- Email: [email protected]
Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers
Soybeans Transformed with a Fungal Phytase Gene Improve Phosphorus Availability for Broilers D. MICHAEL DENBOW,*,1 ELIZABETH A. GRABAU,† GEORGE H. LACY,† E. T. KORNEGAY,* DAVID R. RUSSELL,‡ and PAUL F. UMBECK‡ *Department of Animal and Poultry Sciences, †Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, and ‡Agracetus, Middleton, Wisconsin 53562 nP linearly increased body weight gain, feed efficiency, feed intake, toe ash weight and percentage, and tibia shear force and energy. Phosphorus digestibility decreased linearly as nP level increased, but P excretion increased. Dietary phytase linearly increased growth rate, feed intake, toe ash weight and percentage, tibia shear force and energy, and P digestibility, whereas excretion was decreased. Except for P digestibility, there was no difference in efficacy of responses for performance, bone mineralization, and P excretion between the two sources of phytase. It appears from this study that phytase can improve growth performance of broilers fed low nP diets when provided either as a commercial supplement or in the form of transformed seeds.
ABSTRACT Male broilers (n = 416) were used to compare the efficacy of providing dietary phytase either as a commercial supplement or as a recombinant protein in transformed soybean. From 7 to 21 d of age, broilers were fed a basal diet containing 0.20% nonphytate P (nP) with additional supplementation by fungal phytase as Natuphos or as raw transformed soybeans expressing recombinant phytase at 400, 800, or 1,200 U/kg. For comparison, broilers were also fed the basal diet containing 0.08, 0.16, or 0.24 added nP. The basal diet was fed as the negative control. Diets were consumed ad libitum as a mash. All excreta were collected from each pen from 18 through 20 d of age, and the birds were killed at 21 d of age. Supplementing the basal diet with
(Key words: phytase, broiler, phosphorus availability, transgenic soybean) 1998 Poultry Science 77:878–881
present study was to determine whether soybeans, a common feed component, engineered to express fungal phytase can serve as an effective supplement to broiler diets to enhance P availability.
INTRODUCTION Although P is plentiful in meal prepared from plant seeds, the majority of this P is in the form of phytic acid (myo-inositol 1,2,3,4,5,6 hexakis dihydrogen phosphate; IUPAC-IUB, 1977) and largely unavailable to the bird (Ravindran et al., 1994, 1996). The addition of phytase prepared from Aspergillus ficuum (now classified as Aspergillis niger) to a corn-soybean meal diet was shown to make P more available to birds (Nelson et al., 1971). This result has been recently confirmed in poultry and swine diets (Jongbloed et al., 1992; Cromwell et al., 1993; Denbow et al., 1995; Yi et al, 1996). Transgenic tobacco seeds expressing the A. niger phytase have been shown to enhance P availability when included in poultry diets (Pen et al., 1993); however, unlike soybeans or corn, tobacco is not a normal constituent of poultry diets. The purpose of the
Received for publication September 23, 1997. Accepted for publication January 20, 1998. 1To whom correspondence should be [email protected]
MATERIALS AND METHODS On the day of hatch, 416 male broiler chicks (Ross × Arbor Acres), vaccinated for Marek’s disease only, were equally divided among 52 electrically heated pens with raised wire floors. Chicks were exposed to continuous fluorescent light. All birds were fed a standard cornsoybean meal starter diet (3,146 mcal ME/kg, 24% CP, 0.45% available P, and 1.0% Ca) for the 1st wk. Beginning at 1 wk of age, each of 12 dietary treatments were randomly assigned to four pens except for the basal diet (Table 1), which was fed to eight pens. All diets, except Diets 11 and 12, contained 21.44% of a 1:1 mixture of corn and ground raw soybeans from either transformed or nontransformed soybeans. Diet 1,
addressed:
Abbreviation Key: AOAC = Association of Official Analytical Chemist; nP = nonphytate phosphorus.
