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ENVIRONMENT, WELL-BEING, AND BEHAVIOR |
* Department of Animal Science, C206j Animal Sciences, University of Nebraska, Lincoln 68583; and Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63167
1 Corresponding author: sscheideler1{at}unl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: laying hen • genetically modified • corn • Cry3Bb1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was conducted in accordance with the University of Nebraska Animal Care Committee. In experiment 1, 96 Dekalb (Centurian Poultry Inc., Bogart, GA) White Leghorn laying hens (24 wk of age) that had been laying for 4 wk before the start of the study were used. Birds were housed in cages (45.7 cm x 50.8 cm) with 4 birds/cage in an environmentally controlled facility starting at 16 wk of age and used in the study when they were 24 wk old. Assignment of treatments to cages was conducted using a computer random number generator utilizing a randomized complete block design. Blocks were 12 consecutive cages on 1 row and deck in a 4-tiered Farmer Automatic (Farmer Automatic of America, Register, GA) stacked deck caging system.
In experiment 2, twenty-four Dekalb White Leghorn laying hens were used. Birds were housed individually in cages (45.7 cm x 50.8 cm) in an environmentally controlled facility. Assignment of treatments to cages was conducted using a computer random number generator utilizing a randomized design. There were 12 consecutive cages on 1 row and deck in a 4-tiered Farmer Automatic stacked deck caging system.
Environmental conditions for the hens (i.e., floor space, temperature, lighting, animal density, feeder and water space) were similar for all experimental groups. Feed and water were provided ad libitum throughout the trial.
Experimental Design
Experiment 1. A randomized complete block design was used with 2 dietary treatments with 12 replicate cages containing 4 birds each (48 birds/treatment). The diets were fed for 8 wk.
Experiment 2. A completely randomized design was used with 2 dietary treatments with 12 replicate cages containing 1 bird/cage (12 birds/treatment). The diets were fed for 4 wk.
Corn
The MON 863 (corn grain lot GLP-0303–13756-S) and the near-isogenic control (lot 13758-S) were obtained from Monsanto Company (St. Louis, MO) and shipped to the University of Nebraska. Before the start of the study, MON 863 and control corn were sampled and checked for Cry3Bb1 protein using Cry3Bb1 lateral flow test strips (Strategic Diagnostics Inc., Newark, DE).
Diets
Diets were formulated to contain similar concentrations of corn and conform to industry standards or meet or exceed NRC (1994) values for laying hens, or both. The diet compositions are presented in Table 1
. The same diets were used in both experiments.
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Measurements
Experiment 1. Hens were weighed biweekly, and egg production was measured daily. All feed added and removed from cages was weighed daily. The test facility, cages, and birds were observed twice daily for general bird condition, water, feed, and any unanticipated events. Feces were collected 1 d before the start of study (designated as time zero), and then feces and eggs were collected at 4 and 8 wk for a 24-h test period and then saved for Cry3Bb1 protein analysis. At the end of the 8-wk feeding period, 12 hens per treatment were killed and hepatic and pectoralis tissue samples were taken and tested for the presence of Cry3Bb1 protein using Cry3Bb1 lateral flow test strips.
Experiment 2. Feed intake was recorded daily. After 7 d, 6 hens per treatment were sampled for blood, killed by cervical dislocation, and surgically eviscerated. Blood samples were drawn from the wing using a 20-gauge needle and a 3-mL syringe. Samples were injected into collection tubes containing EDTA. Immediately after collection, test strips were inserted directly into the blood tubes. The crop, small intestine, cecum, and large intestine were excised from each hen, and digesta samples were taken from each section. Digesta samples were tested for the presence of Cry3Bb1 protein using Cry3Bb1 lateral flow test strips. The remaining birds were sampled and tested, as described, at the end of the 4-wk period.
Samples and Processing
Corn Grain Extraction. Samples of grain used to formulate the test and control diets were ground in a mortar and pestle, transferred to tared vials, and mixed with PBS at a rate of 3 mL/g of corn. Each vial was mixed for 20 min on a shaker. Extracts were clarified by centrifugation for 10 min at 4°C. The resulting supernatants were transferred to fresh vials and analyzed using Cry3Bb1 lateral flow test strips.
