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ANIMAL NUTRITION |
* Department of Animal Science, Food, and Nutrition, Southern Illinois University, Carbondale 62901;
and
Department of Animal Sciences, University of Kentucky, Lexington 40546; and
and
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
Abstract
Eight female PIC Line 42 pigs (initial BW = 47.5 ± 1.8 kg) were used in a two-period switchback design (n = 4 per treatment per period) to evaluate the nutritional difference between a genetically modified corn and a similar nontransgenic corn. The genetically altered corn (gdhA+) contained a glutamate dehydrogenase gene isolated from Escherichia coli. The nontransgenic corn was the same variety lacking the transgenic cassette, grown at the same two locations. Pigs were surgically fitted with steered ileocecal valve cannulas for collection of ileal digesta. Diets were made up of primarily one of the two corn sources. Dietary AA profiles were adjusted using crystalline AA to match Illinois Ideal Protein Ratios. Pigs were limit-fed at 8% of metabolic body weight (BW0.75) in two equal feedings at 0600 and 1800 daily throughout the experiment. The study consisted of two 15-d periods. Each period consisted of a 7-d acclimation period, a 3-d total collection of feces and urine, two 12-h ileal collections, and a 3-d adjustment period between ileal collections to ensure adequate hydration. Crude protein, leucine, methionine, alanine, aspartic acid, glutamic acid, and tyrosine concentrations were greater (P < 0.05) in the gdhA+ corn than in the nontransgenic variety. The presence of the gene did not alter (P > 0.17) BW gain. Similarly, DM digestibility, fecal N excretion (grams per day), apparent total-tract N digestibility, N balance, net protein utilization, and N retained as percentages of absorbed were not affected (P 
0.32) by the gene modification. Apparent ileal AA digestibility values did not differ (P > 0.31) between the two dietary treatments. Results of this study suggest corn that contains the E coli. gene for glutamate dehydrogenase was nutritionally equivalent to the unaltered variety.
Key Words: Digestibility • Maize • Nutrients • Pigs • Transgenics
Introduction
Biotechnology is being used to alter and potentially improve the nutrient composition of plants. Creation of genetically modified corn can have major development and profit potential (Johnson et al., 2001
) when targeted for use in animal feeds. A genetically altered corn containing a glutamate dehydrogenase (gdhA+) gene isolated from the bacterium Escherichia coli has been propagated at Southern Illinois University. This genetically modified corn had altered metabolic properties as compared with a nontransgenic corn (Lightfoot et al., 1999). Studies have shown that the presence of the gdhA+ gene can increase NH4+ assimilation 2,000-fold over resident glutamine synthase (Lightfoot et al., 1998). Plants transfected with the gdhA+ gene show a five- to tenfold increase in glutamate dehydrogenase activity over that found in the resident host E coli. (Lea et al., 1990). This gene enables the plant to increase N uptake from the soil in a manner not dependent on energy. A comparison of AA concentration of gdhA+ corn compared with an unaltered variety showed that the gdhA+ gene increases concentrations of certain AA (Lightfoot et al., 1999), which may increase the value of the gdhA+ corn as a feedstuff in swine diets.
Corn is the primary component of most U.S. swine diets. Therefore, relatively modest AA profile alterations may have a significant impact on nutritional quality of the diet. Nutritional quality of corn has been reviewed (Burgoon et al., 1992; NRC, 1998) and specific studies have been conducted to evaluate nutritional equivalence (O’Quinn et al., 2000) and differences among varieties (Snow et al., 1998). The gdhA+ corn was evaluated in vitro (Apgar et al., 2001) but not in vivo. Therefore, the objective of this study was to evaluate the nutritional equivalency of a genetically modified gdhA+ corn in growing pigs fitted with steered ileocecal valve cannulas (SICV).
Materials and Methods
Animals and Housing
This study was approved by the Southern Illinois University Laboratory Animal Care Committee (Protocol No. 2000-14). Eight female pigs (PIC Line 42; initial BW 47.5 ± 1.8 kg) were used in a two-period switchback design (n = 4 pigs per treatment per period) for this study. Pigs were surgically fitted with SICV cannulas as described by Mroz et al. (1996) and Radcliffe et al. (1999). Pigs were individually housed in 1.22-m x 2.43-m smooth-walled metabolism crates in a temperature-controlled room. Each crate contained Tenderfoot flooring (Tandem Prod. Inc., Minneapolis, MN), a low-pressure drinking nipple, a stainless steel feeder, and a 0.608-m2 rubber mat under the feeder to minimize contamination of urine by feed. Feces were collected on screen material suspended between the Tenderfoot flooring and the fiberglass urine collection pans. Crates and feeders were cleaned at the initiation of each experimental period and 12 h before total collection periods. Cleaning metabolism crates 12 h before total collection periods allowed fiberglass urine collection pans to dry and decreased potential urine dilution by H2O. To alleviate boredom, pigs were allowed a 5.08-cm x 30.48-cm polyvinyl chloride pipe toy for the duration of the study.
