Influence of Phytase Transgenic Corn on the Intestinal Microflora and the Fate of Transgenic DNA and Protein in Digesta and Tissues of Broilers

An experiment was conducted to investigate the effect of phytase transgenic corn (PTC) on intestinal microflora, and the fate of transgenic DNA and protein in the digesta and tissues of broilers. A total of 160 1-day-old Arbor Acres commercial male broilers were randomly assigned to 20 cages (8 chicks per cage) with 10 cages (replicates) for each treatment. Birds were fed with a diet containing either PTC (54.0% during 1–21 days and 61.0% during 22–42 days) or non-transgenic isogenic control corn (CC) for a duration of 42 days. There were no significant differences (P>0.05) between birds fed with the PTC diets and those fed with the CC diets in the quantities of aerobic bacteria, anaerobic bacteria, colibacillus and lactobacilli, or microbial diversities in the contents of ileum and cecum. Transgenic phyA2 DNA was not detected, but phyA2 protein was detected in the digesta of duodenum and jejunum of broilers fed with the PTC diets. Both transgenic phyA2 DNA and protein fragments were not found in the digesta of the ileum and rectum, heart, liver, kidney, and breast or thigh muscles of broilers fed with the PTC diets. It was concluded that PTC had no adverse effect on the quantity and diversity of gut microorganisms; Transgenic phyA2 DNA or protein was rapidly degraded in the intestinal tract and was not transferred to the tissues of broilers.


Introduction
Phosphorus (P) sources in the world are limited, and overconsumption of P could result in a crisis in P supply [1]. However, phytate-P is poorly available to poultry from their diet [2], due to the low activity of phytase in their digestive tracts [3], and thereby inorganic P must be supplemented in poultry diets to meet their P requirements. Consequently, the unabsorbed treatment diets were formulated to meet the requirements of broilers recommended by the National Research Council (Table 1) [22].

Animal housing and management
One hundred and sixty 1-day-old Arbor Acres commercial male broilers were assigned to 1 of 10 replicate cages (8 chicks per cage) for each of 2 treatments in a completely randomized design. Experimental diets contained either CC or PTC (54.0% during 1 to 21 days and 61.0% during 22 to 42 days). Broilers were raised in stainless cages coated with plastic (100×50×45 cm) and equipped with fiberglass feeders and waterers. During 42 days, birds were allowed ad libitum access to experimental diets and tap water, and maintained on a 24-h light schedule. The management of the birds was in accordance with guidelines approved by Arbor Acres Breeding Company in Beijing.

Sample collections
At 42 days of age, 2 birds from each cage (20 birds/group) were sacrificed by cutting the carotid arteries and bled after a 12-h fast. The heart, liver, kidney, breast and leg muscles were removed, and the contents of rectum, ileum, jejunum, and duodenum were also collected for DNA and protein analysis. At the same time, 2 birds from each cage (20 birds/group) were anesthetized by intravenous injection of Sumianxin II, and then killed by cutting the carotid arteries and bled. Ileal and cecal contents of the birds were collected under sterile conditions for the measurement of microbial population and diversity. All the above samples were snapfrozen in liquid nitrogen and then frozen at -80°C until analysis.

Incubation and enumeration of intestinal microorganisms
After the intestinal samples were thawed at 4°C, one gram of the ileal or cecal contents was diluted with 9 mL of sterile water and mixed on a vortex. The suspensions were diluted by 10-7-times, and then the dilutions were plated on nutrient agar medium, eosin methylene blue medium, GMA broth medium and LBS medium for the culture of aerobic bacteria, colibacillus, anaerobic bacteria and lactobacilli, respectively. All the media were obtained from Beijing Land Bridge Technology Co., Ltd. Aerobic bacteria and colibacillus were cultured in an incubator (HPS-250, Harbin, P. R. China) at 37°C for about 30 h. Anaerobic bacteria and lactobacilli were cultured in an anaerobic (28% CO2, 15% N2, and 25%H 2 ) incubator (YQX-2, Shanghai, P. R. China) at 37°C for about 48 h. Viable counts of microbials in the ileal and cecal samples were then conducted immediately after removal from the incubator.