Diet Extraction. Samples of the formulated test and control diets were collected, as well as the basal diet fed to hens before the start of the study. Diet samples (0.5 to 2 g) were transferred to tared vials and mixed with PBS at a rate of 3 mL/g of diet. Each sample was then processed as described for corn grain and analyzed using Cry3Bb1 lateral flow test strips. The control diet contamination level was semiquantitatively estimated by comparison to extracts containing known amounts of test diet spiked into basal diet. A titration curve containing 0, 0.5, 1.0, and 2.0% (wt/wt) test diet spiked into basal diet was prepared and analyzed using Cry3Bb1 lateral flow test strips.
Collection of Feces. To ensure that the collected feces were not contaminated with diet(s), diets were removed and clean collection trays placed under each cage approximately 24 h before feces collection. On the day of collection, feathers were removed from the trays, and feces were transferred into labeled containers. The feces were mixed and representative samples (12 control and 12 test samples) transferred to labeled collection tubes and promptly frozen on dry ice. All samples were shipped frozen on dry ice from the University of Nebraska to Monsanto Company and stored in a –80°C freezer until extracted.
Feces and Digesta Extraction. The fecal and digesta collection tubes were placed on dry ice, and a portion (approximately 1 g) of each was transferred to tared vials. Deionized water was added at a rate of 1.5 mL/g of feces, and the vials were placed at room temperature. Each vial was mixed for 20 min on a shaker. Extracts were clarified by centrifugation for 30 min at 4°C. The resulting supernatants were transferred to fresh vials and stored, or mixed with 5 x SDS-PAGE sample buffer [312 mM Tris, pH 6.8, 10% (wt/vol) SDS, 25% (vol/vol) 2-mercaptoethanol, 50% (vol/vol) glycerol, and 0.5% (wt/vol) bromophenol blue] at a ratio of 1 part 5 x sample buffer to 4 parts extract and stored frozen in a –20°C freezer until analysis.
Hepatic Samples. One hen was randomly selected from each pen and killed. Fresh liver samples were manually extracted from the killed hens. The samples were immediately placed in Whirl-Pak bags and frozen. Before analysis, samples were thawed and homogenized in 125 mL of distilled water using a blender. A portion of the homogenate (5 mL) was centrifuged for 20 min, and 0.5 mL of supernatant was transferred to a microcentrifuge tube with a Cry3Bb1 lateral flow test strip.
Breast Muscle Samples. One hen was randomly selected from each pen and killed. Fresh breast muscle samples were manually extracted from the killed hens. The samples were immediately placed in Whirl-Pak bags and frozen. Before analysis, samples were thawed and homogenized in 140 mL of distilled water using a blender. A portion of the homogenate (5 mL) was centrifuged for 20 min, and 0.5 mL of supernatant was transferred to a microcentrifuge tube with a Cry3Bb1 lateral flow test strip.
Egg Samples. Two eggs per replicate pen were sampled at 4 and 8 wk. Eggs were homogenized, and 0.5 mL of the liquid egg sample was transferred directly to a microcentrifuge tube with a Cry3Bb1 lateral flow test strip.
Reference Protein. The Cry3Bb1 reference protein (Monsanto lot 20–100044) was produced and isolated from E. coli by Monsanto Company. The lot was previously characterized (7.6 mg/mL of total protein, 83% pure) and was used as a quantitative molecular weight marker on Western blots. The trypsin-resistant core was produced by incubating Cry3Bb1 reference protein (2 µg) with fecal extract (50 µL) obtained from hens fed control diet (time 0) and diluted to 800 µL with water. The mixture was incubated overnight at room temperature and diluted to a final total protein concentration of 0.04 ng/µL in 1 x SDS-PAGE sample buffer. The purity correction factor of 0.83 was applied after loading all gels.
Sample Analysis
Cry3Bb1 Lateral Flow Test Strips. Extracts were evaluated for the presence of Cry3Bb1 protein using double antibody sandwich format lateral flow test strips (part number 7000041, Strategic Diagnostics Inc.). Test strips used to evaluate gastrointestinal tract digesta samples, feces, liver, blood, and egg samples were developed for 10 min. Test strips used to evaluate corn and diet extracts (400 µL) were developed for approximately 20 min.