878
879
RESEARCH NOTE TABLE 1. Composition of basal diet Ingredients
Percentage
Ground corn (8.8% CP) Soybean meal (48% CP) Defluorinated phosphate Limestone Stabilized fat Vitamin premix1 Trace mineral premix2 Iodized salt DL-methionine MBD (22.7 g/kg)3 Total
39.57 30.84 0.41 1.78 3.00 0.50 0.20 0.40 0.20 0.05 76.95
1Supplied per kilogram of diet: retinyl acetate, 1,816 mg; cholecalciferol, 132 mg; dl-a-tocopheryl acetate, 53 mg; menadione sodium bisulfate complex, 1.5 mg; riboflavin, 15 mg; dl-calcium pantothenate acid, 19.4 mg; niacin, 52.8 mg; cyanocobalamin, 22 mg; choline chloride, 2,025 mg; biotin, 0.62 mg; folic acid, 6.2 mg; thiamin-HCl, 16 mg; pyridoxine-HCl, 6.2 mg; ethoxyquin, 100 mg; virginiamycin, 5.8 mg. 2Supplied per kilogram of diet: manganese, 88 mg; zinc, 95 mg; iron, 100 mg; copper, 12.5 mg; and selenium, 0.6 mg. 3Bacitracin methylene disalicylate.
the basal diet, contained 0.20% nonphytate P (nP) and 0.46% total P. Diets 2 to 4 had increasing levels of nP (0.08, 0.16, or 0.24%) added as defluorinated phosphate to provide diets with 0.28, 0.36, and 0.44% nP. Diets 5 to 7 had varying levels of supplemental phytase (400, 800, and 1,200 U/kg) provided by substituting transformed soybeans for nontransformed soybeans. Diets 8 to 10 were similar to Diets 5 to 7 except that the supplemental phytase was supplied as Natuphos2 . Diets 11 and 12 contained processed soybeans in place of raw soybeans, and contained nP levels of 0.20 and 0.44%, respectively. A 2:1 calcium:total P ratio was maintained in all diets, and diets were fed as a mash. The care and treatment of birds followed published guidelines (Consortium, 1988). The soybean expression plasmid pWRG2787, containing A. niger phytase sequence amplified by PCR, was stably introduced into soybean (Asgrow A5403) by the method of McCabe et al. (1988). Amplification resulted in two changes in the phytase sequence, the substitution of a methionine for leucine as the first amino acid of the mature phytase sequence, and an amino acid change from asparagine to glycine due to a single nucleotide substitution at position 1374 (GenBank Accession number M94550). These changes did not significantly interfere with enzyme activity. The mature phytase coding sequence was inserted downstream of the cauliflower mosaic virus 35S promoter (Gardner et al., 1981) and tobacco (Nicotiana plumbaginifolia) extension leader and signal peptide (De Loose et al., 1991). The termination signal was provided as the Agrobacterium tumefaciens nopaline synthase polyadenylation sequence (Depicker et al., 1982).
2BASF Corp., 3Model 1123,
Mount Olive, NJ 17828-1234. Instron Corp., Canton, MA 02021.
From Day 18 through 20, all the excreta from each pen were quantitatively collected and stored at –20 C, and feed intake measured. The samples were dried at 55 to 60 C, then ground to pass through a 1-mm sieve. Dry matter was determined according to AOAC (1990) procedures. Following a nitric-perchloric acid wet digestion, P concentrations were determined colorimetrically (AOAC, 1990). At 3 wk of age, all surviving birds were killed by cervical dislocation. Toe samples were obtained and ashed as described by Potter (1988). The left tibia was removed from each bird, stripped of all soft tissues, and frozen. Shear force and shear energy of the tibias were determined using an Instron Universal Testing Machine3 at a loading rate of 2 mm/min. Linear and quadratic contrasts were used to evaluate statistical differences between dietary combinations. Also, phytase sources, transformed soybeans and Natuphos, were compared using linear contrasts and their interactions. The effect of raw soybeans was compared to soybean meal diets (Diets 1 and 4 vs 11 and 12). Nonlinear and linear functions were derived for P levels (Diets 1 to 4) and phytase addition from transformed soybeans (Diets 1 and 5 to 7) and from Natuphos (Diets 1 and 8 to 10) with the nonlinear model: Y = a(1 – be – kX) and linear model: Y = a + bX; where Y = the response measurements; X = nP (percentage) or phytase added (units per kilogram of diet). The calculation of equivalency values of phytase for nP was described in detail by Denbow et al. (1995). Statistical significance implies P ≤ 0.05 unless otherwise noted.
