Four research experiments were conducted to evaluate the influence of dietary enzyme inclusion (phytase and NSPase) on broiler performance parameters, processing yields, and bone mineralization. In Experiment 1, a 35-day grow out trial was conducted to investigate the effect of three commercially available phytase enzymes on growth performance and bone mineralization in phosphorus deficient corn/soy based diets. Increasing the level of available phosphorus (aP) in the control diets resulted in improved bird performance and bone ash data. The presence of dietary phytase in phosphorus deficient diets resulted in improvements in growth parameters and bone mineralization. Regression analysis confirmed that phytase supplementation can potentially increase the bioavailability of phosphorus in broiler diets up to 0.15 to 0.20 percent, however, the responses varied according to the enzyme used and inclusion level. In Experiment 2, a 42-day grow out trial was conducted to analyze the effects of NSPase inclusion on broiler performance and processing parameters when supplemented in diets with varying protein and energy concentrations. Reduced protein and energy levels reduced bird performance throughout the trial. The inclusion of both NSPase enzymes resulted in improvements in feed conversion throughout the starter and grower periods (day 26 of age). The results from this trial showed that NSPase inclusion can improve broiler performance and processing parameters. In Experiments 3 and 4, a battery trial and a floor trial were conducted to determine the effects of phytase and NSPase enzyme co-administration on growth and bone ash in low phosphorus diets. Increasing the level of available phosphorus resulted in increased bird performance and bone ash. The inclusion of phytase enhanced bird performance and bone mineralization. NSPase inclusion in diets containing low levels of phytase had improvements in bird performance during early stages of growth. The enhanced effects associated with dual administration of phytase and NSPase were not observed in a full grow out trial during later stages of growth. These four experiments indicate that phytase and NSPase enzyme inclusion in broiler diets have the ability to enhance bird performance, processing yield, and bone mineralization.

Growing interests in dietary manipulation to reduce P excretion in animal manure and P loss from agricultural soils to the environment have led to strategies such as the use of phytase in monogastric animal diets. The efficacy of phytase has been confirmed by several studies that reported its ability to hydrolyze phytate P present in grain feeds and thus, reduce manure total P. However, the solubility of manure P from phytase supplemented diets in soils is not well known, and as thus, the environmental implications of dietary P manipulation require further investigation. Two related studies were carried out in the laboratory to investigate the fate of manure phosphorus (P) from pigs fed phytase supplemented diets in Manitoba soils. The first study characterized the forms of manure P from phytase supplemented swine diets to evaluate their potential environmental impact. The seven dietary treatments fed randomly to a total of 28 growing pigs were: a positive control that contained P at the NRC (1998) recommendations (NRC), a negative control (RED) containing 0.1 percentage units reduction (about 33%) in available P from 1998 NRC recommendations, RED with 500 U of phytase kg-1 of diet (RED + P1), RED with 1000 U of phytase kg-1of diet (RED + P2), a double negative control with no added inorganic P (DNC), DNC plus 2000 U of phytase kg-1 of diet (DNC + P3) and DNC plus 4000 U of phytase kg-1 of diet (DNC + P4). The second study examined the solubility of manure P from the manure collected from the first study. Manure collected from the first study were applied at a rate of 75 kg of total P ha-1 of soil to surface samples from four Manitoba soils (0-15 cm); Osborne clay (Rego Humic Gleysol/Gleysolic Humic Vertisol), Red River clay (Gleyed Rego Black Chernozem/Gleyed Humic Vertisol), Ladywood very fine sandy loam (Gleyed Dark Gray Chernozem), and Glenhope loamy fine sand (Gleyed Rego Black Chernozem). In the first experiment, total P in feces and manure were significantly reduced (p < 0.05) with phytase addition to the diets. The labile P concentration (sum of H2O–P and NaHCO3-P) was about 71 to 89% and 77 to 89% of total P in both feces and manure, respectively. Phytase addition to the diets reduced the labile P in feces. The solubility of P was greatest in the calcareous soils amended with the manure from the DNC diets and solubility of P varied with time and extracting solutions. Although, a combination of physico-chemical properties (e.g. CEC, Exchangeable Ca2+), texture seems to play a significant role, as P solubility increased in coarse textured soils after longer period of incubation (16wks). However, our results showed that phytase supplementation in the diets of pigs did not affect the solubility of manure P in amended soils.