SDS-PAGE and Western Blotting. Extracts containing SDS-PAGE sample buffer (20 µL) were subjected to gel electrophoresis in reducing conditions, according to the method of Laemmli (1970), using 12-well, 1-mm, 4 to 20% polyacrylamide gradient Tris-glycine mini gels (In-vitrogen, Carlsbad, CA). Colored markers (Bio-Rad, Hercules, CA) were included in all gels and used to verify electrotransfer of protein and to calibrate the blots. Electrotransfer to polyvinylidene fluoride membranes was performed for 75 min at 300 mA in Hoefer Inc. (San Francisco, CA) transfer units chilled to 4°C. All incubations and wash steps were performed at room temperature with gentle shaking. Membranes were blocked by incubation in 5% (wt/vol) nonfat dry milk (NFDM) in PBS containing 0.05% (vol/vol) Tween-20 (PBST) detergent for 1 h. Polyclonal goat anti-Cry3Bb1 IgG (Monsanto lot 6844572) was used to probe the membrane for 1 h at a dilution of 1:3,000 in 1% (wt/vol) NFDM in PBST. Excess serum was removed by four 5-min washes with PBST. The secondary antibody solution was preincubated with rabbit serum for 1 h to reduce background signals. To probe for bound IgG, the membranes were incubated with a solution containing rabbit anti-goat IgG, peroxidase conjugate (Sigma, St. Louis, MO) at a dilution of 1:5,000, 10% (vol/vol) rabbit serum (Sigma), and 1% (wt/ vol) NFDM in PBST, for 1 h, and again washed with PBST (four 5-min washes). Immunoreactive bands were visualized using the enhanced chemiluminescence detection system (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) and exposed (1 and 4 min) to Hyperfilm enhanced chemiluminescence high performance chemiluminescence film (Amersham Biosciences). Films were developed using a Konica SRX101A automated film processor (Tokyo, Japan). All protein estimates were made from using films exposed for 1 min.
Densitometric Estimation of Cry3Bb1 Protein in Fecal Extracts. Immunoreactive signals were quantitated using a Bio-Rad GS-800 calibrated densitometer. Densitometric quantities of unknowns were selected as rectangles encompassing the 57-kDa trypsin-resistant band (when observed) or the region of the lane corresponding to 57 kDa (when no band was observed). An empty region of each blot was selected as background, and subtracted from all unknown and known sample values. All densitometric values were expressed as adjusted optical density per mm2.
Fecal Dry Matter Determination. Four clean 50-mL Pyrex beakers were placed in a forced-air oven set at 60°C to thoroughly dry. Fecal samples (2 control diet and 2 test diet groups, ranging from 2.9 to 3.2 g) were weighed into tared beakers and heated at 60°C until the weight of the container was constant (approximately 24 h). The percentage of dry matter was calculated as an average of the 4 sample values.
Statistical Analysis
Statistical analyses were conducted using the repeated measures MIXED model procedure of SAS (SAS Institute Inc., Cary, NC). Parameters included feed intake (g/hen per day), egg production (%hen/day), and BW (kg).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Corn and diet samples were evaluated for the presence of the Cry3Bb1 protein using Cry3Bb1 lateral flow test strips. The results are summarized in Table 2. No Cry3Bb1 protein was detected in the basal diet fed to hens before the start of the study. The conventional grain used to formulate the control diet was negative for the Cry3Bb1 protein. The MON 863 grain used to formulate the test diet was positive for the Cry3Bb1 protein. The test diet was observed to be positive, as expected. However, control diet was observed to be weakly positive for Cry3Bb1 protein apparently from contamination during the mixing process.
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Results of the testing for the presence of Cry3Bb1 protein in liver, breast meat, and feces are presented in Table 3. All eggs appeared to test positive regardless of the diet. Eggs were subsequently purchased from a supermarket (Lincoln, NE) and also all appeared to test positive, indicating that an interfering substance was present in the egg. Therefore, the results for detection of Cry3Bb1 protein in egg were inconclusive. All hepatic and breast muscle samples were negative for Cry3Bb1 protein (Table 3). Feces from birds fed the control corn were 100% negative for the presence of Cry3Bb1 protein at 0 and 4 wk; fecal samples from the birds fed the MON 863 corn containing Cry3Bb1 protein were negative at 0 wk but were positive at the 4- and 8-wk sampling times.
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). Quantitative Western blots were used to assess the amount and intactness of the Cry3Bb1 protein in fecal extracts.
The Cry3Bb1 reference protein was used as a quantitative molecular weight marker on Western blots. The Cry3Bb1 reference protein was mixed with feces extract from hens fed control diet in an attempt to eliminate any effect(s) the extract may have on the mobility of the reference protein. Interestingly, in the presence of feces extract, the reference protein was observed to degrade from the full-length size of 74 kDa to smaller fragments with approximate molecular weights of 68 and 57 kDa (Figure 1).