RESULTS AND DISCUSSION Supplementing the low P basal diet with 0.08, 0.16, and 0.24% nP linearly increased feed intake and BW gain during Weeks 2 and 3 (Table 2). Phosphorus digestion coefficients were linearly decreased as the dietary nP level increased, but P excretion was linearly increased with dietary additions of nP. Increasing nP also linearly increased toe ash weight and percentage, and tibia shear force and energy (Table 2). Similar results have previously been reported (Potter et al., 1995; Yi et al., 1996). Previous reports have shown that 0.20% nP (0.13% nP from plant ingredients + 0.07% P from defluorinated phosphate) would reduce growth and bone development without causing high mortality (Denbow et al., 1995; Potter et al., 1995). Across sources of phytase, supplementing the low P basal diet with 400, 800, or 1,200 U/kg of phytase linearly increased BW gain (Table 2) while having no effect on feed efficiency and feed intake during Weeks 2 and 3. Dietary phytase linearly increased P digestibility and decreased P excretion. Phytase also significantly improved the toe ash weight and percentage, and tibia shear force and energy (Table 2). These results support numerous reports showing that supplementing low nP broiler diets with phytase improves P availability
880
DENBOW ET AL.
resulting in enhanced growth and toe ash percentage, and improves bone parameters (Nelson et al., 1971; Denbow et al., 1995; Kornegay et al., 1996; Yi et al., 1996). The enhanced growth appeared to be mediated primarily by increased feed intake because phytase addition had little effect on feed efficiency. With the exception of P digestibility, which was less for Natuphos than for transformed soybeans, and a phytase source by quadratic effect for tibia force in which the response was linear for Natuphos and quadratic for transformed soybeans, there were no differences in the responses to phytase sources for several measurements. When added on an equal activity basis, phytase was equally effective in enhancing BW gain, feed intake, feed efficiency, toe ash, tibia shear force, and energy when transformed soybeans containing a fungal phytase gene or a commercial microbial phytase (Natuphos) were fed. Eeckout and DePaepe (1996) reported that phytase activity of wheat middlings was only 74% as efficient as microbial phytase (Natuphos) when fed to pigs at equal in vitro activities (500 U/kg). However, one previous report in which tobacco seeds transformed with the phytase gene were fed to broilers showed equivalent activity (Pen et al., 1993).
In order to determine whether the raw soybeans affected any of the measurements, the responses to Diets 1 and 4 were compared to those of Diets 11 and 12. Both sets of diets contained 0.20 and 0.44% nP, respectively, but Diets 11 and 12 contained only processed soybeans (soybean meal). As shown in Table 2, including raw soybeans in the diet resulted in a 3.9% decrease in BW gain (448 vs 459 g) and a 7% loss in feed efficiency (654 vs 700 g/kg); however, including raw soybeans had no effect on the other measurements. The response to added nP was similar for diets with raw soybeans or soybean meal. The present results confirm that supplementing low nP broiler diets with phytase can improve P availability and thus increase growth and improve bone strength. Adding 1,200 U/kg phytase as transformed seeds to the basal diet provided BW gain similar to that of the basal diet supplemented with 0.24% nP (474 vs 488 g). However, the basal diet supplemented with phytase also resulted in a 50% reduction in P excretion. Poultry waste is typically applied to the ground. Agriculturally produced P waste is a major environmental concern because this P can leave the site of application and cause contamination of water supplies (Sharpley et al., 1993, 1994). Therefore, reducing excretion of P in poultry production has obvious environmental implications.
