Hukuki Sorunlar

José Bento Sterman Ferraz1_{; Joanir Pereira Eler}1_{; Flávio Vieira Meirelles}1_{; Júlio César Carvalho Balieiro}1 1_{Grupo de Melhoramento Animal e Biotecnologia, Departamento de Ciências Básicas, Faculdade de Zootecnia e}

Engenharia de Alimentos da Universidade de São Paulo, Pirassununga, SP, Brasil. [email protected]

Abstract

Selection of zebu (Bos indicus) beef and dairy cattle in Brazil and the validation process of genetic markers for growth, carcass and meat quality traits and also for milk production, fat and protein milk content are discussed as concerned to the concepts and details of their use as auxiliary tools in selection processes. It is highlighted, also, the importance of right selection of ova donor cows for production of embryos to be transferred. Key-words: molecular markers, genetic markers, selection, animal breeding, Bos indicus

Introduction

A recurrent question is always in dairy and beef producers: how to select seedstock? To answer that question, it is necessary, previously, to answer another critical question on beef and dairy business: what is the real use and value, for our herds, of cows and bulls?

Cows and bulls are the producer’s “machines” and their value is associated to the amount of product they generate: the calves. Like any other animal, they deserve our respect and producers should give them the appropriate environment and welfare. But they are, really, our machines, are worth by their progeny, male and female and contribute with the same amount of nuclear genetics for that progeny, through their gametes (sperm and ova cells). Cows and bulls are worth, therefore, exactly the average value of their gametes. That concept is necessary to figure out the right value of the genetic material producers buy for their business. But how can producers know that value?

The genetic evaluations

Unfortunately, it is not possible to know precisely the genetic value of an animal. The problem is quite simple: animal’s performance, also named phenotype, is the result of the joint action of the genetic background of that animal, added with environmental effects and another term, genotype x environment interaction, that in fact mean that some genotypes, that have better performance in some environments, do not repeat that performance in other ones.

The figure that follows represents the main factors that can affect performance and production of animals.

To make it easy to understand, the figure can be put into an equation, where the letter P means the phenotype, the letter G represents the genotype, E represents the environmental effects and GE is concerned to genotype x environment effects. That way, the production of an animal, e.g. growth, milk production, fertility, puberty, etc., can be symbolized by the simple equation:

P = G + E + GE

That equation led us to understand that, unfortunately, when the phenotype is observed or measured, it does not, automatically, represents the genetic potential of the animal. Phenotypes (P), or performance of animals, always Ferraz J.B.S., Eler J.P., Meirelles F.V., Balieiro, J.C.C. 2008. Aplicações práticas dos marcadores moleculares em zebuínos de...

will be influenced by environment (E) and genotype x environment interaction (GE), besides genetics (G). The previous equation became more complicated when the term G is divided in its three components, resultant of gene action, or ways genes are expressed. The model assumes that every gene has its expression, or action and the effects of genes are additive. This component is called the additive effects of genes, represented by A. But genes, in diploid organisms, like mammals, are located in pairs of chromosomes that come one from the female parent and the other from the male parent. Genes can have different compositions, in terms of nucleotide sequences in the same locus (called alleles) and those different alleles can interact and the effect of one can overcame the effect of the other allele of the pair. The model calls that dominance deviation, represented by (D). Finally, genes from different loci can interact and modify the phenotype, in an effect called gene interaction ( I ). Thus, the equation becomes a little more complicated:

P = (A + D + I) + E + GE

However, the only one effect that does not depend on interactions, that are not predictable, because genes come from different gametes, is the term A. That can be transmitted through gametes and the effect can be predicted. Complicated statistical models are used to estimate, from phenotypic and pedigree data, the term A.

So, genetic evaluation of animals is the process of estimation of the A term for each animal that is in the pedigree of a population. It’s impossible to know the true value for A, the prediction of what an animal can transmit to its progeny, also called breeding value. It will, never, be precisely known. However, the models and genetic evaluations can predict that term, free of the effects of environment and GE interactions.

Genetic or breeding value depends of gene action, number of information about targeted animals and related animals, relationship among targeted animals and sources of information, and permanent environmental effects.

