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Effects of application variation and side activities on the efficacy of phytase in broiler diets

Published: May 30, 2007
By: JEANNINE HARTER-DENNIS, JENNIFER TIMMONS and JOHN DRIVER - University of Maryland/University of Pretoria (Courtesy of Alltech Inc.)

At last year’s Alltech Symposium (Harter-Dennis, 2000) we reported results of work in our laboratory conducted to determine the possible effects of a high coefficient of variation (CV) of phytase application in commercial male broilers from 0-21 days of age. It was reported that coefficients of variation in feed sampling as high as 103% did not adversely affect efficacy of Allzyme Phytase when used at recommended levels in a post-pellet spray application system.

While the young broiler is an excellent tool to use in nutritional research, it is necessary to confirm such results in the ‘target’ animal before making recommendations for field use. In the case of commercial broiler production, the target animal will most likely be the older broiler between 21 days of age and market weight. This is the rearing period when normal meattype broilers will consume approximately 70-80% of their total feed and therefore represents the time period when the majority of benefit of using phytase can be experienced.

While the potential effect of high mixing CVs in older broiler diets could be extrapolated from the results with young birds, the results need to be confirmed due to the difference in patterns of growth between the younger and older birds. It has also been demonstrated that age has an effect on endogenous intestinal phytase activity. Maenz and Classen (1998) reported that under assay conditions, the total brush border phytase activity of the small intestine was 5.2 mmol P/minute for laying hens and 3.8 mmol P/minute for broiler chicks.

While this age difference may actually benefit the older broiler compared to the young chick, it may reduce the older bird’s ability to benefit from exogenous phytase, which may also affect its tolerance to high dietary coefficients of variation.


A NEW URGENCY FOR WORK WITH PHYTASE

During the summer of 1997 the state of Maryland experienced outbreaks of Pfiesteria in several of its rivers and tributaries. These dinoflagellate microorganisms were implicated in several fish kills, which closed the rivers to fishing and tourism. Similar outbreaks were experienced in North Carolina and other east coast areas; and although not proven there was thought to be a link between these outbreaks and nutrient over-enrichment of the soil and subsequent runoff. Phosphorus runoff from animal manure has been identified as one of the possible causative agents.

In the fall of 1997 the governor established a Blue Ribbon Citizens Pfiesteria Action Commission to study the problem and make recommendations to the legislature. In the spring of 1998, the Water Quality Improvement Act of 1998 passed the Maryland Senate and House and was signed into law by the governor. This bill mandated that anyone using sludge or animal manure have nitrogen- and phosphorus-based nutrient management plans.

The nitrogen plans must be developed by 2001 and implemented by 2002, while the phosphorus plans must be done by 2004 and 2005, respectively. Persons using chemical fertilizers must also have nutrient management plans, but the deadlines are 2001 and 2002, respectively, for both nitrogen and phosphorus. One additional regulation included in the Water Quality Improvement Act of 1998 was that all contract feed manufactured for chickens must contain phytase or some other enzyme or additive that reduces phosphorus excretion. This must be implemented by the end of the year 2000.

The research in this paper addresses two areas. Trial 1 was designed to determine the effect of a high CV of phytase consumption in finishing broilers (21-42 days of age). In addition, Trial 1 was designed to determine the available phosphorus requirement of the type of commercial broiler typically grown in the Delmarva area and compare that to the value published by the National Research Council (1994). Trial 2 was designed to determine the effect of a solid-state fermentation-derived phytase source containing side activities (Allzyme Phytase, Alltech, Inc.) compared to one produced by submerged culture fermentation (Natuphos, BASF, Inc.)


Trial 1: Application of Allzyme Phytase to broiler finisher feeds: effects of high coefficient of variation

The coefficient of variation is a statistical parameter used as an indicator of variability. The CV should be determined by taking a series of samples from the same location at predetermined intervals and analyzing them for the substance of interest, in this case phytase. The CV is then calculated by dividing the standard deviation of all the samples taken by the sample mean and multiplying by 100 to express the value as a percentage (Agricultural Experimentation Design and Analysis, 1978).

The advantage of using the CV as an indicator of variation is that it allows the comparison of mixing variability regardless of the units of measure. This will allow for the comparison of mixing accuracy of phytase (or other materials) that have different units of measure (i.e., PTUs can be compared to FTUs or PUs).

The first trial employed phytase units expressed as PTU while the second trial expressed phytase in terms of PU.