TABLE 2. Effect of feeding phytase transformed soybeans (TSB) or Natuphos phytase on various growth, bone, and phosphorus measurements
BW gain Week 2 to 3
Diets
1. Basal 2. B+0.08P 3. B+0.16P 4. B+0.24P 5. B+400U phytase TSB 6. B+800U phytase TSB 7. B+1200U phytase TSB 8. B+400U phytase Natuphos 9. B+800U phytase Natuphos 10. B+1200U phytase Natuphos 11. Low P with SBM 12. High P with SBM (0.24 %P) Root mean square1 Probabilities of contrast comparisons Linear phosphorus Quadratic phosphorus Natuphos vs TSB phytase Linear phytase Quadratic phytase Source × linear Source × quadratic Diets 1 and 4 vs 11 and 12 Diets 1 and 11 vs 4 and 12 Protein × P 1Standard
(g) 407 436 487 488 435 451 474 416 440 454 400 519 25
0.001 0.006 0.105 0.001 0.314 0.607 0.171 0.007 0.001 0.001
Feed intake Week 2 to 3
(g) 618 669 739 749 643 707 720 616 686 710 590 719 41
0.042 0.350 0.084 0.001 0.128 0.937 0.908 0.367 0.001 0.439
Tibia Gain:feed Week 2 to 3
Digestibility Excretion
(g/kg) 658 650 659 649 678 638 658 677 641 639 679 720 30
(% of intake) 47.2 42.8 44.0 40.0 55.8 60.4 61.6 50.6 53.3 55.1 50.1 41.1 3.9
0.001 0.048 0.672 0.489 0.439 0.534 0.156 0.048 0.395 0.002
Phosphorus
0.024 0.921 0.001 0.014 0.516 0.751 0.705 0.315 0.001 0.669
Toe ash
(g/kg DM intake) 2.56 3.01 3.56 3.90 2.15 1.98 1.82 2.34 2.24 2.10 2.29 3.89 0.20
(g) 0.127 0.160 0.188 0.208 0.137 0.173 0.169 0.123 0.172 0.163 0.101 0.210 0.017
(% of dried toes) 8.88 10.66 11.82 12.86 9.65 10.86 11.45 9.92 10.62 10.77 8.12 12.40 0.47
0.001 0.874 0.005 0.009 0.932 0.659 0.866 0.079 0.001 0.385
0.001 0.469 0.415 0.001 0.004 0.890 0.624 0.160 0.001 0.099
0.001 0.128 0.279 0.001 0.166 0.054 0.943 0.014 0.001 0.518
Shear force
Shear energy
(kg) 287 359 466 539 336 402 385 346 369 425 294 509 31.2
(kg/cm2) 547 631 659 778 607 685 699 647 661 807 581 845 98.1
0.001 0.983 0.672 0.001 0.370 0.351 0.042 0.086 0.001 0.252
0.002 0.728 0.316 0.015 0.691 0.492 0.259 0.308 0.001 0.745
error of a treatment mean equals the root mean square/√n, where n = 4 for all treatments except the basal, where n = 8.
RESEARCH NOTE
ACKNOWLEDGMENTS Appreciation is expressed to BASF Corp., Mount Olive, NJ 17828-1234 for supplying commercial phytase, to Agracetus, Middleton, WI 53502 for supplying the transformed soybeans, to E. Mullaney for supplying the phyA clone, and to Barbara Self for technical help. This study was partially supported by UDSA NRI Award Number 94-37500-0681.
REFERENCES AOAC, 1990. Official Methods of Analysis. 15th ed. AOAC, Arlington, VA. Consortium, 1988. Guide For the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Consortium For Developing a Guide For the Care and Use of Agricultural Animals in Agricultural Research and Teaching, Champaign, IL. Cromwell, G. L., R. D. Coffey, and H. J. Monegue, 1993. Efficacy of phytase in improving the bioavailability of phosphorus in soybean meal and corn-soybean meal diets for pigs. J. Anim. Sci. 71:1831–1840. Denbow, D. M., V. Ravindran, E. T. Kornegay, Z. Yi, and R. M. Hulet, 1995. Improving phosphorus availability in soybean meal for broilers by supplemental phytase. Poultry Sci. 74: 1831–1842. Depicker, A., S. Stachel., P. Dhaese, P. Zambryski, H. M. Goodman, 1982. Nopaline synthase: Transcript mapping and DNA sequence. J. Mol. Appl. Genet. 1:499–512. De Loose, M., G. Gheysen, J. Gielen, R. Vallarroel, C. Genetello, M. Van Montagu, A. Depicker, and D. Inze´. 1991. The extension signal peptide allows secretion of a heterologous protein from protoplasts. Gene 99:95–100. Eeckout, W., and M. de Paepe, 1996. In vitro and in vivo comparison of microbial and plant phytase. Pages 237–241 in: Phytase in Animal Nutrition and Waste Management. M. B. Coelho and E. T. Kornegay, ed. BASF Corp., Mount Olive, NJ. Gardner, R. C., A. J. Howarth, P. Hahn, M. Brown-Luedi, R. J. Shepherd, and J. Messing, 1981. The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun cloning. Nucleic Acids Res. 9: 2871–2888. IUPAC-IUB: Commission on Biochemical Nomenclature, 1977. Nomenclature of phosphorus containing compounds of biochemical importance. Eur. J. Biochem. 79:1–11. Jefferson, R. A., T. A. Kavanagh, and M. W. Bevan, 1987. GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6:3901–3907.