Duration
The study consisted of two 15-d periods. Each period comprised a 7-d dietary acclimation, a 3-d total collection of feces and urine, a 12-h ileal collection, and a 3-d rest period followed by the final 12-h ileal collection. Pigs were given the 3-d rest period to prevent dehydration. No physical signs of dehydration were observed during the trial.
Diets and Treatments
Pigs were limit-fed daily at 8% of metabolic body weight (BW0.75), which was divided into two equal feedings occurring at 0600 and 1800 throughout the experiment. Experimental diets were primarily composed (approximately 94%) of a genetically modified gdhA+ corn created at Southern Illinois University (Lightfoot et al., 1999) or the same corn (gdhA–) lacking the transgene cassette. Composite corn used within this experiment was grown in two locations in Southern Illinois. The gdhA+ and gdhA– varieties were grown on similar plots in both locations. Before formulating diets for the current study, four composite samples were obtained from each test corn. The four composite corn samples of each corn variety were analyzed for AA concentrations (Table 1). These AA profiles were used to formulate the experimental diets. Dietary nutrient profiles were adjusted using crystalline AA to meet or exceed Illinois Ideal Protein Ratios (Baker, 1997) and crude protein was adjusted to 10% using crystalline glutamate (Table 2). Calculated and analyzed AA compositions of experimental diets are presented in Table 3. All other nutrients met or exceeded NRC (1998) requirements. Chromic oxide was used as an indigestible marker for digestibility estimates. Diets were stored and weighed in separate rooms, and mixed with 450 mL of deionized water before feeding. Offering experimental diets mixed with water reduced feed dust and the incidence of feed refusal.
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Urine and Fecal Collection
During each total collection, urine and feces were collected every 12 h for 3 d. Urine was collected from fiberglass basins (Palco Manufacturing, Belle Plaine, IA) residing under the pens, strained through cheesecloth, and collected into plastic containers with sulfuric acid. Samples were stored in 6-L carboys. Every 12 h, pH was checked using Alkacid test paper (A-980; Fisher Scientific, Pittsburgh, PA) and maintained 
2.0. If the pH was >2, H2SO4 was added to produce a final concentration of 10% vol/vol.
Feces were collected in 12-h intervals from the screens below the floor and the interior of the pen if applicable. Samples were placed in sealable bags and stored at –20°C.
Measurement, Sampling, and Analysis
Feed and fecal samples were dried (60°C), ground through a 1-mm screen (Thomas-Wiley mill, stainless steel) and subsampled. Ileal samples were partially thawed, placed by collection and pig into large plastic bowls, and thoroughly mixed with a hand mixer. Digesta samples were freeze-dried and subsampled for analyses.
Total N was determined on feed, feces, and urine using the Kjeldahl procedure (AOAC, 1990) with automated equipment (Tecator, Hoganas, Sweden). Feed, feces, and ileal digesta were wet-ashed using nitric and perchloric acid (Sandel, 1959) and chromium concentration was quantified by flame atomic absorption spectrophotometry (AAnalyst, Model 300; Perkin Elmer Corp., Wellesley, MA). Amino acid profiles of feed and ileal samples were determined by high-performance liquid chromatography at the University of Missouri, Agric. Exp. Stn, Chemical Laboratories (Columbia, MO) after acid hydrolysis (AOAC, 1990), whereas total sulfur AA content was determined after performic acid oxidation followed by acid hydrolysis (AOAC, 1990). Tryptophan content was determined after alkaline hydrolysis (AOAC, 1990). Amino acid concentrations were not corrected for incomplete recovery resulting from hydrolysis.
Calculations and Statistical Analysis
The AA composition of the two corn samples were compared using a t-test. Apparent indirect AA digestibility was calculated using the following equation: 100 – { [{AA in digesta/AA in feed) x (Cr in feed/Cr in digesta)] x 100%}. Nitrogen data were calculated as described by Adeola (2001). Apparent AA digestibility and N balance data were analyzed by ANOVA using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC). Pig was the experimental unit. The model for the apparent AA digestibility and the N balance data included the effects of dietary treatment, pig, and period. An alpha level of 0.05 was used for determination of statistical significance. A trend toward significance was considered when P 0.10. Estimates of power were calculated for the apparent AA digestibility data. Apparent AA digestibility values from the two ileal collections in each period were averaged and used as a single observation. Values obtained from two pigs from the first ileal collection of Period 1 were excluded. The first pig, receiving the gdhA– treatment, did not produce a sufficient amount of ileal digesta compared with all other pigs during the first ileal collection (134.9 vs. 1,089.8 g). The second pig, receiving the gdhA+ treatment, appeared to be lethargic and feed intake decreased dramatically, which may have affected the apparent ileal digestibility data. Hence, for these pigs only values from the second collection during Period 1 were used as opposed to an average, resulting in a total of n = 16 observations for apparent AA digestibility estimates. The second pig was also excluded from the final analysis for N utilization in the first period. Means reported for N utilization included the remaining observations (n = 15).