By definition, expected breeding value (EBV) of an animal is the value that animal has a breeder. That is what seedstock herds sell, because that is the prediction of what can be transmitted to progeny. That value indicates, in average, how much progeny production would be deviated from average of a reference population. When EBV is divided by two, because sires (or dams) transmit only half of their genes through gametes, expected progeny difference (EPD) is obtained in beef cattle or predicted transmitted ability (PTA) in dairy cattle. Those concepts mean the fraction of difference from the mean, due to the effect of genes of a given animal.

The concept of EPD is usually used by beef breeders, while PTA is used in the dairy industry. Both have the same meaning and indicate the value of the average gamete of the animal and estimate half of breeding value. EPD and PTA are powerful auxiliary tools for beef and dairy breeders and that tool should be used for choosing genetic resources for stock herds.

Molecular or genetic markers

Molecular markers are fragments of DNA – deoxyribonucleic acid, the molecular basis of heredity of any living organism, that are close to genes that are important to relevant traits. These fragments become “markers” when their associations with those traits are statistically proved. They have to be located very close to the coding part of genes (introns), so no recombination (crossing over) occurs between marker and gene.

Molecular markers have to:

• Be polymorphic, what means be detectable by proper laboratory tests; • Be close to genes that are important to relevant traits;

• Be easy to use;

• Have low cost of detection;

• Allow detection of multiple markers per laboratory test.

• Molecular markers use in animals is very broad. They, basically, are used to: • Increase production;

• Paternity detection tests, including multi-sire pastures;

• Marker assisted selection (MAS), especially in traits that are not easy to measure; • Auxiliary tools in mating and culling systems;

• Marker assisted management;

• Strategic decisions for sales and acquisitions; • Ideal time to sell animals to slaughterhouses; • Grouping animals based on probable performance; • Group homogeneity;

Ferraz J.B.S., Eler J.P., Meirelles F.V., Balieiro, J.C.C. 2008. Aplicações práticas dos marcadores moleculares em zebuínos de...

• Strategic decisions of milking cows;

• Obtain bonus for protein, fat, total solids, in milk; • Oriented mating;

• Add value to final product; • Traceability tool.

There are many molecular markers, like DNA microsatellites, mitochondrial DNA markers, SCAR, SSCP, RFLP, RAPD and, the newer ones, SNP, single nucleotide polymorphisms, where only one nucleotide changes.

As molecular markers are additional tools that can improve efficiency of selection, when choosing sources of genetic material, with similar EPD, the ones that have favorable markers will be preferred. That will be even more useful in traits like carcass and meat quality traits, hard or expensive to measure.

In the case of marker assisted management, molecular markers will be very useful helping to sort animals based on their production potential. The new science, nutrigenomics, will use that to orient technicians to give group of animals the right nutrition, based on their classification by molecular markers. Molecular markers will increase the speed of genetic progress.

The validation of genetic markers in Bos indicus in Brazil.

Since 2006, Merial, one of the largest animal pharmaceutical companies in the world, incorporated Igenity, a new company dedicated to the application of genetic markers to beef and dairy cattle business. The company’s R&D resulted in commercial panels of genetic markers for Bos taurus and Bos indicus cattle.

The Brazilian branch of that corporation, Merial Saúde Animal Ltda., established two important partnerships in Brazil, to lead the validation process of use of genetic markers, developed around the world, mostly in Bos taurus, in the Brazilian Bos indicus. The first partnership where with the Animal Breeding, Genetic and Biotechnology Group of Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo (GMAB/FZEA), located in Pirassununga, State of São Paulo, that would work with the validation of close to one hundred markers in markers in close to 6,000 Nellore beef cattle. The other partnership was with Embrapa Gado de Leite, located in Juiz de Fora, State of Minas Gerais, that used close to 3,500 lactations to validate markers in milking Gyr (a Bos indicus breed) and Girolando, a synthetic breed with Holstein and Gyr breeds in its composition.

The major problem in a validation process is to collect good quality data on phenotypes, and, obviously, collect DNA samples. That’s why partnerships with Universities and Research Centers are very useful in such kind of validation processes.