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Jongbloed, A. W., Z. Mroz, and P. A. Kemme, 1992. The effect of supplementary Aspergillus niger phytase in diets for pigs on concentration and apparent digestibility of dry matter, total phosphorus, and phytic acid in different sections of the alimentary tract. J. Anim. Sci. 70:1159–1168. Kornegay, E. T., D. M. Denbow, Z. Yi, and V. Ravindran, 1996. Response of broilers to graded levels of microbial phytase added to maize-soyabean-meal-based diets containing three levels of non-phytate phosphorus. Br. J. Nutr. 75: 839–852. McCabe, D. E., W. F. Swain, B. J. Martinell, and P. Christou, 1988. Stable transformation of soybean (Glycine max) by particle acceleration. BioTechnology 6:923–926. Nelson, T. S., T. R. Shieh, R. J. Wodzinski, and J. H. Ware, 1971. Effect of supplemental phytase on the utilization of phytate phosphorus by chicks. J. Nutr. 101:1289–1293. Pen, J., T. C. Verwoerd, P. A. van Paridon, R. F. Beudeker, P.J.M. van den Elzen, K. Geerse, J. D. van der Klis, H.A.J. Versteegh, A.J.J. van Ooyen, and A. Hoekema, 1993. Phytase-containing transgenic seeds as a novel feed additive for improved phosphorus utilization. BioTechnology 11:811–814. Potter, L. M., 1988. Bioavailability of phosphorus from various phosphates based on body weight and toe ash measurements. Poultry Sci. 67:96–102. Potter, L. M., M. Potchanakorn, V. Ravindran, and E. T. Kornegay, 1995. Bioavailability of phosphorus in various phosphate sources using body weight and toe ash as response criteria. Poultry Sci. 74:813–820. Ravindran, V., G. Ravindran, and S. Sivalogan, 1994. Total and phytate phosphorus contents of various foods and feedstuffs of plant origin. Food Chem. 50:133–136. Ravindran, V., W. L. Bryden, and E. T. Kornegay, 1996. Phytates: Occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol. Rev. 6:125–143. Sharpley, A. N., T. C. Daniel, and D. R. Edwards, 1993. Phosphorus movement in the landscape. J. Prod. Agric. 6: 492–500. Sharpley, A. N., S. C. Chapra, R. Wedepohl, J. T. Sims, T. C. Daniel, and K. R. Reddy, 1994. Managing agricultural phosphorus for protection of surface waters: Issues and options. J. Environ. Qual. 23:437–451. Yi, Z., E. T. Kornegay, V. Ravindran, and D. M. Denbow, 1996. Improving phytate phosphorus availability in corn and soybean meal for broilers using microbial phytase and calculation of phosphorus equivalency values for phytase. Poultry Sci. 75:240–249.
ABSTRACT Male broilers (n = 416) were used to compare the efficacy of providing dietary phytase either as a commercial supplement or as a recombinant protein in transformed soybean. From 7 to 21 d of age, broilers were fed a basal diet containing 0.20% nonphytate P (nP) with additional supplementation by fungal phytase as Natuphos or as raw transformed soybeans expressing recombinant phytase at 400, 800, or 1,200 U/kg. For comparison, broilers were also fed the basal diet containing 0.08, 0.16, or 0.24 added nP. The basal diet was fed as the negative control. Diets were consumed ad libitum as a mash. All excreta were collected from each pen from 18 through 20 d of age, and the birds were killed at 21 d of age. Supplementing the basal diet with
(Key words: phytase, broiler, phosphorus availability, transgenic soybean) 1998 Poultry Science 77:878–881
present study was to determine whether soybeans, a common feed component, engineered to express fungal phytase can serve as an effective supplement to broiler diets to enhance P availability.