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). Densitometric data from lanes D5 and D6 were omitted from analysis, because they were outside the linear range of the film. Data from lanes D1 through D4 were used to generate a standard curve that yielded an R2 value of 0.97, when fit to a linear equation. The amount of Cry3Bb1 protein observed in fecal extracts was calculated from the standard curve. The limit of detection for Cry3Bb1 reference protein spiked into a fecal extract obtained from hens fed control diet was defined as the lowest amount loaded, or 0.02 ng. To compensate for blot-to-blot variability, a known amount of Cry3Bb1 reference protein was included on all Western blots. The data were normalized to the Cry3Bb1 reference protein (loaded at 0.3 ng).
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. Consistent with the lateral flow test strip results, no immunoreactive band corresponding to the trypsin-resistant core (57 kDa) was observed at time 0 for extracts of test diet feces. All fecal extracts collected at 4 and 8 wk contained some level of the Cry3Bb1 trypsin-resistant core. No full-length Cry3Bb1 protein or intermediately stable 68-kDa Cry3Bb1 fragment was observed in fecal extracts from hens fed test diet. The amount of Cry3Bb1 protein detected in these samples was estimated by comparison to the titration curve generated using the reference protein (Figure 2). Two test diet fecal extracts were omitted from the densitometric analysis (Figure 3, T10/8 wk, and T11/time 0) due to the high background levels observed in these samples.
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) were consistent with the lateral flow test strip results. No detectable Cry3Bb1 trypsin-resistant core was observed in fecal extracts at time 0. The 2 lateral flow test strips samples that were positive for Cry3Bb1 protein correspond to the Western blot lanes designated as C8/8-wk and C9/8-wk extracts (Figure 4). A faint band is observed at approximately 57 kDa in these 2 lanes, consistent with the lateral flow test strip results. No detectable Cry3Bb1 trypsin-resistant core was observed in fecal extracts from all other hens fed the control diet. All control diet fecal extracts were analyzed in the same manner as the test diet samples. Two control diet fecal extracts were omitted from the densitometric analysis (C6/time 0 and C8/time 0) due to the high background levels observed in these samples.
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summarizes the amount of detectable trypsin-resistant core Cry3Bb1 protein observed in fecal extracts. Variable amounts of trypsin-resistant core Cry3Bb1 protein were detected in fecal extracts from hens fed the test diet. The amount ranged from approximately 0.1 to 0.6 ppm. The overall average amount of trypsin-resistant core Cry3Bb1 protein detected in all fecal extracts from hens fed test diet was 0.22 ppm.
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Feed intake, egg production, and BW for each of the 8 wk of experiment 1 are presented in Table 4. No differences (P > 0.05) were detected in feed intake, egg production, or BW between birds fed corn grain containing the Cry3Bb1 protein versus birds fed the control corn grain during the 8-wk trial.
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Results of the detection of Cry3Bb1 protein in gastrointestinal tract digesta samples and feces are presented in Table 5. At d 7 and 28 sampling periods, Cry3Bb1 protein or its fragments were detected in each of the sections in the digestive tract for the birds fed test diet, whereas none were detected in the digesta from birds fed the control diet. All blood samples, regardless of diet, appeared to test positive for the Cry3Bb1 protein or its fragment(s) indicating the presence of an interfering substance with the assay (similar to the egg protein detection results). Therefore, the blood sample results were inconclusive and not included in Table 5.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Production Performance.
Feed intake, egg production, or BW were not different between laying hens fed diets containing Cry3Bb1 protein versus conventional corn grain. Similarly, in broilers, Taylor et al. (2003) reported no differences in performance or carcass parameters. Others feeding soybean meal containing the CP4 EPSPS protein (Ash et al., 2003) to laying hens or corn grain containing Bacillus thuringiensis proteins such as Cry34Ab1 and Cry35Ab1 (Jacobs et al. 2007) to chickens or Cry1Ab over 10 generations to quail (10 x 12 wks) and 4 generations to laying hens (4 x 31 wk; Flachowsky et al., 2007) reported no significant effects on animal health, feed intake, feed efficiency, laying performance, or hatch-ability. Meat and egg quality were also not affected.
Tissue Analyses.
No Cry3Bb1 protein was detected in hepatic or breast muscle samples in laying hens in this study. The results for the egg and blood were inconclusive, because there appeared to be an interfering substance with the assay. Jennings et al. (2003) reported no detection of Cry1Ab protein in the breast muscles of broilers fed diets containing MON 810 corn grain. Ash et al. (2003) reported no detection of CP4 EPSPS protein in the whole egg, albumin, or liver of laying hens fed diets containing Roundup Ready soybean meal.
Fecal Analyses.
