Hello, my name is Charmaine Espinosa, and I’ll be presenting the results of the experiments we have conducted at the University of Illinois which is the effect of a novel consensus bacterial 6-phytase variant on mineral digestibility and bone ash in young growing pigs.

A traditional US swine diet mainly consists of corn and soybean meal, and these feed ingredients contain phytate, which consists of 6 phosphate molecules bound to a myo-inositol ring. The problem with that is pigs do not synthesize adequate amounts of endogenous phytase to liberate the Phosphorus (P) that is bound to phytate; thus, majority of P in plant sources is not available for absorption and is the reason for the low digestibility of Ca and P by pigs. Therefore, microbial phytase is usually included in diets for pigs to increase P absorption and utilization by hydrolyzing phytic acid within the gastrointestinal tract of pigs.
However, pig diets may include feed ingredients such as canola meal and rice bran due to availability of these feed ingredients to some areas. Corn and soybean meal contain 0.5-1.7% phytate and these concentrations are less compared with canola meal and rice bran that typically contain 3 to 8% phytic acid. Therefore, if we include a significant portion of these feed ingredients in the diet, it is expected that the concentration of phytate will increase in the diets. With that in mind, we wanted to determine if supplementation of high doses of phytase is needed to reduce the negative impact of high concentration of phytate in the diets.
High doses of phytase is hypothesized to increase degradation of phytate in the intestinal tract of pigs, and therefore, increases the release of nutrients other than Ca and P. Phytate acid is unstable in the free acid form and occurs mainly as a complex with metal cations; thereby, the formation of these complexes reduces the digestibility of minerals in the gastrointestinal tract of pigs. Therefore, the use of high doses of phytase in diets may exert positive effects on nutrient digestibility due to increased reduction of the anti-nutritional effects of phytate. However, the effect of phytase in high-phytate containing diets are limited.
A next generation biosynthetic bacterial 6-phytase has been developed and it is believed to increase P availability in diets for pigs. In an in-vitro study, it was demonstrated that this novel consensus phytase has high thermostability, and also has high relative activity over a wide pH range. However, there are limited data to demonstrate the efficacy of this phytase.
Therefore, an experiment was conducted to test the hypothesis that the negative impact of phytate is reduced at higher phytase doses. 
It was also the objective of this research to test the hypothesis that inclusion of the novel phytase to diet increases bone ash and apparent total tract digestibility (ATTD) of minerals in diets containing varying phytate concentrations, 
And also to test the hypothesis that the novel phytase is as effective as the Buttiauxella phytase in exerting positive effects on nutrient digestibility in bone ash in young growing pigs.
For this experiment, a total of 18 diets were prepared. Three diets based on corn, soybean meal, and canola meal were formulated. The first basal diet was formulated to contain 0.23% phytate-bound P, whereas the second and third basal diets were formulated to contain 0.29 and 0.35% phytate-bound P, respectively, by increasing the concentration of canola meal at the expense of soybean meal and cornstarch. Twelve additional diets were formulated by adding 500, 1,000, 2,000, or 4,000 phytase units of a novel consensus bacterial 6-phytase variant or PhyG to the 3 basal diets. In addition, 3 reference diets were formulated by adding a commercial Buttiauxella phytase or PhyB at 1,000 FTU/kg to the 3 basal diets containing no PhyG.
A total of 144 growing pigs with an initial body weight of 12.7 kg were randomly allotted to 18 diets with 8 replicate pigs per diet. Pigs were assigned to treatment groups using a randomized complete block design with 4 blocks of 36 pigs and 2 replicate pigs per diet in each block.
Pigs adapted to the diets for 15 days and fecal samples were collected on day 16 to day 19, and on the last day of the experiment, pigs were euthanized and the right femur was collected immediately. Diets and fecal samples were analyzed for minerals to be able for us to calculate for mineral digestibility, whereas femurs were defatted, dried, and then ashed at 600 ºC for 16 h.
