Hello everyone, my name is Vanessa Lagos and today I will be sharing some data from one of the experiments that will be part of my PhD dissertation looking at the effects of reducing de concentration of calcium and phosphorus and increasing microbial phytase on gastric pH, fecal score, growth performance, and bone ash of weanling pigs. 
Weaning is a very stressful period for pigs because of environmental, nutritional, physiological, and immunological changes. This, along with the restriction on the use of antibiotic growth promoters or AGP results in morbidity, most specifically diarrhea, and mortality.

Therefore, alternatives to AGP have been studied and include: direct fed microbial, prebiotics, acidifiers and plant extracts. But today I will focus on the reason for the use of acidifiers. Weanling pigs are believed to be unable to secrete enough hydrochloric acid (HCl) in the stomach to provide an appropriate pH for proper digestion of proteins. However, the inability to reach a low gastric pH can also be attributed to the inclusion of limestone and monocalcium phosphate in phase 1 diets, because these ingredients have a high acid binding capacity. Therefore, it is possible that lowering limestone and monocalcium phosphate MCP in these diets will decrease stomach pH and will reduce diarrhea. 
On the other hand, the inclusion of microbial phytase results in reduced concentration of limestone and MCP in diets due to the increase in Calcium (Ca) and Phosphorus (P) digestibility. Phytase also reduces the anti-nutritional factors of phytate and in high doses, phytase also increases the concentration of inositol in blood, which is believed to have a positive effect on intestinal integrity and immune system. Therefore, phytase may contribute to the reduction of gastric pH and diarrhea, and maintaining the immune response of weanling pigs, this would result in improved growth performance without affecting bone ash.
With that in mind, I will move on to the objective of this experiment, which was testing the hypothesis that reducing Ca and P in diets for weanling pigs reduces stomach pH and therefore diarrhea. The second hypothesis is that inclusion of microbial phytase partly overcomes negative effects of low Ca and P by increasing plasma inositol, which prevents reduction in growth performance and bone ash.
Moving on to the materials and methods, 320 pigs with an average BW of 6.4 kg were allotted to 2 blocks and 8 experimental diets. There were 5 pigs per pen and 8 replicate pens per diet. The experimental diets included 2 levels of Ca and P. 100 or 50 % of the NRC requirement. Within each level of Ca and P, there was a control diet with no phytase and 3 diets with 500, 2,000, and 16,000 FTU of quantum blue, in these diets 0.16% total Ca and 0.11% digestible P was reduced to account for the release of Ca and P by phytase. Here I would like to point out that although inclusion of phytase at 16,000 FTU is not used in commercial conditions, we had hypothesized that using extremely high levels of phytase will result in additional benefits to weanling pigs. 
To better understand the timeline of the experiment, this study started on the day of weaning and was divided into 3 phases, pigs were weighed at the beginning and end of each phase to calculate growth performance parameters, and a blood sample was collected to measure plasma inositol. The experimental diets were only fed in phase 1, whereas during phase 2 and 3, a common diet without phytase was provided. During phase 1, every other day fecal score was visually assessed and by the end of this phase, 1 pig per pen was euthanized to measure gastric pH and to collect the right femur. At the conclusion of the experiment, again, 1 pig per pen was euthanized to collect the femur and measure bone characteristics. 
Moving on to the results, I would like to point out that we used contrast statements to analyze these data, and we will start with growth performance parameters. 
I would like first to set up the slide, blue colors in the left represents diets formulated to have 100% of the Ca and P requirement whereas orange colors to the right represent diets formulated to have 50% of the Ca and P requirement. The darkest color in each group represents the control diet, and inclusions of phytase from 500 to 16,000 FTU are represented by the increase in darkness in the bars. 
Here we observe a reduction in average daily gain (ADG) when diets are formulated with 50% of Ca and P compared with 100% which indicates that growth is compromised by limiting Ca and P.
Now, looking only at the diets with 100% Ca and P, the ADG of pigs tended to be greater with the diet containing 500 FTU compared to the control diet, and pigs had greater ADG with diets containing 2,000 or 16,000 FTU grouped as High Phytase Levels or “H” diets than with the control diet. This indicates that the inclusion of phytase in diets with 100% Ca and P is beneficial to pigs growth. 