Results and Discussion
Presence of the transgene altered the CP concentration and AA profile of the gdhA+ corn. Crude protein, leucine, methionine, alanine, aspartic acid, glutamic acid, and tyrosine concentrations were greater (P < 0.05) in the gdhA+ corn when compared with the nontransgenic corn (Table 1
).
Feeding diets containing the nontransgenic or the genetically modified corn affected neither BW gain nor ADG (P > 0.17; Table 4). Additionally, corn source produced no differences (P > 0.30) in DM digestibility and N utilization (Table 5). The similarity in the various N responses would indicate these two gdhA corn varieties were equivalent for these aspects of nutritional value. Other measured parameters, such as apparent total-tract N digestibility, N balance, net protein utilization, or N retained as a percentage of that absorbed did not differ between the two respective corn sources (P 0.30). Hansen and Lewis (1993) reported values of apparent total-tract N digestibility of 86.8% for gilts (47.3 to 58.5 kg) fed a 13% CP diet composed of corn and SBM. These reported apparent total-tract N digestibility values are comparable to the values reported for this study.
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0.31; Table 6). However, power analysis suggests the study lacked adequate observations for conclusive findings regarding apparent AA digestibility. Introduction of the E coli. gdhA gene into the corn genome allows increased uptake of N into corn (Lightfoot et al., 1999) and alters the AA profile of the corn grain. However, the actual nutritional value of the altered AA profile is highly dependent on the digestibility estimates of each AA.
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). Haydon et al. (1984) reported that nutrient digestibilities determined at the end of the small intestine of pigs were not greatly affected by feeding level. In general, the values for the apparent ileal AA digestibility ascertained in this study are somewhat lower than the values summarized for corn by Sauer and Ozimek (1986) and Stein et al. (1999). The latter researchers reported apparent ileal AA digestibility coefficients of 68.9% for indispensable AA and 63.7% for dispensable AA (Stein et al., 1999). Our values were lower (66.6 and 55.9%, respectively) and only the gdhA+ dispensable AA digestibility values approached those reported by Stein et al. (1999). Although our values differ from those in the literature, our objective to evaluate nutritional equivalence was fulfilled as we used the same varieties of corn containing or lacking the gdhA+ transgene. Amino acid digestibility of corn has been shown to be influenced by varieties (Snow et al., 1998). In this study, corn variety was the same although one corn source was genetically modified. The apparent ileal AA digestibility values of lysine obtained from the gdhA+ corn (73.0 ± 1.48) and nontransgenic corn (72.8 ± 1.48) fall within the range (71 to 82%) of apparent ileal digestibilities of lysine that have been reported (Sauer et al., 1977; Adeola et al., 1986; Lin et al., 1987). O’Quinn et al. (2000) reported the apparent ileal digestibility of lysine to be 65.2% in high-oil corn and 71.2% in high-lysine, high-oil corn. The apparent ileal digestibility values of lysine for the gdhA+ corn also compare with values from Burgoon et al. (1992) of quality protein maize (72%) and normal corn (67%) and Snow et al. (1998) of normal yellow dent corn (66.1%). Tryptophan, which is arguably the second-limiting AA in corn, exhibited the highest digestibility of the indispensable AA. When comparing the apparent ileal digestibility values of threonine to those of the remaining indispensable AA, threonine had the lowest digestibility in both corn sources. Threonine digestibility values of the gdhA+ corn (59.0%) and nontransgenic corn (58.4%) also compare with values (53.8 to 78.9%) that have been reported (Sauer et al., 1977; Lin et al., 1987; Stein et al., 1999). The remaining indispensable AA values from this study are low compared with those summarized by Sauer and Ozimek (1986) and those reported by Stein et al. (1999) for corn. Furuya and Kaji (1991) reported the apparent ileal digestibility of glycine (22%) to be low in corn. Glycine values of both sources of corn are the lowest of the dispensable AA. Overall, the genetically altered gdhA+ corn variety has equivalence to the unaltered variety and the apparent ileal AA digestibilities approach values reported in the literature. Implications
The presence of the E. coli gdhA transgene alters the composition of the corn by increasing the concentration of certain AA. In this study, the genetically modified gdhA+ corn proved to be utilized as efficiently as the unaltered, nontransgenic corn variety. Apparent ileal amino acid digestibility coefficients in the gdhA+ corn were comparable to those obtained in nontransgenic corn. Likewise, N retention and excretion were similar between the two sources.
Footnotes
1 This research was partially supported by a USDA NRI Strengthening Grant (9903665), the Illinois Corn Marketing Board, and the Illinois Council for Food and Agriculture Research Internal Competitive Grant 98I-15. Appreciation is expressed to J. Usry, Ajinomoto USA, for crystalline amino acids; C. Parsons for freeze-drying ileal digesta; D. A. Lightfoot for providing both corn varieties for this experiment; and Memorial Hospital of Carbondale for sterilization of rings and cannulas. 
2 Correspondence-phone: 618-453-1765; fax: 618-453-5231; e-mail: pigguy{at}siu.edu.
Received for publication June 21, 2003. Accepted for publication February 16, 2004.
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