**4. Nutritional assessment of genetically modified soybean**

In animal nutrition, many studies with GM plants were carried out in target species using the substantial equivalence method. The application of this method to animal studies led to the development of the concept of nutritional equivalence which implies specific measurements regarding animal production. The European Commission has a combined safety approach that requires an assessment of risks for humans, animals, and the environment prior to approval of importation or cultivation of a novel crop (European Commission, 2001). An integral part of the safety evaluation of GM plants is to test for "substantial equivalence". The concept of substantial equivalence is the starting point and guiding concept for safety assessment (Food and Agriculture Organization/World Health Organization, 2000). It is not the conclusion, but it is part of the safety assessment (Konig et al., 2004; Kuiper & Kleter, 2003). The aim of such a test is to determine whether a transgenic plant is substantially equivalent to its conventional counterpart at a chemical and nutritional level. While the parameters to be measured have not been formally defined, minimal analyses performed should determine whether the major nutritional components (i.e., lipids, carbohydrates, proteins, vitamins, minerals, trace elements) and known antinutrients and

transfer of the enolpyruvyl group from phosphoenol pyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P), thereby yielding inorganic phosphate and 5 enolpyruvylshikimate-3-phosphate (Alibhai and Stallings, 2001). Shikimic acid obtained is a substrate for the biosynthesis of aromatic amino acids (phenylalanine, tryptophan and tyrosine) as well as many secondary metabolites, such as tetrahydrofolate, ubiquinone, and vitamin K. Importantly, the shikimate pathway and, hence, EPSPS proteins, are absent in mammals, fish, birds, reptiles and insects (Alibhai and Stallings, 2001). In contrast, it has been estimated that aromatic molecules, all of which are derived from shikimic acid,

Fig. 1. Schematic representation of glyphosate mode of action and mechanism of CP4 EPSPS

In animal nutrition, many studies with GM plants were carried out in target species using the substantial equivalence method. The application of this method to animal studies led to the development of the concept of nutritional equivalence which implies specific measurements regarding animal production. The European Commission has a combined safety approach that requires an assessment of risks for humans, animals, and the environment prior to approval of importation or cultivation of a novel crop (European Commission, 2001). An integral part of the safety evaluation of GM plants is to test for "substantial equivalence". The concept of substantial equivalence is the starting point and guiding concept for safety assessment (Food and Agriculture Organization/World Health Organization, 2000). It is not the conclusion, but it is part of the safety assessment (Konig et al., 2004; Kuiper & Kleter, 2003). The aim of such a test is to determine whether a transgenic plant is substantially equivalent to its conventional counterpart at a chemical and nutritional level. While the parameters to be measured have not been formally defined, minimal analyses performed should determine whether the major nutritional components (i.e., lipids, carbohydrates, proteins, vitamins, minerals, trace elements) and known antinutrients and

mediated tolerance (Center for Environmental Risk Assessment, 2010).

**4. Nutritional assessment of genetically modified soybean** 

represent 35% or more of the dry weight of a plant (Franz et al. 1997).

toxins of transgenic plants are equivalent to those in conventional varieties that have a history of safe use. Guidelines have been established by several organizations regarding assessment of the allergenic risk of each novel protein expressed in a GM plant, prior to market approval (FAO/WHO, 2000; Konig et al., 2004; Martens, 2000). These typically include comparison of amino acid sequence homology of the novel protein to known allergens and digestion of the protein in simulated gastric environments. While allergic reactions are primarily a concern for human consumption of GM foods, certain proteins in soybean have been shown to elicit allergenic reactions in calves and piglets. The assessment of the safety of GM organisms addresses both intentional and unintentional effects that may result as a consequence of genetic engineering of the food source. Future transgenic crops are expected to contain fewer or no marker genes in the final products since marker free insertion techniques or methods to eliminate marker genes from transgenic plants are being improved. The assessment of safety measures are a lengthy and tedious process (Figure 2). The nutritional aspects, risk characterization and exposure assessment are preliminary steps being taken. Before hitting the market, all GM products have to pass all the allergic tests and provide the details. Only those products find as possessing no harmful or allergic effects are only recommended.

Fig. 2. Pre-market safety and nutritional testing of genetically modified plant derived food and fed (EFSA, 2008).