Under normal feed mixing of dry ingredients, a CV of 10% is usually considered as to be the benchmark for adequate mixing performance (Wicker and Poole, 1991). Coefficients higher than this desired level may have a negative effect on performance. McCoy et al. (1994) reported that the growth rate and feed efficiency of young broilers was significantly depressed by 21.3% and 6.0%, respectively, when CV was 40.5% compared to 12.1% in a nutrient deficient diet.

This is an appropriate comparison to consider given that phytase will be added to diets otherwise marginal to deficient in available phosphorus. The post-pellet application of phytase may result in even higher CVs due to the nature of the spray application where a liquid is being sprayed on a dry feed that is frequently traveling past the applicator at a non-uniform rate.

Work in our laboratory as well as others (Johnson and Southern, 2000) suggests that CVs of supplemental phytase as high as 69-103% are well tolerated by broilers from 0-21 days of age. Young broilers showed no decrease in performance as measured by growth rate, feed efficiency, tibia ash content and bone breaking strength (BBS). The work presented here was designed to determine if older broilers (21-42 days of age) would also tolerate high CVs without reductions in performance.


EXPERIMENTAL PROCEDURES: TRIAL 1

Approximately three hundred one-day old commercial male broilers were purchased from a commercial hatchery on Delmarva. Birds were vaccinated in ovo for Marek’s disease at 18 days of incubation and spray vaccinated for Infectious Bronchitis and Newcastle diseases at one day of age. Birds were fed a standard commercial starter diet adequate in all nutrients from 0-20 days of age. On day 21, following an overnight fast, birds were wingbanded and 210 were allotted to treatments. Birds were allotted in a manner resulting in a similar average weight and weight range for all pens. Birds were housed in floor pens with wood shaving/sawdust litter. Temperature and ventilation requirements were similar to commercial conditions.

The experiment consisted of seven treatments (Table 1) with six replicate pens/treatment and five male broilers/pen. Treatments were blocked by location in the research facility and treatments were randomly assigned within block. Treatments 1–5 included graded levels of available phosphorus to establish the standard response curve while Treatments 6 and 7 were the test diets. The CVs of the phytase treatments were calculated based on daily phytase levels in the feed. The CV associated with Treatment 6 was calculated to be 0% while that associated with Treatment 7 was 103%.


Table 1.Dietary treatments: Trial 1.

Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 1



The experimental diets were based on corn and soybean meal supplemented with vitamins and minerals (Table 2). All diets were fed in mash form.

Dicalcium phosphate and calcium carbonate were added to the diets as sources of available phosphorus and calcium. The test diets were formulated to contain 0.90% Ca and graded levels of available phosphorus ranging from 0.15% to 0.30% (Table 2). All diets contained the NRC-recommended crude protein level of 20% and ME level of 3200 kcal/kg.

Treatments 1-5 contained 0.15%, 0.20%, 0.25%, 0.30% and 0.35% available phosphorus to form a standard response curve. Treatments 6 and 7, respectively, contained the 0.15% AP diet supplemented with either the recommended level of 11,500 PTU Allzyme Phytase fed daily or double the recommended level (23,000 PTU) fed on alternate days. Feed and water were offered ad libitum. Data were analyzed using the General Linear Model with treatment and replicate as sources of variation. Tukey’s HSD test was used to separate treatment means where appropriate.

Birds were fed their respective diets from 21-42 days of age. Any mortality was weighed and recorded daily. Following an overnight fast, the birds and feeders were weighed on day 42. At this time the birds were humanely sacrificed and the right tibias removed and frozen for further analysis of bone breaking strength and % bone ash determinations.

Bone breaking strength was determined using an Instron machine. All right tibias were thawed, then flesh and tibial caps removed. Once the bones reached room temperature they were individually placed on the rollers of the Instron for breaking strength determination.

A constant pressure was applied and the bone breaking strength was determined to be the pressure required to break the center of the bone. After the bone breaking strength was determined, the pieces of bone were recovered and pooled by pen for bone ash content determinations using AOAC procedures.


Table 2.Composition of experimental diets (%) used in Trial 1.

Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 2


Comparison of phytase sources: submerged culture vs solid state fermentation

Enzyme preparations from SSF are likely to contain detectable amounts of side activities including protease, cellulase and xylanase in addition to phytase while LF products do not. It is possible that these side activity enzymes may provide a nutritional benefit to the bird. Trial 2 was conducted to determine the effects of pelleting on the thermostability of two different types of phytase products, a solid state fermentation (SSF) product and a submerged culture liquid fermentation (LF) product.