INTRODUCTION Although P is plentiful in meal prepared from plant seeds, the majority of this P is in the form of phytic acid (myo-inositol 1,2,3,4,5,6 hexakis dihydrogen phosphate; IUPAC-IUB, 1977) and largely unavailable to the bird (Ravindran et al., 1994, 1996). The addition of phytase prepared from Aspergillus ficuum (now classified as Aspergillis niger) to a corn-soybean meal diet was shown to make P more available to birds (Nelson et al., 1971). This result has been recently confirmed in poultry and swine diets (Jongbloed et al., 1992; Cromwell et al., 1993; Denbow et al., 1995; Yi et al, 1996). Transgenic tobacco seeds expressing the A. niger phytase have been shown to enhance P availability when included in poultry diets (Pen et al., 1993); however, unlike soybeans or corn, tobacco is not a normal constituent of poultry diets. The purpose of the
Received for publication September 23, 1997. Accepted for publication January 20, 1998. 1To whom correspondence should be [email protected]
MATERIALS AND METHODS On the day of hatch, 416 male broiler chicks (Ross × Arbor Acres), vaccinated for Marek’s disease only, were equally divided among 52 electrically heated pens with raised wire floors. Chicks were exposed to continuous fluorescent light. All birds were fed a standard cornsoybean meal starter diet (3,146 mcal ME/kg, 24% CP, 0.45% available P, and 1.0% Ca) for the 1st wk. Beginning at 1 wk of age, each of 12 dietary treatments were randomly assigned to four pens except for the basal diet (Table 1), which was fed to eight pens. All diets, except Diets 11 and 12, contained 21.44% of a 1:1 mixture of corn and ground raw soybeans from either transformed or nontransformed soybeans. Diet 1,
addressed:
Abbreviation Key: AOAC = Association of Official Analytical Chemist; nP = nonphytate phosphorus.
878
879
RESEARCH NOTE TABLE 1. Composition of basal diet Ingredients
Percentage
Ground corn (8.8% CP) Soybean meal (48% CP) Defluorinated phosphate Limestone Stabilized fat Vitamin premix1 Trace mineral premix2 Iodized salt DL-methionine MBD (22.7 g/kg)3 Total
39.57 30.84 0.41 1.78 3.00 0.50 0.20 0.40 0.20 0.05 76.95
1Supplied per kilogram of diet: retinyl acetate, 1,816 mg; cholecalciferol, 132 mg; dl-a-tocopheryl acetate, 53 mg; menadione sodium bisulfate complex, 1.5 mg; riboflavin, 15 mg; dl-calcium pantothenate acid, 19.4 mg; niacin, 52.8 mg; cyanocobalamin, 22 mg; choline chloride, 2,025 mg; biotin, 0.62 mg; folic acid, 6.2 mg; thiamin-HCl, 16 mg; pyridoxine-HCl, 6.2 mg; ethoxyquin, 100 mg; virginiamycin, 5.8 mg. 2Supplied per kilogram of diet: manganese, 88 mg; zinc, 95 mg; iron, 100 mg; copper, 12.5 mg; and selenium, 0.6 mg. 3Bacitracin methylene disalicylate.
the basal diet, contained 0.20% nonphytate P (nP) and 0.46% total P. Diets 2 to 4 had increasing levels of nP (0.08, 0.16, or 0.24%) added as defluorinated phosphate to provide diets with 0.28, 0.36, and 0.44% nP. Diets 5 to 7 had varying levels of supplemental phytase (400, 800, and 1,200 U/kg) provided by substituting transformed soybeans for nontransformed soybeans. Diets 8 to 10 were similar to Diets 5 to 7 except that the supplemental phytase was supplied as Natuphos2 . Diets 11 and 12 contained processed soybeans in place of raw soybeans, and contained nP levels of 0.20 and 0.44%, respectively. A 2:1 calcium:total P ratio was maintained in all diets, and diets were fed as a mash. The care and treatment of birds followed published guidelines (Consortium, 1988). The soybean expression plasmid pWRG2787, containing A. niger phytase sequence amplified by PCR, was stably introduced into soybean (Asgrow A5403) by the method of McCabe et al. (1988). Amplification resulted in two changes in the phytase sequence, the substitution of a methionine for leucine as the first amino acid of the mature phytase sequence, and an amino acid change from asparagine to glycine due to a single nucleotide substitution at position 1374 (GenBank Accession number M94550). These changes did not significantly interfere with enzyme activity. The mature phytase coding sequence was inserted downstream of the cauliflower mosaic virus 35S promoter (Gardner et al., 1981) and tobacco (Nicotiana plumbaginifolia) extension leader and signal peptide (De Loose et al., 1991). The termination signal was provided as the Agrobacterium tumefaciens nopaline synthase polyadenylation sequence (Depicker et al., 1982).