An extant model was evaluated to assess its adequacy for nutrient management planning for swine operations in Manitoba with regards to phosphorus (P) and nitrogen (N) excretion and the land base for the optimum spreading of manure based on P requirement of crops. Two dietary treatments were used, control diet formulated to meet the requirement of pigs for nutrients as per the recommendations of NRC (No-phytase) and a diet formulated with P level in the No-phytase diet reduced by an average of 0.1 percentage units and amended with microbial phytase at 500 FTU/kg (Phytase). Data was generated from starter to finisher pigs (10 per dietary treatment) and sows (9 per dietary treatment) to evaluate the model. The model was found to be adequate for the prediction of P outputs from starter to finisher but not sows. Model was found to be inadequate for prediction of N output.

Growing interests in dietary manipulation to reduce P excretion in animal manure and P loss from agricultural soils to the environment have led to strategies such as the use of phytase in monogastric animal diets. The efficacy of phytase has been confirmed by several studies that reported its ability to hydrolyze phytate P present in grain feeds and thus, reduce manure total P. However, the solubility of manure P from phytase supplemented diets in soils is not well known, and as thus, the environmental implications of dietary P manipulation require further investigation. Two related studies were carried out in the laboratory to investigate the fate of manure phosphorus (P) from pigs fed phytase supplemented diets in Manitoba soils. The first study characterized the forms of manure P from phytase supplemented swine diets to evaluate their potential environmental impact. The seven dietary treatments fed randomly to a total of 28 growing pigs were: a positive control that contained P at the NRC (1998) recommendations (NRC), a negative control (RED) containing 0.1 percentage units reduction (about 33%) in available P from 1998 NRC recommendations, RED with 500 U of phytase kg-1 of diet (RED + P1), RED with 1000 U of phytase kg-1of diet (RED + P2), a double negative control with no added inorganic P (DNC), DNC plus 2000 U of phytase kg-1 of diet (DNC + P3) and DNC plus 4000 U of phytase kg-1 of diet (DNC + P4). The second study examined the solubility of manure P from the manure collected from the first study. Manure collected from the first study were applied at a rate of 75 kg of total P ha-1 of soil to surface samples from four Manitoba soils (0-15 cm); Osborne clay (Rego Humic Gleysol/Gleysolic Humic Vertisol), Red River clay (Gleyed Rego Black Chernozem/Gleyed Humic Vertisol), Ladywood very fine sandy loam (Gleyed Dark Gray Chernozem), and Glenhope loamy fine sand (Gleyed Rego Black Chernozem). In the first experiment, total P in feces and manure were significantly reduced (p < 0.05) with phytase addition to the diets. The labile P concentration (sum of H2O–P and NaHCO3-P) was about 71 to 89% and 77 to 89% of total P in both feces and manure, respectively. Phytase addition to the diets reduced the labile P in feces. The solubility of P was greatest in the calcareous soils amended with the manure from the DNC diets and solubility of P varied with time and extracting solutions. Although, a combination of physico-chemical properties (e.g. CEC, Exchangeable Ca2+), texture seems to play a significant role, as P solubility increased in coarse textured soils after longer period of incubation (16wks). However, our results showed that phytase supplementation in the diets of pigs did not affect the solubility of manure P in amended soils.

An extant model was evaluated to assess its adequacy for nutrient management planning for swine operations in Manitoba with regards to phosphorus (P) and nitrogen (N) excretion and the land base for the optimum spreading of manure based on P requirement of crops. Two dietary treatments were used, control diet formulated to meet the requirement of pigs for nutrients as per the recommendations of NRC (No-phytase) and a diet formulated with P level in the No-phytase diet reduced by an average of 0.1 percentage units and amended with microbial phytase at 500 FTU/kg (Phytase). Data was generated from starter to finisher pigs (10 per dietary treatment) and sows (9 per dietary treatment) to evaluate the model. The model was found to be adequate for the prediction of P outputs from starter to finisher but not sows. Model was found to be inadequate for prediction of N output.