EXPERIMENTAL PROCEDURES: TRIAL 2

One hundred and seventy-five one-day old commercial male broilers were purchased from a commercial hatchery on Delmarva. Birds were vaccinated in ovo for Marek’s disease at 18 days of incubation and spray vaccinated for Infectious Bronchitis and Newcastle diseases at one day of age. Birds were housed in stainless steel batteries in an environmentally controlled room. Temperature and ventilation requirements were similar to commercial conditions.

The trial was conducted as a randomized complete block design with five treatments (Table 3). There were five replicate pens/treatment and seven male broilers/pen. Treatments 1-3 formed a standard curve with three dietary AP levels: 0.25%, 0.30% and 0.35% AP, respectively. Treatments 4 and 5 included 300 PU Allzyme Phytase/kg and 300 PU Natuphos, respectively, to the 0.25% AP basal diet. (1 PU = 1 micromole of phosphorus released/ minute under assay conditions).


Table 3.Dietary treatments: Trial 2.

Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 3



A standard corn-soybean meal based starter diet containing 0.5% AP was fed from 0-3 days of age as a pre-test diet. Following an overnight fast, birds were weighed, wing-banded and allotted to pens in a manner that ensured a similar average weight and weight range per pen. Birds were fed their respective diets from 4-18 days of age. Any mortality was weighed and recorded daily.

On the 18th day birds and feeders were weighed, the birds were humanely sacrificed and the right tibias removed and frozen for further analysis of bone breaking strength and bone ash content determinations using methods similar to Trial 1.

The experimental diets were based on corn and soybean meal supplemented with vitamins and minerals (Table 4). Dicalcium phosphate and limestone were added to the diets as sources of available phosphorus and calcium.

The test diets were formulated to contain 1.00% Ca and graded levels of available phosphorus ranging from 0.25% to 0.35%. All diets contained the NRC-recommended crude protein level of 23% and ME level of 3200 kcal/kg. Feed and water were offered ad libitum. Data were analyzed using the General Linear Models procedures with treatment and replicate as sources of variation. Tukey’s HSD test was used to separate treatment means where appropriate.


Results

TRIAL 1: EFFECTS OF COEFFICIENT OF VARIATION, DETERMINATION OF AVAILABLE PHOSPHORUS REQUIREMENTS

As seen in Table 5, weight gain, bone breaking strength (BBS) and bone ash (% BA) of the birds responded to increasing levels of dietary available phosphorus. Both BBS and % BA responded in a linear fashion up to the level of the bird’s requirement. Figure 1 is a graph of the response curve using % BA as the response parameter. Using breakpoint analysis, the AP requirement was determined to be 0.288%, which is lower than the value of 0.35% established by the NRC (1994). This value suggests that the NRC recommendation for older broilers may need to be adjusted downward slightly to reflect the actual requirement of the modern broiler.


Table 4.
Composition of experimental diets (%): Trial 2.

Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 4
1Phytase was added to provide 300 PU/kg feed of either Allzyme Phytase or Natuphos.
21 PU = 1 micromole of phosphorus released/minute under assay conditions
.



Table 5.Performance of male broilers fed phosphorus deficient diets with phytase (days 21-42): Trial 1.

Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 5



Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 6

Figure 1.Determination of the available phosphorus requirement of the 21-42 day old male broiler.



Adding Allzyme Phytase to the diet containing 0.15% AP significantly improved both BBS and % BA. Using the standard curve generated for % BA, we are able to calculate the phosphorus sparing value of adding dietary phytase to a phosphorus deficient diet (Figure 2). Birds fed diet 6 (0.15% AP + phytase) had a % BA of 49.18% compared to 45.46% for birds fed Diet 1 (0.15% AP).

The only difference between these diets was the addition of phytase. By plotting the 49.18% BA on the Y-axis and finding the point where it intersects the standard curve, we are able to determine the % AP equivalency on the X-axis. When the recommended level of 11,500 PU/kg of Allzyme Phytase was added to a diet deficient in available phosphorus, it had a sparing effect of 0.071% AP (0.221%-0.150%). This is also in agreement with data from our laboratory which suggests a 0.060% AP sparing effect in 0-21 day old broilers.

Comparing the performance of birds fed phytase either daily or every other day, we found no significant differences in either BBS or %BA. The phosphorus sparing value of every-other-day phytase supplementation was 0.60%, which was not significantly different from that of birds fed phytase every day. This is also in agreement with the data from our laboratory which found similar results with 0-21 days old broilers.