Data were analyzed in a 3 × 5 factorial arrangement with the pig as the experimental unit. The model included the amount of phytate-bound P, phytase which is PhyG, and the interaction between phytate-bound P and PhyG as the main effects. Contrast statements were used to determine the effects of PhyG (1,000 FTU/kg) on mineral digestibility and bone ash by comparing the diet with the diet supplemented with 1,000 FTU/kg from the Buttiauxella phytase.
Moving on with the results:
This graph shows the digestibility of Ca in experimental diets. The x-axis represents the dose of the novel phytase, whereas the y-axis represents the response parameter. The blue line represents the diet containing 0.23% phytate-bound P; orange, the diet with 0.29% phytate-bound P; and the green line represents the diet with 0.35% phytate bound-P.  No interaction between phytate and PhyG was observed for Ca digestibility. Diets containing 0.35% phytate-bound P had reduced digestibility of Ca compared with diets containing 0.23 or 0.29% phytate-bound P, inclusion of PhyG to diets increased ATTD of Ca in all experimental diets. 
An interaction between phytate and PhyG was observed for P digestibility, where PhyG increased the ATTD of P, but to a greater extent in diets with 0.23 or 0.29% phytate-bound P, than in diets with 0.35% phytate-bound P. The observed improvement in Ca and P digestibility upon PhyG inclusion indicates than the novel phytase was able to hydrolyze the Ca-phytate complexes, as well as some of the ester bonds between P and the inositol ring of phytate. The observation that diets containing higher levels of phytate had reduced digestibility of Ca and P indicates that a significant amount of these minerals are bound to phytate, and this increases the proportion of unavailable minerals for pig utilization in diets with higher levels of phytate.
Inclusion of PhyG to diets increased bone ash, but to a greater extent if there was 0.35 or 0.29% rather than 0.23% phytate-bound P in the diets. This is likely do to greater concentration of apparent total tract digestible P that was liberated from phytate present in diets containing 0.35 or 0.29% phytate-bound P.
In this experiment, we also wanted to determine the efficacy of PhyG by comparing it with an established commercial Buttiauxella phytase. Blue bars represent PhyG, whereas orange bars represents PhyB. Here we can observe that at 0.35% phytate-bound P, diets containing PhyG had greater Ca and P digestibility compared with diets supplemented with the Buttiauxella phytase. 
Diets containing PhyG had also had greater P digestibility in diets with 0.35% phytate-bound P compared with diets supplemented with the Buttiauxella phytase. And overall, PhyG increased P digestibility more than the Buttiauxella phytase. This indicates that the novel consensus phytase was more effective in liberating the P bound to phytate. PhyG was produced via fermentation with a fungal production strain, and it was demonstrated from previous research that the novel phytase had high activity in a wider pH range than the Buttiauxella phytase. This indicates that PhyG could efficiently hydrolyze phytate and subsequently reduce the negative effect of phytate on nutrient digestibility.
At 0.35% phytate-bound P, pigs fed diets containing PhyG tended to have greater bone ash compared with diets supplemented with the Buttiauxella phytase indicating that greater P was available for bone mineralization in pigs fed the novel phytase.
In summary, we have demonstrated that phytate reduced mineral digestibility indicating the significant amount of minerals are bound to phytate, which increases the proportion of unavailable minerals for pig utilization. We have also demonstrated in this experiment that novel phytase was able to efficiently increase the ATTD of minerals and also increased the bone ash of young growing pigs.
The novel phytase also increased P digestibility more than the Buttiauxella phytase, which can be attributed to its high activity over a wide pH range and subsequently reducing the negative effect of phytate on nutrient digestibility.
We would like to take this opportunity to acknowledge Dupont for their inputs and financial support, and also to the whole team of Stein Monogastric Nutrition Laboratory. Thank you for your interest in this presentation and please feel free to visit our website at nutrition.ansci.illinois.edu. Thank you.