Genetically Modified Soybean in Animal Nutrition 135

interfering with nutrient absorption or natural toxicants typically present in the genus (Padgette et al., 1996). Trypsin inhibitors are similarly destroyed during heating associated with oil extraction and preparation of soybean meal. In addition, the activity of urease measured by the variation in pH is efficiently reduced by heat treatment whatever the

Lectins (HU/mg sample) 1.2 - 1.0 -

Table 2. Main anti-nutritional factors in raw soybean and soybean meal (parental vs. RR): absence of effect of genetic modification for glyphosate resistance (Adapted from Padgette

Additional nutritional data can be of importance in the case of oil seeds or oil-rich cereals such as corn because they can markedly affect the composition of fatty tissues when fed to farm animals. Data obtained from the analysis of corn kernels demonstrated similar proportions of fatty acids in oil of kernels of insect resistant and herbicide-resistant corn. Similarly, and in the majority of cases, it has also been observed that insect resistant and herbicide-tolerant corn kernels contain similar proportions of amino acids. The introduction of Bt and herbicide tolerance genes in corn has never been found to create starch modifications expressed as the proportion of amylose and amylopectin. Thus similar proportions of 21.5 and 21.0% of amylose have been found in Bt and herbicide-tolerant modified corn, respectively, compared to 22.4 and 22.7% of amylose in starch of the isogenic varieties, respectively (Benetrix, 2000). Data from the literature have many times corroborated the substantial equivalence in major nutrients and minerals and trace elements in corn and kernels of GM compared to isogenic control corn (Brake & Vlachos, 1998; Sidhu et al., 2000). The whole modified corn plant (Clarke & Ipharraguerre, 2001; Faust, 2000) has also been found to be substantially equivalent in composition to isogenic plants. All these data suggest a similar nutritional value for the feed material derived from the modified

In order to evaluate the nutritive value of feeds for ruminants, nowadays the in vitro gas production technique (Theodorou et al. 1994) (IVGPT) is commonly used. IVGPT is based on the assumption that the accumulated gas production by a substrate, incubated in with rumen liquor, is proportional to the amount of digestible carbohydrates, and thus highly correlated to the energy value of feeds. In addition, IVGPT allows to study also the fermentation kinetics of feeds. Tudisco et al. (2004) in a research, aimed to compare the fermentation kinetics of Roundup Ready defatted soybean to its conventional counterpart by the IVGPT, found that the genetic modification, although did not affect the chemical composition, led to a significantly lower cumulative gas production and volume per gram of incubated organic matter. It could be hypothesized that the genetic modification may lead to pleiotropic effects (effect of a single gene on multiple phenotypic traits) that may alter the starch and/or protein structure. Alternatively, the results could be explained with a plant

DNA transfer to ruminal bacteria which may modify their fermentation activity.

Parental soybean RR

Raw Meal Raw Meal

sample) 22.6 3.4 23.7 3.3 Urease (pH) 2.18 0.03 2.17 0.04

genotype of the soybean kernel (Table 2).

Trypsin inibithor (TIU/mg

plants (Aumaitre et al., 2002).

et al., 1996).

In most countries authorities and agencies involved in feed/food safety assessment have based their safety assessment strategies and guidelines on this approach. To provide consumers the opportunity for choice, in 2004 the European Union (EU) has extended regulations concerning GM foods to include animal feeds and feed additives. According to Regulation (EC) No. 1829/2003 (European Commission, 2003), all foods and feeds containing or derived from approved GM products in amounts greater than a 0.9% threshold are subject to labelling rules. Labelling of feeds containing GM ingredients informs farmers and gives them the choice of using such feed for their livestock. However, products such as milk, meat, and eggs, that are derived from livestock fed transgenic feeds are exempt from EU-labelling laws. One of the controversies, important for safety aspects of GM feeds, is a potential possibility of transfer of the transgenic DNA to animal tissues, and in consequence its negative effect on consumers of such products originating from animals fed diets containing GM plants. Detailed studies of the feeding qualities of GM plants for livestock and their nutritional evaluation have been reviewed previously (Aumaitre et al., 2002; Flachowsky et al., 2005a; Flachowsky & Aulrich, 2001). A lot of animal studies with GM plants aimed to evaluate the compositional and nutritional equivalency of transgenic feeds and their conventional counterparts.

Accordingly to the substantial equivalence theory, feed chemical analyses were performed to determine whether the macronutrients, vitamins, minerals and even trace elements were found at the same level as in the conventional or isogenic plants. The composition studies confirmed the substantial equivalence of genetically modified (GM) soybean to conventional counterpart (Table 1) (Cromwell et al., 2001; Padgette et al., 1996).


Table 1. Chemical composition (% dry matter) of genetically modified soyabean (RoundUp Ready, RR) and its conventional counterpart (Adapted from Cromwell et al., 2001). NDF: neutral detergent fiber.