The Cry3Bb1 protein was not detected in feces collected at time 0, using lateral flow test strips. Two extracts collected at 8 wk tested positive from hens fed the control diet. This observation was attributed to contamination, perhaps due to spillage of test diet from a neighboring cage. The Cry3Bb1 protein was detected in the feces of all hens fed test diet collected at 4 and 8 wk. These data suggested that some level of Cry3Bb1 protein, which retained immunological reactivity with the detection antibodies in the lateral flow test strips, escaped digestion in hens fed the test diets. Quantitative Western blot analyses were performed to further evaluate the abundance and intactness of the Cry3Bb1 protein in fecal samples.
The Cry3Bb1 reference protein, when mixed with feces extract from hens fed control diet, was observed to partially degrade (Figure 1). The in vitro degradation pattern of Cry3Bb1 protein in simulated intestinal fluid has been previously established by Monsanto Company as part of the safety assessment studies submitted to regulatory agencies (OECD, 2007). The 68-kDa fragment is known to be intermediately stable to trypsin, and the 57-kDa fragment corresponds to the trypsin-resistant core. This indicates that the fecal extracts contain active trypsin or trypsin-like proteases. Although the Cry3Bb1 reference protein was readily detected using Western blot analysis, high background levels were present in lanes containing fecal extracts (Figure 1, panel A). As demonstrated in panel B, the origin of the background signal is due to nonspecific interaction(s) with the secondary detection antibody (rabbit anti-goat horseradish peroxidase-conjugate). Unsuccessful attempts to reduce the background level were made, including preincubation of the secondary antibody with rabbit serum. Although the high background level was not desirable, the remaining fecal extracts were analyzed using the same Western blot conditions as shown in Figure 1, panel A. In one instance, and further confounding interpretation of the Western blots, a dark band was observed in control diet extract very near the size expected for intact Cry3Bb1 protein (Figure 4, C11/8 wk). However, this band was attributed to a nonspecific interaction, because the lateral flow test strip result was negative, and the band was present at a position smaller than the intact protein and greater than the intermediately stable trypsin fragment.
The Cry3Bb1 protein detected in fecal extracts from hens fed test diet corresponded to the trypsin-resistant core (57 kDa). The amount detected ranged from approximately 0.1 to 0.6 ppm and averaged 0.22 ppm. To estimate the percentage of Cry3Bb1 protein digestion, the values were expressed on a dry matter basis. The wet feces collected in this study were 15 ± 2% dry matter. Therefore, the amount of Cry3Bb1 protein in dry feces is actually 6.7 times greater than what is observed in the wet fecal extracts (i.e., 1 ÷ 0.15 = 6.7). That is, the detectable amount of Cry3Bb1 protein averaged 1.5 ppm and ranged to 4 ppm on a dry matter basis.
The amount of Cry3Bb1 protein in the test diet can also be calculated on a dry matter basis. The expression level of Cry3Bb1 protein in MON 863 is 70 ppm in the grain on a fresh weight basis or about 80 ppm on a dry matter basis. The test diet was formulated to contain 51% (wt/ wt) grain and therefore contains 41 ppm Cry3Bb1 protein.
The percentage of Cry3Bb1 protein digestion was calculated assuming that 77% of the organic matter (Aulrich et al., 2001) and ash were digested leaving 23% of the total feed consumed as fecal dry matter. That is, for each 100 g of diet dry matter consumed, we expect 23 g of fecal dry matter. Using this information and the concentration of Cry3Bb1 protein in test diet (41 ppm) and in feces (1.5 to 4.0 ppm), the digestibility was calculated as follows:
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Therefore, the Cry3Bb1 protein was calculated to be 98 to 99% digested.
The digestibility of proteins in corn-based poultry diets has been previously reported to range from 84 to 90% (Johnson et al., 1999; Aulrich et al., 2001). The digestibility of the Cry3Bb1 protein observed in this study was estimated to be from 98 to over 99%, indicating the Cry3Bb1 protein is highly digestible compared to typical dietary protein. The low levels of Cry3Bb1 protein detected in feces were not unexpected in light of the fact that corn is not 100% digested by the bird.
In conclusion, the feeding of laying hens with diets containing MON 863 did not affect the production parameters: feed intake, egg production, or BW compared with birds fed control diets. The Cry3Bb1 protein or protein fragment(s) were detected in digesta and feces but not in hepatic or breast muscle tissues. The small amount of detected protein was expected due to the fact that corn in itself is not totally digested in the laying hen.
Received for publication October 18, 2007. Accepted for publication February 20, 2008.
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