For average daily feed intake (ADFI), a similar response was observed. Pigs fed diets with 50% of Ca and P had reduced feed intake compared with pigs fed diets with 100% of Ca and P. This was expected because data demonstrated that P deficiency results in depression in feed intake.
For the diets with 100% Ca and P, feed intake tended to increase in the diets with phytase.
Finally for gain to feed ratio (G:F) in phase1, regardless of the concentration of Ca and P in the diet, super-dosing of phytase resulted in improved G:F compared with the control diet. Again indicating a beneficial effect of including high levels of phytase in diets for weanling pigs. In addition to this, in the diets with 100% of Ca and P, there was a tendency for a linear increase in feed efficiency as phytase increased in the diets. This indicates that there could be an effect of more inositol being present in blood as more phytase is included in the diet. 
Now I will jump into the results for G:F ratio for the overall experimental period because we did not observe any effect of the diet on growth performance parameters during phases 2 and 3. Here we observe again that G:F tend to increase as phytase level increases. So the positive effect of phytase remains after 4 weeks, even under diets without phytase.
But we also observe in the diets with 50% Ca and P a reduction in G:F in the diet with 500 FTU compared with the control diet, which was not expected. 
We will now look at the results for inositol in plasma of pigs. For this variable, data from day 14, 27, and 42 were analyzed using day negative 1, the day before weaning, as a covariate.   
These are the results for day 14 and we can observe that regardless of the concentration of dietary Ca and P, inclusion of phytase results in increased concentration of inositol in plasma. For diets containing 50% of Ca and P, there was a tendency for a linear increase in plasma inositol, whereas in diets with 100% Ca and P, plasma inositol linearly increased as more phytase was included in the diets. These results support the hypothesis that in diets with 100% Ca and P, increasing levels of phytase increases the concentration of plasma inositol, and results in improved feed efficiency. 
Data from day 27 and 42 will not be showed because none of the contrasts evaluated were significant. 
I will however share the results obtained when the effect of day was taken into account. 
As we observe here, there was an interaction between diet and day. In this graph, the horizontal axis represents the day and the vertical axis represents the response variable and the diets are represented by the same colors as in the previous results. So, if we focus our attention on day negative 1, we can observe some variability, more than expected, but there is no specific pattern regarding the diets. Now if we look at day 14 we observe that the 2 diets without phytase represented by colors dark blue and brown result in reduced inositol compared with the diets with phytase. At day 27 and 42, we observe the same pattern in all diets, at day 27, the concentration of inositol in plasma is around 15 μM and is increased to around 45 μM at day 42. We can conclude from these data that in early phases, inclusion of phytase is important to maintain plasma inositol, but, after 6 weeks pigs are able to increase plasma inositol back to the concentration observed before weaning. 
Now we are going to move into the results for gastric pH. 
Surprisingly, we did not observe any differences when 50 and 100% Ca and P diets were compared, but we did observe a tendency for a reduction in stomach pH in diets with high phytase levels compared with the control diet. And this is regardless of the concentration of dietary Ca and P. These results then reject our hypothesis that reducing limestone and MCP in diets results in lower stomach pH. 
For fecal score we used a 1 to 5 scale, where 1 is normal feces and 5 watery diarrhea. And I will show data indicating the frequency of which values of 3 or above were observed. 
As we can appreciate, there was no effect of dietary treatment on diarrhea frequency. Therefore, the improvement in G:F cannot be attributed to a reduction in diarrhea. 
Lastly, we are going to look at the results for bone ash. 
As we expected at day 14 bone ash expressed as grams per femur, was grater in diets with 100% Ca and P, compared with 50% diets.
At the end of the experiment there was still a lower concentration of bone ash in diets with 50% Ca and P than in 100% diets, indicating that pigs will not recover if diets are limited in Ca and P.

In conclusion, lowering Ca and P reduces growth performance and bone ash but does not reduce gastric pH or fecal score. But, increasing levels of phytase linearly increase plasma inositol which results in improved G:F ratio.
With this I would like to acknowledge AB Vista for the financial support. 
The members of the Stein Lab for their support, and all of you for your attention. Thank you.