The concerns that have been raised with respect to the potential risk associated with the use of GM plant products in animal feed are related to the possible unintended effects of inserting novel DNA into the plant by biotechnology. The possible "side effects" of the genetically modication is often termed unintended effects and may result from the random integration in the genome of the novel DNA which may result in an over-expression in the plant of inherently toxic substances such as anti-nutritional factors (ANFs), silencing of endogenous plant genes (e.g essential nutrients), or alterations in host metabolic pathways (Novak & Hasleberger 2000; Saxena & Stotzky, 2001). The possible combination of an unexpected increase in expression of endogenous ANFs and the presence of new exogenous toxicants have been of particular concern as these could compromise the quality of feedstuffs and may affect animal health and nutrition (Francis et al., 2001). Studies also include any known anti-nutritional factors (ANFs), such as trypsin inhibitors in soybean,

In most countries authorities and agencies involved in feed/food safety assessment have based their safety assessment strategies and guidelines on this approach. To provide consumers the opportunity for choice, in 2004 the European Union (EU) has extended regulations concerning GM foods to include animal feeds and feed additives. According to Regulation (EC) No. 1829/2003 (European Commission, 2003), all foods and feeds containing or derived from approved GM products in amounts greater than a 0.9% threshold are subject to labelling rules. Labelling of feeds containing GM ingredients informs farmers and gives them the choice of using such feed for their livestock. However, products such as milk, meat, and eggs, that are derived from livestock fed transgenic feeds are exempt from EU-labelling laws. One of the controversies, important for safety aspects of GM feeds, is a potential possibility of transfer of the transgenic DNA to animal tissues, and in consequence its negative effect on consumers of such products originating from animals fed diets containing GM plants. Detailed studies of the feeding qualities of GM plants for livestock and their nutritional evaluation have been reviewed previously (Aumaitre et al., 2002; Flachowsky et al., 2005a; Flachowsky & Aulrich, 2001). A lot of animal studies with GM plants aimed to evaluate the compositional and nutritional equivalency of transgenic

Accordingly to the substantial equivalence theory, feed chemical analyses were performed to determine whether the macronutrients, vitamins, minerals and even trace elements were found at the same level as in the conventional or isogenic plants. The composition studies confirmed the substantial equivalence of genetically modified (GM) soybean to conventional

Dry Matter 90.30 91.00 Crude Protein 51.50 51.20 NDF 4.95 4.85 Lysine 3.16 3.09

Methionine+Cysteine 1.47 1.51

Table 1. Chemical composition (% dry matter) of genetically modified soyabean (RoundUp Ready, RR) and its conventional counterpart (Adapted from Cromwell et al., 2001). NDF:

The concerns that have been raised with respect to the potential risk associated with the use of GM plant products in animal feed are related to the possible unintended effects of inserting novel DNA into the plant by biotechnology. The possible "side effects" of the genetically modication is often termed unintended effects and may result from the random integration in the genome of the novel DNA which may result in an over-expression in the plant of inherently toxic substances such as anti-nutritional factors (ANFs), silencing of endogenous plant genes (e.g essential nutrients), or alterations in host metabolic pathways (Novak & Hasleberger 2000; Saxena & Stotzky, 2001). The possible combination of an unexpected increase in expression of endogenous ANFs and the presence of new exogenous toxicants have been of particular concern as these could compromise the quality of feedstuffs and may affect animal health and nutrition (Francis et al., 2001). Studies also include any known anti-nutritional factors (ANFs), such as trypsin inhibitors in soybean,

Conventional soybean GM soybean

feeds and their conventional counterparts.

neutral detergent fiber.

counterpart (Table 1) (Cromwell et al., 2001; Padgette et al., 1996).

interfering with nutrient absorption or natural toxicants typically present in the genus (Padgette et al., 1996). Trypsin inhibitors are similarly destroyed during heating associated with oil extraction and preparation of soybean meal. In addition, the activity of urease measured by the variation in pH is efficiently reduced by heat treatment whatever the genotype of the soybean kernel (Table 2).


Table 2. Main anti-nutritional factors in raw soybean and soybean meal (parental vs. RR): absence of effect of genetic modification for glyphosate resistance (Adapted from Padgette et al., 1996).

Additional nutritional data can be of importance in the case of oil seeds or oil-rich cereals such as corn because they can markedly affect the composition of fatty tissues when fed to farm animals. Data obtained from the analysis of corn kernels demonstrated similar proportions of fatty acids in oil of kernels of insect resistant and herbicide-resistant corn. Similarly, and in the majority of cases, it has also been observed that insect resistant and herbicide-tolerant corn kernels contain similar proportions of amino acids. The introduction of Bt and herbicide tolerance genes in corn has never been found to create starch modifications expressed as the proportion of amylose and amylopectin. Thus similar proportions of 21.5 and 21.0% of amylose have been found in Bt and herbicide-tolerant modified corn, respectively, compared to 22.4 and 22.7% of amylose in starch of the isogenic varieties, respectively (Benetrix, 2000). Data from the literature have many times corroborated the substantial equivalence in major nutrients and minerals and trace elements in corn and kernels of GM compared to isogenic control corn (Brake & Vlachos, 1998; Sidhu et al., 2000). The whole modified corn plant (Clarke & Ipharraguerre, 2001; Faust, 2000) has also been found to be substantially equivalent in composition to isogenic plants. All these data suggest a similar nutritional value for the feed material derived from the modified plants (Aumaitre et al., 2002).