Bielefeld, Universiẗat, Diss., 2002.

The objective of this research was to isolate and characterize soybean genes involved in phytic acid metabolism for use in genetic engineering strategies to improve phosphorus utilization. A soybean phytase from germinated cotyledons was purified 28,000-fold to apparent homogeneity and was determined to be a glycosylated homodimer with 70 kD subunits. Soybean phytase preferred phytate as substrate (Km = 60 mM) and was capable of removing of all six phosphate groups from phytate. The pH and temperature optima for soybean phytase activity were 4.5 and 58*C, respectively. The N-terminus and four internal peptides from the purified soybean phytase were sequenced by Edman degradation. The amino acid sequence data were used to design degenerate oligonucleotide primers for PCR amplification of the soybean phytase coding sequence. A protein 547 amino acids in length was predicted from the 1641 bp coding sequence. The phytase protein showed significant similarity to plant purple acid phosphatases (PAPs) and contained the conserved metallo-phosphomonoesterase active site motif. The soybean phytase coding sequence was placed under the control of a constitutive 35S CaMV promoter in a soybean biolistic transformation vector and was introduced into "Williams 82" suspension culture cells by particle bombardment. Stably transformed cell suspension lines were recovered. DNA blot analysis demonstrated that the recombinant soybean phytase coding sequence had integrated into the genomes of two cell lines. Expression of the transgene was confirmed by RNA blot analysis. Phytase activity was three to four fold higher in these two lines compared to control non-transformed cultures. A soybean L-myo-insoitol-1-phosphate synthase (MIPS) cDNA was isolated from total RNA from developing seeds. The protein encoded by the soybean MIPS cDNA showed 87-91% homology to MIPS protein sequences from other plant species. RNA blot analysis of staged developing soybean seeds revealed that MIPS is transcribed early in the cotyledonary stage of development. Compared to other soybean tissues, MIPS expression levels were highest in developing seeds. DNA blot analysis demonstrated that multiple copies of the MIPS gene are present within the soybean genome. / Ph. D.

Previous research shows that dissolved P losses in runoff are well related to soil test P (STP), thus, various P loss prediction models incorporate the use of a STP vs. runoff DRP relationship. However, the relationship between STP and runoff DRP will vary based on soil type due to differences in soil properties. The purposes of the first two studies were to (i) investigate the effect of soil mineralogy on P sorption behavior and dissolved P in runoff and (ii) determine if any simple soil test extractions could indirectly take into account this effect of mineralogy. Nine soil types from the Virginia Piedmont, Coastal Plain, and Ridge and Valley were collected and used in a rainfall simulation study. Phosphorus retention among separated clay fractions and whole soils were related to Al bearing minerals such as hydroxy-interlayered vermiculite (HIV), gibbsite, and amorphous Al. Samples dominated by kaolinite retained very little P. Application of these results to runoff data showed that soil types with a HIV:kaolinite ratio > 0.5 caused significantly less DRP in runoff for a given soil WSP level compared to soils with a ratio < 0.5. The second study showed that the soil P vs runoff DRP relationships varied between physiographic provinces. Generally, the Coastal Plain soils resulted in a higher runoff DRP concentration for a given soil P level compared to Piedmont and Ridge and Valley soils. However, soil M3-P/Al resulted in one relationship with DRP for all three groups of soils. Results from the incubation study suggested that Al related P is more easily desorbed into solution compared to Fe related P. The final study demonstrated that phytase enzyme and high available P corn supplements in poultry diets can reduce manure WSP and total P. Results from the runoff study showed that DRP losses were related to sediment losses which consisted of > 90% manure particles. Manure particles were directly deposited into the collection container followed by desorption of P based upon the WSP content of that manure type. The results emphasized best management practices that prevent direct loss of manure particles from soil into surface waters. / Ph. D.

