be necessary to wait for your steak to reach the table to find out whether the meat is tender. Through biotechnology tests of blood, skin and even semen samples, it will become possible to forecast whether a given animal of the Nelore breed will or will not produce tender meat, while it is still a calf of only a few months. The new technology uses molecular markers, which are variations in the DNA sequence that differentiate the individuals of a species, to identify the animals that are genetically predisposed to produce tender meat. Developed by a network under the coordination of researchers from Embrapa (the Brazilian Company of Research into Crop and Livestock Farming), in São Carlos, in inner-state São Paulo, and from the Federal University of São Carlos (UFSCar), the research resulted in a request for an international patent to identify animals with this potential quality. Nelore cattle accounts to roughly 60% of the Brazilian herd, which amounted to 205 million bovines at the end of 2009, according to IBGE, the Brazilian Geography and Statistic Institute. Brazil has the second largest herd in the world, behind India, and is the second largest producer of beef, immediately after the United States, with 6.6 million metric tons of beef produced in 2009. Of this total, 14% was exported. According to the Ministry of Development, Industry and Foreign Trade, in the first 10 months of 2010, just the exports to Europe totaled US$ 548 million.

These important results for Brazil’s livestock farming as regards molecular markers are the outcome of a prior study conducted by Embrapa Pecuária Sudeste with the Canchim breed, which has some 90 thousand registered animals and is important principally for crossing with other breeds in the production of beef. “We’d been working since 1998 on the development of molecular markers to help improve selection in this breed”, explains the researcher Luciana Correia de Almeida Regitano, the leader of the two projects. “We had found markers for animal weights of different ages, but only in 2003 did we manage to gather information on the thickness of the subcutaneous fat, an important feature for a breed to perform better in financial terms, because it helps to protect the beef while it is in cold storage.”

The choice of the Canchim breed is a natural evolution of the studies conducted in São Carlos. That was where this breed was created. Its development started in the 1940s in the old Breeding Farm of the Ministry of Agriculture, where Embrapa Pecuária Sudeste now operates. This breed is 5/8 Charolais (originally from France) and 3/8 cattle of Zebu origin, such as Nelore and Indubrasil. The aim of this was to bring together in one breed the qualities of the other two, such as the sturdiness and capacity to adapt to Brazil’s tropical condition of the Zebu animals (Bos taurus indicus), originally from India, and the productivity and tender beef of European cattle (Bos taurus taurus), such as the Charolais animals. Thus, researchers from this Embrapa unit have always maintained close ties with the improvement program and with the cattle breeders’ associations. Luciana explains that the molecular markers are variations in the sequence of the DNA bases, which occur naturally among individuals of a species and are passed on from parent to offspring. However, these variations don’t always produce visible differences between individuals, but they can be used like “road signs” on a highway, helping to locate the genome regions that produces these differences. In our case, we are interested in locating the genes that contribute to these differences in the production and the quality of the cattle created in tropical conditions.”

In the research with Canchim cattle, during the doctoral studies of Gisele Batista Veneroni, from UFSCar, conducted under Luciana’s guidance and with FAPESP support from 2007 to 2010, some of the genes that might be linked to the animal’s capacity to produce and store body fat were studied. For one of these genes, the cell differentiation factor of DDEF1 – a protein that transmits signals to the cell and that is involved in the differentiation process – there were reports in the literature indicating that in mice and humans it transformed fibroblasts (conjunctive tissue cells) into adipocytes, the cells that store fat in animals. However, there were no such studies of bovines.

EmbrapaNelore cattle: the largest number in the Brazilian herdEmbrapa

Keeping an eye on fat
Luciana explains that the group decided, at this point, to look for variations in this DDEF1 gene, also known as Arf-GAP or Asap1, and located in chromosome 14 of bovines, which produces the protein of the same name (DDEF1), to then connect it with the amount of subcutaneous fat in Canchim cattle. She explains that to identify the associations between the markers and the characteristic that are of interest, one must have phenotype information, such as the thickness of the fat, the loin (section of the rear muscle that corresponds to the tenderloin) and the tenderness of the beef in a large number of animals. The eye of the loin is used as a measure of the animal’s production of beef. The more muscular it is, the more meat it will have, compared to bones and fat for example.