16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) analysis
Genomic DNA extraction. Genomic DNA was extracted from ileal and cecal samples using QIAamp DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's protocol, and then stored at -20°C.
DGGE. DGGE was performed using the method as previously described [23]. Up to 35% to 65% linear DNA-denaturing gradients were formed in [15]10% polyacrylamide gels. Low and high concentrations of the denaturing gradient gel solutions were then prepared ( Table 2). Bacterial V 3 16S PCR products were loaded in each lane, and electrophoresis was conducted in 1× TAE Buffer at a voltage of 200 V at 60°C for 10 min, and then at 80 V for 16 h. After electrophoresis, the gels were silver-stained and scanned using a GS-800 Calibrateda Densitometer system [24,25].

DNA analysis
DNA extraction. DNA from the CC, PTC, diets, intestinal contents, or tissues was extracted using the method reported by Ma et al. [15], and then stored at -80°C.
PCR analysis. The ivr (a specific invertase gene of corn, 226 bp) and phyA2 (a foreign gene in the PTC, 678 bp) were used to monitor corn DNA and transgenic DNA, respectively. Furthermore, the chicken ovalbumin gene (ov, 396 bp) in tissues was determined to validate the DNA extraction for PCR. The ivr, phyA2 and ov genes were acquired from AuGCT, Beijing, China. The primers and PCR conditions were showed in a previous study [15]. Separation of products of the PCR and analysis of the gel images were performed according to the method as previously described [15].

Western blot analysis
Samples from the corn, diets, digesta and tissues were homogenated and cleaved using RIPA lysis buffer (Beyotime Institute of Biotechnology, Beijing, P. R. China). After centrifuged at 12,000×g for 5 min at 4°C, the supernatants were collected for SDS-PAGE and western blotting analysis. Protein concentrations were determined by Bradford Protein Assay Kit (Beyotime Institute of Biotechnology, Beijing, P. R. China). Other procedures for western blot analysis were carried out as described by Ma et al. [15].

Statistical analyses
The data were analyzed by one-way ANOVA using the GLM procedure of SAS (SAS Institute, Inc., 2001). Differences between the two treatments were analyzed using t-tests. Differences were considered significant if P < 0.05. The data were shown as mean ± SD.

Influence of PTC on microbial population in the intestine of broilers
There were no significant differences (P>0.05) between the birds fed with the PTC and CC diets in the populations of aerobic bacteria, anaerobic bacteria, colibacillus, or actobacilli in ileal or cecal contents (Table 3).

Microbial diversity in the intestine of broilers
Birds fed with the PTC and CC diets had similar bands (Figs 1 and 2) in theirileum and cecum, and the band numbers in the ileum or cecum were also similar (P>0.05) ( Table 4), indicating that dominant microfloras in the ileum or cecum were similar between the two treatments. In addition, the band numbers were higher (22.1 VS 10.4, P<0.05) in cecum than in ileum. This represents the diversity of microflora in different segments of the intestinal tracts of broilers. The similarity coefficient (Cs) values of ileal microflora patterns for broilers fed with the same diets were numerically higher than those of cecal microflora patterns. Additionally, the Cs of ileal and cecal microflora patterns for broilers fed with different diets ranged from 59.4% to 34.6%, and 76.4% to 29.1%, respectively. However, different groups were not formed in the band patterns of ileum or cecum of birds fed with different diets. This suggested that PTC may not have an adverse effect on microflora in ileum and cecum.

Detection of DNA fragment in corn, diets, digesta and tissues of broilers
Fragments of the ivr gene were detected in all the corn and diet samples, whereas the transgenic phyA2 gene fragments were only found in PTC and PTC diets (Fig 3). The ivr gene was also detected in all of the intestinal sectors from broilers fed with either a CC diet or a PTC diet. (Fig 4), whereas the phyA2 gene was not detected in all of the intestinal sectors of broilers fed with the PTC diet ( Table 5). The ivr gene was detected in a relatively high to low frequency as the extension of the intestine from broilers fed with both the CC and PTC diets (duodenum, 100% and 100%; jejunum, 80% and 90%; ileum, 50% and 60%; rectum, 10% and 20%, respectively). No transgenic phyA2 gene fragments were detected in the heart, liver, kidney, breast muscle, and leg muscle (Fig 5, Table 6). Nevertheless, the endogenous chicken gene (ov) was detectable in all of the tissue of broilers fed with both the CC and PTC diets (Fig 5).