As environmental concerns increase in the U.S., there is a push to go "green". This has fueled the development of ethanol production in the U.S. as a renewable source of energy. In the U.S., corn is used to produce ethanol. Therefore, displacement of corn used in the animal industry to ethanol production has, inevitably caused an increase in the price of corn. Replacing conventional ingredients, such as corn, in monogastric diets with lower cost by products can be attractive economically. The objective of this study was to determine if exogenous enzymes that free reducing sugars from fiber will increase the dispensability of bound nutrients in the economically favorable by-product of ethanol production, distillers dried grains with solubles (DDGS). Effects of non-starch polysaccharidase (NSPase) inclusion rates and combinations with phytase on the ability to release reducing sugars were investigated. Distillers dried grains with solubles was tested in vitro in combination with hemicellulase (0, 0.1, 1, and 10 U/g of DDGS), cellulase (0, 1.5, 15, and 150 U/g of DDGS), xylanase (0, 2.5, 25, and 250 U/g of DDGS), ï ¢ glucanase (0, 0.006, 0.06, and 0.6 U/g of DDGS), and phytase (0, 250, 500, and 1,000 FTU/kg of DDGS). Results indicated an inhibitory effect of phytase on the ability of NSPases to release reducing sugars from DDGS (P = 0.0004). A second study was conducted to determine individual enzyme kinetics of NSPase on pure substrates when combined with increasing concentrations of phytase. Cellulase (P = 0.03), xylanase (P = 0.09), and ï ¢-glucanase (P = 0.06) combined with increasing concentrations of phytase showed a reduction in the velocity of reducing sugars release from pure substrate. However, we speculate that due to the inability to reach Vmax, the Lineweaver-Burk results were inconclusive. The 1/Km was calculated for all enzymes, and the presence of phytase increased 1/Km for xylanase (P = 0.006). However, -1/Km did not change for cellulase (P = 0.62), ï ¢-glucanase (P = 0.20), and hemicellulase (P = 0.13). In addition, the 1/Vmax was calculated for all enzymes, and the presence of phytase decreased 1/Vmax for cellulase (P = 0.03) and ï ¢ glucanase (P = 0.01). However, the presence of phytase resulted in no change for the 1/Vmax for xylanase (P = 0.81) and hemicellulase (P = 0.14). The slopes of the regression lines for the Lineweaver-Burk plots showed no effect of the presence of phytase for cellulase (P = 0.40) and hemicellulase (P = 0.27). However, the presence of phytase decreased the linear slope for xylanase (P = 0.006), and increased the linear slope for ï ¢ glucanase (P = 0.006). In summary, phytase appears to act as an inhibitor of NSPase activity in the in vitro digestion on DDGS; however, inconsistent results from the inhibition curves preclude us from determining the type of inhibition. / Master of Science

The role of dietary phytate (inositol hexaphosphate) in the regulation of intestinal mucosa phytase was investigated in chicks. Seven-day-old chicks were grouped by weight into six blocks of three cages with six birds per cage. Three purified diets [a chemically defined casein diet, a chemically defined casein diet plus sodium phytate (20 g/kg diet) and a chemically defined casein diet plus sodium phytate (20 g/kg diet) and microbial phytase (1000 units/kg diet)] were randomly assigned to cages within each block. Chicks were fed experimental diets from 8 to 22 days of age then killed, and duodenal mucosa and left tibia removed. Phytase activity in duodenal mucosa, growth performance and bone ash content were determined. Addition of phytate to the chemically defined casein diet reduced (p < 0.05) the Vmax of the duodenal brush border phytase, but the Km of the enzyme was not affected. Addition of phytate also reduced (p < 0.05) weight gain, feed intake, feed efficiency and percentage ash. Addition of microbial phytase fully restored the feed efficiency (p < 0.05), but Vmax and body weight gain were only partially restored (p < 0.05). In conclusion, it would seem that dietary phytates non-competitively inhibit intestinal mucosa phytase.