In the case of the Canchim, 750 animals from several ranches were studied. The researchers submitted the data to mathematical models that allowed them to include the environmental and genetic effects connected with the production characteristic. “In these models, we included the molecular marker effect”, explains Luciana. “With the advance of marker analysis methodologies, we will soon be able to include in these models the effects of a large number of markers, covering the entire genome, which will enable us to forecast a genomic value for each animal”. This value indicates, more precisely, the weight and the influence of the genes in the animal’s characteristics. The other source of influence is the environment. According to her, the mapping of the bovine genome – the result of the work of a consortium of 300 researchers from 25 countries, including Brazil, which took six years and was completed in April of 2009 – was an important stage in the development of the marker for beef quality characteristics, because it provided information about the sequence and location of the DDEF1 gene in bovines. “This information was essential for us to begin our studies”, says Luciana. Gisele’s project led to the initial filing of a patent. In this case, a method and a kit for early identification of fat deposits in cattle, using the variation in the sequence of the gene DDEF1 as an indicator of the genetic fat accumulation potential of the Canchim breed. Besides Gisele, from the Graduate Program in Genetics and Evolution of UFSCar, researchers from Embrapa Pecuária Sudeste and from the School of Veterinary Medicine of Botucatu at Paulista State University also took part (UNESP).

In 2007, the group started another project: To form a network of research (the Bifequali network) into genetic studies connected with beef production or the Bifequali network. This network is comprised of six Embrapa units, the University of São Paulo (through Esalq, the Luiz de Queiroz College of Agriculture), Unesp at Jaboticabal, the State University of Campinas, the State University of Santa Cruz, in Ilheus, in Bahia state, and the Zootechnical Institute of São Paulo State. In this network, the researchers assess, among other characteristics, the genetic variability of Nelore animals regarding the quality and efficiency of their transformation of feed into beef, besides their temperament. To this end, they are raising, confining and slaughtering approximately 250 males a year. This will be done for a three-year period.

The right variation
The first slaughter was conducted in 2009, when the researchers investigated the relation between the variation of the sequence of the DDEF1 gene and several production characteristics assessed in the projects. They did not find in the Nelore breed any association with the amount of fat, but they did discover a connection to the variation of the weight of the animal at 18 months, in the area of the loin. However, the most surprising finding, according to Luciana, was that the studies also showed an association with the tenderness of the beef, one of the most important qualities for the product’s acceptability, especially in foreign markets. “The interesting thing is that the variant connected to a larger tenderloin area is also connected to the beef being more tender”, says the researcher. “So selecting Nelore cattle based on this variation should increase the amount of muscle and the tenderness of the beef”. This discovery was protected by an international patent request. The project that gave rise to this was part of the master’s degree research, conducted from 2009 to 2010, by Polyana Tizioto, a FAPESP grant holder, at UFScar.

According to Luciana, who is also Polyana’s advisor, this work is merely the first step toward unveiling other bovine genes that might be of interest. The research that Luciana plans to start in early 2011 will make this task easier. “I intend to use the DNA of 600 Nelore animals produced during the three years of the project in a research study to be conducted jointly with the Bovine Functional Genomics Laboratory of the Agricultural Research Service in the United States. There, she plans to analyze the animals’ markers known as single nucleotide polymorphism (SNPs), which can be in any part of the genome, within the gene sequence or in non-coding sequences, which do not produce protein. There are 700 thousand markers in the Bovine HD chip, a board containing genetic data”, she reveals. The technology that Luciana will use in the USA is to be implemented in Brazil thanks to the purchase of equipment with funds provided by the FAPESP Multiusers Equipment Program, recently approved and under the coordination of professor Luiz Lehmann Coutinho, from Esalq.

The technology developed by the researchers is not available for producers yet. The next stage of the group’s work is to identify potential partners for marketing this technology. “The selection of more efficient animals does not provide merely financial benefits”, Luciana reminds us. “Feed alone can account for 5 to 26 percent of the costs of beef cattle production. Additionally, more efficient animals release less greenhouse gases and require a smaller area for production.”

The projects
Molecular markers applied to the improvement program for Canchim breed bovines (n° 2001/10036-5); Type Regular Research Awards; Coordinator/Advisor Luciana Correia de Almeida Regitano – Embrapa; Grant Holders Gisele Batista Veneroni-UFSCar; Investment R$ 17,641.35 and US$ 6,029.00 (FAPESP)
2. Association of SNPs in candidate genes and in chromosome regions with the thickness of the subcutaneous fat layer in Canchim breed cattle (n° 2006/06237-9); Type Doctoral Grant; Coordinator/Advisor Luciana Correia de Almeida Regitano – Embrapa; Grant Holders Polyana Cristine Tizioto – UFSCar; Investment R$ 99,864.96 (FAPESP)
3. Prospecting and validating SNPs in candidate genes for beef tenderness in families of the Nelore breed (nº 2010/06515-4); Type Doctoral Grant; Coordinator/Advisor Luciana Correia de Almeida Regitano – Embrapa; Investment R$ 111,318.48 (FAPESP)