The validation of molecular markers in beef cattle in Brazil was not an easy task. Brazil has close to 200 million heads of cattle, 150 million of zebu origin, especially from Nellore origin. Within that breed there are several different populations: purebred with both parents originated from purebred from India, purebred not controlled by breeders association, polled and horned animals, herds raised under pasture conditions and steers fed in feedlots, etc. The first step was to define populations and sample size.

There were defined populations that could represent the large variation in the Nellore population in Brazil. From those populations there were collected phenotypes, DNA samples from blood, hair or semen. The partner herds were very representative of very important bulls, leaders in semen sales in the country. Data on close to 6,000 Nellore animals were collected on growth, reproduction, carcass and meat quality traits.

As concerned to dairy cattle, the partnership Merial-Embrapa Gado de Leite resulted in analysis of more than 3,000 cows, from purebred Gyr and Girolando ( a synthetic breed originated from different proportions of Holstein and Gyr breeds). Molecular markers were validated for milk, fat and protein production.

The validation process occurs in the sequence that follows:

• Selection of herds that represent the variation of animals in the country, considering the relationship among animals;

• Collection of phenotype data on animals chosen, and biological material samples to extract DNA (usually blood, hair or semen);

• DNA extraction;

• Genotyping in DNA sequencing platforms, in specialized laboratories; • Statistical analysis, in proper models.

• Grouping of markers for those that were statistically significant, and definition of commercial panels, easy to be interpreted by the ma rket.

Ferraz J.B.S., Eler J.P., Meirelles F.V., Balieiro, J.C.C. 2008. Aplicações práticas dos marcadores moleculares em zebuínos de...

The panels developed in Brazil for Bos indicus

After two years of intense work on data collection and analysis, with thousands of animals, there were developed commercial panels, validated with Brazilian data, off cattle raised under Brazilian environment. The validation process is continuous and more animals are added daily to the databank, so validation can be re-evaluated, or because of new data and/or because new markers are available.

The commercial panels developed for Nellore beef cattle were: • Yearling weight

• Carcass weight

• Loin eye area, a trait related to beef yield in the carcass;

• Backfat, a very important traits in Brazil, due to packing plant necessities; • Tenderness

• Animals that are sensitive to bovine viral diarrhea • Paternity determination.

• New markers are being developed, especially for traits linked to reproduction, temperament, sexual precocity, longevity, carcass yield, resistance to ticks, fat profile of beef, feed efficiency and others.

• As related to zebu dairy cattle, the result of the partnership with Embrapa – Gado de Leite, was the following commercial panels:

• Dairy production; • Fat production; • Protein production; • Kappa-Casein; • Beta-casein; • Beta-lacto globulin; • BLAD • DUMPS • VMC • Paternity determination.

The use of those Igenity commercial panels is, like the variety of traits that validated their use, very broad. The same company commercializes in Brazil other panels, developed for Bos taurus animals, used, mostly, in southern Brazil.

How the effect of molecular markers is expressed?

The analysis of Brazilian beef and dairy zebu data indicated that several markers affect different traits, as it happened in American and European data. Besides that, there were detected interactions among some markers. So, Igenity decided to express the effect of significant markers in traits in scores, varying from 1 (animals with a markers combination that would cause low productivity in that trait) to 10 (animals that carry the best combination of markers for that trait). For example, a bull that has the score 8 to tenderness will produce progeny with more tender meat that another bull that has the score 5, when they breed similar sample of cows. Another bull, with score 7 for protein production in milk is more interesting for a cheese production company than another bull, with score 4, because it’s daughters will produce milk with larger protein content, which will give more yield in cheese production. The interpretation of those scores can be considered quite linear. Cows score 4, when bred by bulls score 8, will produce progeny, in average, score 6. That is a concept that can be very easy to use by the breeders.

Why to use molecular markers?