The phyA2 protein detection in corns, diets, digesta and tissues of broilers
PhyA2 protein was not detected in all the CC and CC diet samples, but was shown in PTC and PTC diet samples (Fig 6). In addition, the phyA2 protein was not observed in all the digesta samples from broilers fed with the CC diets (Fig 7). However, the phyA2 protein was detected in the 70% of duodenum samples and 30% of jejunum samples taken from broilers fed with PTC ( Table 7). The PhyA2 protein was not detectable in the ileal and rectal contents from broilers fed with the PTC diets. The phyA2 protein was not detected in the heart, liver, kidney, breast and leg muscles of broilers fed with CC or PTC diets (Fig 8, Table 7), though the internal reference protein (β-actin) was detected in all the tissue samples of broilers fed with CC or PTC diets (Fig 8).

Influence of PTC on microbial microflora in the intestine of broilers
Intestinal microflora plays a critical role in the maintenance of health, the digestion, absorption, and metabolism of nutrients [26,27]. Diet has significant impacts on the intestinal microbiota [28]. Plate culture is a traditional technique to detect intestinal microbial communities. The PCR-DGGE, a molecular biotechnology, provides a rapid, accurate and culture-independent survey of the microbial community, and has been widely used to study the gut microbiota of animals in the recent years. No significant differences in the number and diversity of intestinal microbes between broilers receiving non-GM diet and a GM rice-based diet have been reported [29]. Likewise, a previous report showed that there were no differences in the diversity of intestinal microbes between mice fed Bar-transgenic rice diet and conventional diet [30]. Similar findings in bees were also documented by some researchers [31,32]. The results from the present study indicated that the number and diversity of intestinal microbes from broilers fed with PTC and CC diets were not different, indicating that PTC did not have an adverse effect on the intestinal microflora of broilers.

Detection of phyA2 gene in digesta and tissues of broilers
Foreign DNA fragments are one concern in terms of safety associated with GM crops. Highly sensitive PCR can be used to detect transgenic DNA in animal tissues. The results from the  current study showed that the ivr gene was detected with high efficiency in all the intestinal sectors of non-GM and GM treatments, and no significant differences on the ivr recovery in the gut were found between the two treatments. However, the phyA2 D [20]NA was not detected in the different sections of intestine of the broilers fed with the PTC diets. Similar findings have been reported by Chambers et al. [33], who found that transgenic genes were not detectable in the distal GIT of chickens fed with GM corn. Ma et al. [15] also reported that fragments of the phyA2 genes in the digesta of laying hens fed with a PTC diet reduced as the extension of the GIT (crop, 100%; gizzard, 87.5%; and small intestine or rectum, 0%). Thus, the transgenic phyA2 gene tends to be rapidly degraded as it moves through GIT of poultry. In addition, the phyA2 gene was only detected in the PTC and PTC diets, and not in the CC and CC diets in this study, suggesting that cross-contamination between the two treatments was not found. In general, some foreign DNA in feeds can be absorbed by animals. It is speculated that DNA of GM crops might transfer into tissues of domestic animals. It was reported that foreign DNA fragments could be inserted into immune cells of mice [34]. This makes it necessary to detect the fragments of foreign DNA in the gut and tissues of food-producing animals. Recently, a number of studies have demonstrated that no ingested foreign DNA was found in the blood or tissues of poultry [17,20] and livestock [35] fed with transgenic grains. Similar results were also obtained from studies with other animal species, such as fish [36] and rabbits [37]. A study suggested that the transgenic DNA of GM soya was not detectable as it moved through the GIT of healthy humans [38]. Ma et al. also reported that phyA2 genes were not found in the blood, heart, liver, spleen, kidneys and breast muscle of laying hens fed with a PTC diet [15]. The results from the present study were consistent with the above findings, which showed that none of the transgenic gene (phyA2) was detected in any broiler tissues of the GM group. . Arrows represent the expected length of PCR products. M: marker; 2, 4, 6, and 8: heart, liver, kidney, breast muscle, and leg muscle of CC-fed broilers, respectively; And 3, 5, 7, 9, and 11: heart, liver, kidney, breast muscle, and leg muscle of PTC-fed broilers, respectively. doi:10.1371/journal.pone.0143408.g005 Phytase Transgenic Corn on Broilers Table 6. Detections of the transgenic phyA2 gene in corns, diets, digesta, and tissues of broilers fed with either control corn (CC) or phytase transgenic corn (PTC) 1 .

