All animal handling and experimentation were conducted under the approval of the Bavarian State Research Center (LfL, Grub, Germany) institutional animal care and use committee. To investigate the digestive fate of recombinant Cry1Ab protein from genetically modified (GM) maize MON810 in dairy cow digestion, thirty-six Bavarian Fleckvieh cows were separated into a “transgenic” group (n = 18, nine primiparous and nine multiparous) fed on a diet containing GM maize MON810 and a control or “non-transgenic” group (n = 18, nine primiparous and nine multiparous) fed conventional maize over a period of 25 months. All experimental cows were apparently healthy with the body condition score ranging from 3.3 to 3.9. Cows were housed at the Bavarian State Research Center and fed a partial total mixed ration (pTMR). The diet composition is shown in Table 1. According to the milk yield further concentrates [40.4% maize kernels (transgenic or non-transgenic based on respective diets), 34.4% rapeseed meal, 19.9% molasses dried beet pulp, 3.2% mineral mixture and 2.4% urea] were offered above 22 kg milk yield per day. For pTMR preparation, GM maize MON810 and the non-transgenic variety had been cultivated and harvested in 2004, 2005 and 2006 under similar agronomic conditions at the Bavarian State Research Center for Agriculture (Germany). Nutrients and energy contents of both maize varieties were comparable, ensuring equivalent feed conditions (data not shown). The details of the animal management and sample collection are given somewhere else (Steinke et al. 2009; Guertler et al. 2009a, b).
Table 1 Ingredients and composition of the partial total mixed ration
To evaluate the digestive fate of recombinant Cry1Ab protein during the feeding trial, four samples of each diet (transgenic and non-transgenic maize kernel, cobs, silage, and pTMR) and forty-two feces samples (26 transgenic and 18 non-transgenic) were collected from the subset of cows. To determine the amount and fragmentation pattern of Cry1Ab protein in the GIT digesta, contents of rumen, abomasum, small intestine, large intestine and cecum were collected after the slaughter of six cows of each feeding group at the end of the feeding trial. All samples were stored at −80°C until analyzed for total protein and Cry1Ab protein.
All feed samples were finely ground in liquid nitrogen using mortar and pestle. Total proteins were extracted from the pulverized feed (100 mg), feces (100 mg) and GIT digesta (200 mg) samples after the homogenization with 1 ml ice-cold extraction buffer (PBST; 8 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, 1.5 mM potassium phosphate, 0.1% Tween 20 pH 7.4; containing protease inhibitors) using 300 mg matrix green ceramic beads in a Fastprep homogenization machine. The supernatants were collected in 1.5 ml microcentrifuge tubes after centrifugation at 15,000×g at 4°C for 15 min. A clear extract was collected after re-centrifugation at 15,000×g at 4°C for 10 min and stored at −20°C until used for total protein and Cry1Ab protein quantification.
Total protein concentration in each extract was measured by bicinchoninic acid (BCA) assay (Smith et al. 1985) using BSA as protein standard. For determination of the Cry1Ab protein concentration, a previously developed sandwich ELISA (Paul et al. 2008) was used under slight modifications and matrix specific assay validations (Guertler et al. 2009a, b). Briefly, an assay was performed on Cry1Ab protein affinity purified specific antibody coated 96-well microtiter plates by incubating 50 μl of sample extract or Cry1Ab protein standards (HPLC-purified trypsin-activated Cry1Ab protein calibrator concentrations ranging from 2 to 1,000 pg per 50 μl extraction buffer) along with 100 μl assay buffer (PBST containing 0.1% BSA). After overnight incubation at 6 to 8°C, plates were washed six-times with 300 μl PBST per well. The amount of antibody captured Cry1Ab protein from samples/standards was measured by applying a biotin labeled Cry1Ab specific antibody, streptavidin-peroxidase enzyme conjugate and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reaction. Cry1Ab protein concentrations in unknown samples were interpreted from the Cry1Ab protein calibration curve generated using online software Magellan 6 (Tecan, Austria). The concentrations were finally presented as μg Cry1Ab protein per g total protein and ng Cry1Ab protein per g wet sample.
To monitor the fragmentation of Cry1Ab protein from MON810 in transgenic feed, GIT digesta and feces immunoblot analyses were performed. Total protein (60 μg total protein or 100 pg Cry1Ab protein extracts from T feed and feces or respective equivalent amount of total protein from NT feed and feces) of transgenic and non-transgenic feed, GIT digesta and feces were resolved on 12% SDS–PAGE and transferred to nitrocellulose membranes (Protran AB 85, Whatman, Dassel, Germany). Membranes with blotted protein bands were incubated with affinity purified anti-Cry1Ab protein (rabbit pAb, 0.1 μg/ml) (Paul et al. 2008) and HRP-labeled polyclonal goat anti-rabbit secondary (1:10,000) antibody (Santa Cruz biotechnology, Germany). Antibody binding was visualized by chemiluminescence (Supersignal west pico system, Pierces, USA). In immunoblot analysis, a positive control containing HPLC purified trypsin activated Cry1Ab protein (65 kDa) was used to confirm and verify the positive presence of Cry1Ab protein. Student’s t-test was used to compare the means of Cry1Ab protein concentrations in transgenic feed and feces, and total proteins in GIT digesta of cows fed transgenic and non-transgenic diets. A P-value below 0.05 was considered significant.