2BASF Corp., 3Model 1123,
Mount Olive, NJ 17828-1234. Instron Corp., Canton, MA 02021.
From Day 18 through 20, all the excreta from each pen were quantitatively collected and stored at –20 C, and feed intake measured. The samples were dried at 55 to 60 C, then ground to pass through a 1-mm sieve. Dry matter was determined according to AOAC (1990) procedures. Following a nitric-perchloric acid wet digestion, P concentrations were determined colorimetrically (AOAC, 1990). At 3 wk of age, all surviving birds were killed by cervical dislocation. Toe samples were obtained and ashed as described by Potter (1988). The left tibia was removed from each bird, stripped of all soft tissues, and frozen. Shear force and shear energy of the tibias were determined using an Instron Universal Testing Machine3 at a loading rate of 2 mm/min. Linear and quadratic contrasts were used to evaluate statistical differences between dietary combinations. Also, phytase sources, transformed soybeans and Natuphos, were compared using linear contrasts and their interactions. The effect of raw soybeans was compared to soybean meal diets (Diets 1 and 4 vs 11 and 12). Nonlinear and linear functions were derived for P levels (Diets 1 to 4) and phytase addition from transformed soybeans (Diets 1 and 5 to 7) and from Natuphos (Diets 1 and 8 to 10) with the nonlinear model: Y = a(1 – be – kX) and linear model: Y = a + bX; where Y = the response measurements; X = nP (percentage) or phytase added (units per kilogram of diet). The calculation of equivalency values of phytase for nP was described in detail by Denbow et al. (1995). Statistical significance implies P ≤ 0.05 unless otherwise noted.
RESULTS AND DISCUSSION Supplementing the low P basal diet with 0.08, 0.16, and 0.24% nP linearly increased feed intake and BW gain during Weeks 2 and 3 (Table 2). Phosphorus digestion coefficients were linearly decreased as the dietary nP level increased, but P excretion was linearly increased with dietary additions of nP. Increasing nP also linearly increased toe ash weight and percentage, and tibia shear force and energy (Table 2). Similar results have previously been reported (Potter et al., 1995; Yi et al., 1996). Previous reports have shown that 0.20% nP (0.13% nP from plant ingredients + 0.07% P from defluorinated phosphate) would reduce growth and bone development without causing high mortality (Denbow et al., 1995; Potter et al., 1995). Across sources of phytase, supplementing the low P basal diet with 400, 800, or 1,200 U/kg of phytase linearly increased BW gain (Table 2) while having no effect on feed efficiency and feed intake during Weeks 2 and 3. Dietary phytase linearly increased P digestibility and decreased P excretion. Phytase also significantly improved the toe ash weight and percentage, and tibia shear force and energy (Table 2). These results support numerous reports showing that supplementing low nP broiler diets with phytase improves P availability
880
DENBOW ET AL.
resulting in enhanced growth and toe ash percentage, and improves bone parameters (Nelson et al., 1971; Denbow et al., 1995; Kornegay et al., 1996; Yi et al., 1996). The enhanced growth appeared to be mediated primarily by increased feed intake because phytase addition had little effect on feed efficiency. With the exception of P digestibility, which was less for Natuphos than for transformed soybeans, and a phytase source by quadratic effect for tibia force in which the response was linear for Natuphos and quadratic for transformed soybeans, there were no differences in the responses to phytase sources for several measurements. When added on an equal activity basis, phytase was equally effective in enhancing BW gain, feed intake, feed efficiency, toe ash, tibia shear force, and energy when transformed soybeans containing a fungal phytase gene or a commercial microbial phytase (Natuphos) were fed. Eeckout and DePaepe (1996) reported that phytase activity of wheat middlings was only 74% as efficient as microbial phytase (Natuphos) when fed to pigs at equal in vitro activities (500 U/kg). However, one previous report in which tobacco seeds transformed with the phytase gene were fed to broilers showed equivalent activity (Pen et al., 1993).