The results of this trial confirm that older broilers from 21-42 days of age respond to Allzyme Phytase in a similar fashion to younger birds. Additionally, older birds are able to tolerate a high coefficient of variation (103%) in phytase consumption without any negative effect on performance.


Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 7

Figure 2.Determination of the available phosphorus sparing value of phytase when fed daily or every other day to 21-42 day old male broilers.



RESULTS: COMPARISON OF TWO PHYTASE SOURCES: TRIAL 2

Dietary phosphorus level had a significant effect on weight gain up to the level of 0.30% (Table 6, Figure 3). Increasing the level to 0.35% gave no further response (336, 364 and 365g, respectively). When phytase was added to the 0.25% AP diet, weight gain was significantly (P<0.06) improved, although there was no significant difference detected between Allzyme and the Natuphos phytase sources (362, 352 g, vs. 336, respectively). Feed efficiency was not significantly affected by treatment.


Table 6.Performance of broilers fed two sources of phytase with and without pelleting.

Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 8



Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 9
Figure 3.Effect of phytase source on weight gain: Trial 2.



Bone performance data is presented in Table 7. Tibia breaking strength, % tibia ash and % toe ash all responded in a linear fashion to increasing levels of dietary AP in the standard curve treatments (1-3). Both Allzyme and Natuphos were effective in increasing phosphorus availability when fed to birds receiving the 0.25% phosphorus deficient diet.

As seen in Figure 4, when using bone breaking strength as the response parameter, both phytase products has a phosphorus sparing value of approximately 0.05%. However, when using % tibia ash (Figure 5) or % toe ash (Figure 6) as the response parameters the phosphorus sparing value was closer to 0.075%. There was no significant difference between the two phytase products.


Table 7.Performance of broilers fed two sources of phytase with and without pelleting.

Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 10
1300 PU/kg Allzyme Phytase
2300 PU/kg Natuphos




Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 11

Figure 4. Effect of phytase source on bone breaking strength: Trial 2.



Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 12

Figure 5.Effect of phytase source on tibia bone ash: Trial 2.



Conclusions

The purpose of these experiments was to first; determine the effects on performance of finishing broilers (21-42 days of age) of a wide coefficient of variation of phytase applied using post pellet spray application systems and secondly, to compare a solid state fermentation phytase to one produced by liquid fermentation.

Our results suggest that older broilers from 21-42 days of age are similar to young broilers in their ability to tolerate high coefficients of variation of phytase application without any detriment to performance. Secondly, our results also suggest that the Allzyme Phytase product, produced by solid state fermentation, gave performance equal to that of liquid fermentation. And lastly, our results confirm earlier work showing that the Allzyme Phytase has a phosphorus sparing value in the range of 0.05-0.075%.


Effects of application variation and side activities on the efficacy of phytase in broiler diets - Image 13

Figure 6. Effect of phytase source on toe ash: Trial 2.



References

Agricultural Experimentation Design and Analysis. 1978. John Wiley & Sons, Inc. Publisher. p18.

Harter-Dennis, J.M. 2000. Phytase application variations in broiler diets and legislative update. In: Biotechnology in the Feed Industry. (T. P. Lyons and K. A. Jacques, eds). Nottingham University Press, Nottingham, UK, pp. 163-174.

Inborr, J. and M.R. Bedford. 1994. Stability of feed enzymes to steam pelleting during feed processing. Animal Feed Science and Technology 46:179–196.

Johnson, S. L. and L. L. Southern. 2000. The effect of varying mix uniformity(simulated) of phytase on growth performance, mineral retention, and bone mineralization in chicks. Poultry Sci. 79:1485-1490.

McCoy, R.A., K.C. Behnke, J.D. Hancock and R.R. Mcellhiney. 1994. Effect of mixing uniformity on broiler chick performance. Poultry Sci. 73:443-451.

Maenz, D.D. and H.L. Classen. 1998. Phytase activity in the small intestinal brush border membrane of the chicken. Poultry Sci. 77:557-563.

NRC. 1994. Nutrient Requirements of Poultry (9th Ed.). National Academy Press, Washington, DC.

Wicker, D.L. and D.R. Poole. 1991. How is your mixer performing? Feed Management 42:40-44.


Authors: JEANNINE HARTER-DENNIS1, JENNIFER TIMMONS1 and JOHN DRIVER
2
1
University of Maryland-Eastern Shore, Princess Anne, Maryland, USA,
2 University of Pretoria, South Africa

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