Detection of phyA2 protein in digesta and tissues of broilers
In the present study, the phyA2 protein was detected in 100% of PTC and PTC diet samples, 70% of duodenum samples and 30% of jejunum samples of broilers fed with PTC, but not detected in the lower gastrointestinal tract (ileum and rectum) samples. Similar evidences in laying hens were provided by Ma et al. [15]: the phyA2 protein was found in the crop (100%), gizzard (87.5%), duodenum (37.5%) and jejunum (12.5%), and not found in the ileum, cecum, and rectum of laying hens fed with PTC. These results also are consistent with the results obtained from laying hens [39]. However, Chowdhury et al. [16] demonstrated that Cry1Ab protein was detected at very high concentrations in rectal digesta of pigs. The possible explanation for degradation of the recombinant protein might be that the ingested protein is usually degraded into small peptides and/or free amino acids, which are ingested in the upper intestine [40].
The differences concerning the digestive fate between the DNA and protein in the intestine have been obtained also by Ma et al. [15] and Walsh et al. [41] This might be because extraneous protein has a steadier structure than its gene and could be protected against rapid degradation in the upper GIT.
Proteins produced from foreign genes might be undetectable in the tissues of animals due to their rapid degradation in the gut. Ash et al. [39] demonstrated that CP4 EPSPS protein was not detected in the liver, egg, or feces of laying hens fed with Roundup Ready (RR) soybean meal. Similar results were obtained also in some studies on GM corn. The Cry1Ab protein was not detected in the breast muscle of broilers fed with transgenic corn (MON 810) either [42]. Furthermore, the phyA2 protein was not found in the blood, heart, liver, spleen, kidney, and breast muscle from laying hens fed with the PTC diet [15]. To date, the appearance of transgenic protein in tissues of broilers fed with GM crops has not been reported in studies with broilers. The current results showing that no phyA2 protein was detected in the heart, liver, kidney, breast and leg muscles of broilers are consistent with those previous studies.

Phytase Transgenic Corn on Broilers
In addition, in the present study, we did not measure phytate P, non-phytate P, and phytase activity in the digesta of broilers. However, we got some relative information from the studies of Nyannor et al. [43] and Gao et al. [13]. In their studies, it was reported that the addition of corn expressing phytase to the control diet increased residual phytase activity and decreased phytic acid P concentration in the digesta of proventriculus and gizzard, jejunum, and ileum of broilers and laying hens. It was also observed that residual phytase activity and phytic acid P concentration in the digesta decreased caudally along the GIT. Therefore, we speculate that the similar cases might have occurred in our present study.

Conclusions
In summary, this study shows that PTC had no adverse effect on intestinal microflora of broilers. The phyA2 DNA was not detected in the intestinal digesta of broilers. The transgenic Table 7. Western blot results of the phyA2 protein in corns, diets, digesta, and tissues of broilers fed with either control corn (CC) or phytase transgenic corn (PTC) 1 .

Item
Number   60KD) and β-actin (right, 43KD) protein in tissues of broilers fed with either control corn (CC) or phytase transgenic corn (PTC). 1: phytase transgenic corn (PTC); 2, 4, 6, 8, and 10: heart, liver, kidney, breast muscle, and leg muscle of CC-fed broilers, respectively; And 3, 5, 7, 9, and 11: heart, liver, kidney, breast muscle, and leg muscle of PTC-fed broilers, respectively. phyA2 protein was detected in the digesta of the duodenum and jejunum, but not in the ileal and rectal digesta. PhyA2 DNA or protein was not transferred to the heart, liver, kidney, breast and leg muscles of broilers. These results collectively indicate that the incorporation of PTC in the broiler diets should not be an issue in terms of the accumulation of foreign phytase DNA and proteins in different organs, as well as their influences on intestinal microflora.