In order to determine whether the raw soybeans affected any of the measurements, the responses to Diets 1 and 4 were compared to those of Diets 11 and 12. Both sets of diets contained 0.20 and 0.44% nP, respectively, but Diets 11 and 12 contained only processed soybeans (soybean meal). As shown in Table 2, including raw soybeans in the diet resulted in a 3.9% decrease in BW gain (448 vs 459 g) and a 7% loss in feed efficiency (654 vs 700 g/kg); however, including raw soybeans had no effect on the other measurements. The response to added nP was similar for diets with raw soybeans or soybean meal. The present results confirm that supplementing low nP broiler diets with phytase can improve P availability and thus increase growth and improve bone strength. Adding 1,200 U/kg phytase as transformed seeds to the basal diet provided BW gain similar to that of the basal diet supplemented with 0.24% nP (474 vs 488 g). However, the basal diet supplemented with phytase also resulted in a 50% reduction in P excretion. Poultry waste is typically applied to the ground. Agriculturally produced P waste is a major environmental concern because this P can leave the site of application and cause contamination of water supplies (Sharpley et al., 1993, 1994). Therefore, reducing excretion of P in poultry production has obvious environmental implications.
TABLE 2. Effect of feeding phytase transformed soybeans (TSB) or Natuphos phytase on various growth, bone, and phosphorus measurements
BW gain Week 2 to 3
Diets
1. Basal 2. B+0.08P 3. B+0.16P 4. B+0.24P 5. B+400U phytase TSB 6. B+800U phytase TSB 7. B+1200U phytase TSB 8. B+400U phytase Natuphos 9. B+800U phytase Natuphos 10. B+1200U phytase Natuphos 11. Low P with SBM 12. High P with SBM (0.24 %P) Root mean square1 Probabilities of contrast comparisons Linear phosphorus Quadratic phosphorus Natuphos vs TSB phytase Linear phytase Quadratic phytase Source × linear Source × quadratic Diets 1 and 4 vs 11 and 12 Diets 1 and 11 vs 4 and 12 Protein × P 1Standard
(g) 407 436 487 488 435 451 474 416 440 454 400 519 25
0.001 0.006 0.105 0.001 0.314 0.607 0.171 0.007 0.001 0.001
Feed intake Week 2 to 3
(g) 618 669 739 749 643 707 720 616 686 710 590 719 41
0.042 0.350 0.084 0.001 0.128 0.937 0.908 0.367 0.001 0.439
Tibia Gain:feed Week 2 to 3
Digestibility Excretion
(g/kg) 658 650 659 649 678 638 658 677 641 639 679 720 30
(% of intake) 47.2 42.8 44.0 40.0 55.8 60.4 61.6 50.6 53.3 55.1 50.1 41.1 3.9
0.001 0.048 0.672 0.489 0.439 0.534 0.156 0.048 0.395 0.002
Phosphorus
0.024 0.921 0.001 0.014 0.516 0.751 0.705 0.315 0.001 0.669
Toe ash
(g/kg DM intake) 2.56 3.01 3.56 3.90 2.15 1.98 1.82 2.34 2.24 2.10 2.29 3.89 0.20
(g) 0.127 0.160 0.188 0.208 0.137 0.173 0.169 0.123 0.172 0.163 0.101 0.210 0.017
(% of dried toes) 8.88 10.66 11.82 12.86 9.65 10.86 11.45 9.92 10.62 10.77 8.12 12.40 0.47
0.001 0.874 0.005 0.009 0.932 0.659 0.866 0.079 0.001 0.385
0.001 0.469 0.415 0.001 0.004 0.890 0.624 0.160 0.001 0.099
0.001 0.128 0.279 0.001 0.166 0.054 0.943 0.014 0.001 0.518
Shear force
Shear energy
(kg) 287 359 466 539 336 402 385 346 369 425 294 509 31.2
(kg/cm2) 547 631 659 778 607 685 699 647 661 807 581 845 98.1
0.001 0.983 0.672 0.001 0.370 0.351 0.042 0.086 0.001 0.252
0.002 0.728 0.316 0.015 0.691 0.492 0.259 0.308 0.001 0.745
error of a treatment mean equals the root mean square/√n, where n = 4 for all treatments except the basal, where n = 8.
RESEARCH NOTE
ACKNOWLEDGMENTS Appreciation is expressed to BASF Corp., Mount Olive, NJ 17828-1234 for supplying commercial phytase, to Agracetus, Middleton, WI 53502 for supplying the transformed soybeans, to E. Mullaney for supplying the phyA clone, and to Barbara Self for technical help. This study was partially supported by UDSA NRI Award Number 94-37500-0681.
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