The use of molecular markers will help beef and dairy breeders to identify bulls and cows, at young ages, which have potential to transmit favorable genes to the genetic improvement of herds. The frequency of those favorable genes will increase rapidly, with the decreasing frequency of unfavorable alleles that exist in the herds. So, molecular markers are powerful auxiliary tools for selection processes. The joint use with the traditional genetic evaluation tools, like EPD, will be very useful to:

• Buy of replacement bulls, cows and heifers and even group of finishing animals; • Choice of animals that will be used intensively, like semen or oocytes donors; • Become oriented mating more effective;

Ferraz J.B.S., Eler J.P., Meirelles F.V., Balieiro, J.C.C. 2008. Aplicações práticas dos marcadores moleculares em zebuínos de...

• Group animals in more homogeneous lots, as concerned to performance, helping better definition of diets, in the new science that is just burning, nutrigenomics;

• Identification of bulls that would be used in larger scale to produce progeny with superior beef quality, helping packing plants to gain new, and better, markets and adding value to products;

• Orient buy of genetic material, like semen and embryos;

• Allow decisions on selection to be taken earlier, culling animals that do not have genetic potential for production;

• Speed-up genetic progress;

However, it is necessary to highlight that the use of genetic or molecular markers developed in Bos taurus, in different production systems than the ones used in Brazil can produce very unfavorable results to Brazilian beef and dairy industry.

It is also very important to emphasize that the fact of a bull or cow have a high molecular marker score does not guarantee that progeny will produce what the score previews. All the factors affecting performance, like nutrition and feeding, health, management, environmental conditions, etc. have to be taken care. The optimization of those conditions is essential to accomplish the good results molecular markers help to preview.

Are the other companies working in the molecular markers business?

Different companies present the results of genetic markers in different ways and the user should know those ways to better understand and use the technology. The main international companies that have resources to develop and validate the technology around the world are:

• Bovigen (http://www.bovigen.com/html/genetictechno.html), company that works with the Australian company called Genetic solutions, that uses the brands Genestar, SireTrace and SureTrak, recently incorporated by Pfizer (http://www.geneticsolutions.com.au/content/products_c1.asp?name=Tech_Info). • Igenity, a company owned by Merial (www.igenity.com or http://www.merial.com.br/igenity);

• MMI Genomics, a subsidiary company of MetaMorphix, Inc. (http://www.mmigenomics.com/ products1.html)

Internet brings very interesting and vast material on that subject, as cited in references. Also, a very interesting free book was issued by FAO/UN and it’s a very material to be read (Guimarães et al., 2007).

Molecular markers and embryo transfer

Is there any relationship between molecular markers and embryo transfer? How they can help veterinarians, specialists in embryo transfer?

Like expressed in the text, molecular markers are quite useful as auxiliary tools for selection processes. That’s exactly the point: selection of oocyte donors, based on their genetic potential to increase production, instead of transferring embryos only from “high monetary value” cows and heifers. The intensification of reproductive rates of females in Bos taurus and Bos indicus can cause important impact in production, especially when associated with sexed semen. It will be much larger if molecular markers are used to help choosing genetic material: semen and donor cows. IN near future, there will be available molecular markers to better choice of recipient cows. Veterinarians that are aware of that can give a very good contribution to Brazilian beef and dairy industry.

Next steps

Validation processes of molecular markers always will be continuous , incorporating new information and new scientific discoveries , larger number of information, more accurate in the statistical associations. In the next year, Brazilian marker will experience new panels, new traits with panels, new companies and a large growth of that market. Estimates of production will be more precise and have a bigger impact on genetic gain.

The advance of genetics will bring, very soon, thousands of new markers and molecular markers will explain larger and larger amount of variation and will bring much more useful information to selection.

Genetic material of twenty of the most representative Nellore bulls in Brazil are being studied and sequenced in cooperation with important international research center, searching for genetic markers, specific to Brazilian breed and dairy cattle.

Ferraz J.B.S., Eler J.P., Meirelles F.V., Balieiro, J.C.C. 2008. Aplicações práticas dos marcadores moleculares em zebuínos de...

Conclusions and implications

Genetic markers came to stay. They are sets of tools of great value to speed up genetic gains in herds under selection. Their use will allow more accuracy in the choice of animals that will produce new generations. Molecular