In order to evaluate the nutritive value of feeds for ruminants, nowadays the in vitro gas production technique (Theodorou et al. 1994) (IVGPT) is commonly used. IVGPT is based on the assumption that the accumulated gas production by a substrate, incubated in with rumen liquor, is proportional to the amount of digestible carbohydrates, and thus highly correlated to the energy value of feeds. In addition, IVGPT allows to study also the fermentation kinetics of feeds. Tudisco et al. (2004) in a research, aimed to compare the fermentation kinetics of Roundup Ready defatted soybean to its conventional counterpart by the IVGPT, found that the genetic modification, although did not affect the chemical composition, led to a significantly lower cumulative gas production and volume per gram of incubated organic matter. It could be hypothesized that the genetic modification may lead to pleiotropic effects (effect of a single gene on multiple phenotypic traits) that may alter the starch and/or protein structure. Alternatively, the results could be explained with a plant DNA transfer to ruminal bacteria which may modify their fermentation activity.

Genetically Modified Soybean in Animal Nutrition 137

Finally, the production studies carried out with fish provided similar conclusions to those

**Parameters Results References** 

No significant

No significant differences

No significant differences

differences (Hammond et

al., 1996)

(Cromwell et al., 2001)

(Hammond et al., 1996)

drawn from studies conducted with other livestock species (EFSA, 2008).

Table 3. Some experiments carried out to establish the qualitative and quantitative performance of monogastric or ruminant livestock fed with GM soybean. BWG: Body

**5. Fate of transgenic DNA and new protein in animal organs and products** 

One of the most important questions about the use of GM products in animal nutrition is the possibility that modified DNA could be transferred from plants to animal products or to bacteria, with harmful consequences (FAO, 2004). Other problems regard the ability of transgenic proteins to provoke food intolerance or allergic reaction in susceptible people. Hence, it is necessary to consider the destiny of these molecules within the animal organism

The gastrointestinal tract is constantly exposed to DNA that is released from partially or completely digested food, ingested microbes, and DNA from intestinal microflora. Ingested food is mechanically disrupted and the released DNA, although poorly digested, is cleaved through acid hydrolysis and enzymatic digestion into small DNA fragments. Eventually some of these fragments are converted to single nucleotides. Acid hydrolysis in the gastrointestinal tract is expected to depurinate most adensine and guanine nucleotides of the food DNA (Klinedinst & Drinkwater, 1992). The presence of various phosphatases and deaminases continue to destroy the structural integrity of any free DNA. The breakdown products of DNA are absorbed for using at the cellular level for synthetic processes as they may be found in blood and tissues (McAllan, 1982). All though there were conflicting reports on the fate of GM DNA in the biological system it was observed that DNA could pass through the gut wall into the blood stream and taken up by cells in the blood, liver, spleen and passed through the placenta to the cells of the feoetus and the newborn one

In ruminants, experimental evidence suggests that more than 80% of DNA is completely disrupted after 2 hours (Wiedemann et al., 2006). However, this degradation is not complete

In animal tissues some fragments of chloroplast DNA have been found. The reason why chloroplast DNA is more frequently detected in animal products is the number of the genes involved and the sensitivity of the PCR method. In transgenic plants, every cell contains

hundreds chloroplast genes, but only one transgenic gene (Aumaitre et al., 2002).

Poultry BWG, ADG, FCR, muscle weight

Pig BWG, DMI, FCR, carcass quality,

Cow

(Alexander et al., 2007).

(Doerfler & Schubert, 1998).

and not immediate (van den Eede et al., 2004).

sensory score of meat

Milk yield and composition, DMI, nitrogen balance, rumen VFA composition

Weight Gain; ADG: Average Daily Gain; FCR: Feed Conversion Ratio.

In any case, the results of this research arouse concerns in term of food safety, because other unpredictable metabolic effects, such as metabolic interferences, or direct or indirect insertional mutagenesis cannot be excluded. With this regards Seralini et al. (2011) report that by insertion of the transgene in varieties producing Cry1Ab toxin caused a complex recombination event, leading to the synthesis of new RNA products encoding unknown proteins, or/and to metabolic pathways variations which caused up to 50% changes in measured osmolytes and branched aminoacids.
