Phosphorus, being an essential component of organic compounds, is involved in a wide variety of metabolic functions including energy, carbohydrate, fat, and nervous tissue metabolism. Phosphorus plays a major role as a structural component of skeletal system and is an important part of nucleic acids.
In chickens, phosphorus is the second most abundant element next to calcium on per kg of fat free tissue basis. In a modern integrated poultry production system, dietary P supplementation is the most critical not just from the standpoint of production, but also from the point of its utilization and excretion from the body. Approximately 70% of the phosphorus in plant feedstuff is in the form of phytate P.
Commercially available phytase is presently being added to poultry feed to hydrolyze phytate phosphorus so that the exogenous supply of inorganic P can be minimized to make poultry production cost effective and environmental friendly. Feeding excess NPP has been shown to cause P run off and becomes an environmental concern. There have been several research reports demonstrating how phytase may reduce manure P and feed costs. A few examples of nutrition and management strategies include feeding less P than that recommended by NRC, phase feeding, feeding selected feed ingredients with readily available P, and adding phytase to the feed. Depending on the type of feed ingredients included in poultry diets, the amount of dietary phytase that is added to the feed in combination with dietary NPP is critical because the higher the dose of phytase, the higher the occurrence of phytate hydrolysis. Feeding excess NPP in combination with too much phytase may result in more soluble P in the litter.
Optimizing dietary phytase inclusion based on retainable P levels may help to reduce litter water soluble P. When broilers are provided phosphorus levels that are higher than the physiological threshold needed for maximum utilization and retention, the additional phosphorus will be eliminated most likely through the kidney. Gaining a better understanding of the route of elimination for excess P above the physiological threshold in broilers may give an insight on P utilization.
Feeding large particle calcium to laying birds may be another method of reducing P excretion. Previous research has shown that feeding larger particle size limestone to laying hens increases the retention of limestone particles in the gizzard. Commercial layers and broiler breeder hens fed large particle size Ca (limestone) will produce a slow release of Ca since larger limestone particles remain longer in the gizzard leading to more availability of Ca for intestinal absorption during the period of shell formation. The increased accessibility of a Ca source in the gut at the time of shell formation may reduce bone mobilization there by reducing the P excretion since bone mobilization not only includes Ca mobilization but also its counter ion P. Feeding a range (28 to 1306 microns) of limestone particles to broilers chicks has varied impact on chicks performance and bone ash. The research based on in vivo and in vitro studies indicates that a small calcium particle size (28 microns) limestone with a high solubility (>70.0%) limits phytase hydrolysis and thus decreases potential available P for growth and bone ash formation.
Introduction
In chickens, phosphorus is the second most abundant element next to calcium on per kg of fat free tissue basis (Scott et al., 1982). Phosphorus along with calcium is most critical in maintaining the structural integrity of bone. Understanding total and non-phytate phosphorus retention along with phytate P disappearance in poultry is of both economical and ecological importance. About two-thirds of phosphorus in plant feedstuff is in the form of phytate P. Phytic acid has the ability to form complexes with proteins, starches, and metal ions such as Zn2+, Fe2+, Mn2+, Fe3+, Ca2+, Mg2+, and Co2+ (Cosgrove, 1980; Thomson and Yoon, 1984; Morris, 1986). Poultry and other non-ruminants have either little or no endogenous phytase activity to hydrolyze phytate P (Pointillart et al., 1987; Cromwell et al., 1993). In the past, the poultry industry was limited to feeding diets containing NPP as available P because of poultry’s inability to utilize significant phytate P.
Feeding excess NPP to poultry has been shown to cause P run off in our water supply after applying the litter or poultry waste to the soil. Commercially available phytase is presently being added to poultry feed to hydrolyze phytate phosphorus so that the exogenous NPP can be minimized to make poultry production cost effective and environmental friendly. The environmental concern is evident based on Maryland’s recent state regulatory compulsion to use phytase or other feed additives that decrease excreted P in poultry farming (Angel et al., 2002). The amount of phytate P that is present and the extent of bioavailability of P from phytate hydrolysis with dietary phytase supplementation may vary depending on the type of ingredients (Leske and Coon, 1999; Ravindran et al., 1995). There has been a limited amount of research demonstrating the levels of feed phytase needed for optimum phytate P hydrolysis. Shirley and Edwards (2003) indicated 94.8% phytate P disappearance could be achieved using 12000 units of phytase (Natuphos 5000)/kg diet. Coon and Manangi (2004) indicated 99.5% phytate hydrolysis in broilers fed diets supplemented with 5000 units of phytase (Phyzyme XP) per kg diet.
Although a very high phytate P disappearance with the high levels of added phytase would seem to be very important, it has become evident that an increase in phytate P hydrolysis needs to be correlated with dietary NPP and phytate P to minimize soluble P in the litter. The type of feed ingredients utilized in broiler diets, amount of added NPP, and dietary phytase supplementation are each important in providing optimum retainable P for broilers. Optimizing dietary phytase inclusion based on retainable P may be helpful to reduce litter water soluble P.
When broilers receive phosphorus levels that are higher than the physiological threshold needed for maximum utilization and retention, there is a possibility that the additional phosphorus may be eliminated most likely through the kidney (Leske and Coon, 2002). It is of interest to know if increasing levels of dietary NPP results in increased levels of plasma inorganic P, and whether this excess P due to increased supplementation is likely to be eliminated in the urine. It is also important to determine the physiological P threshold in broilers and to provide integrators this information. If the physiological P threshold in broilers was better understood then integrators could make economic decisions about feeding different P levels for performance enhancement for their specific product and know approximately the loss of dietary P into the litter that will occur.
Feeding layers and broiler breeders the proper amount of Ca and P is a major concern for maintaining optimum eggshell quality for hatching egg production and for commercial production for human consumption. Calcium and phosphorus are also extremely important for maintaining skeletal bone strength and bone ash in these laying birds. Breeders are control fed a restricted amount of feed throughout the rearing and production period and are generally fed one time daily early in the morning. Commercial layers are fed ad libitum throughout the day. Nutritionists recommend feeding more Ca at the beginning of lay to meet the requirement of egg shell calcification while dietary phosphorus is still maintained almost at the same level as used during the rearing period. Common (1932, 1933, and 1936) reported a rise in excreta P corresponding to the period of shell formation, and attributed the excess phosphorus to phosphate set free when the calcium was removed from the bone for shell formation. Research has already shown that feeding commercial layers large particle size calcium improves both the eggshell quality and the bone ash during lay. Breeders are generally not fed large limestone particles for several reasons. It is thought that breeder hens do not produce as many eggs as commercial layers and thus the large particle limestone would not be as critical during a production period. Breeder feed may be pelleted by some integrators, which would require smaller calcium particles. Broiler integrators may also use one feed mill for producing both broiler and broiler breeder feed, which may limit using extra bin space for the different calcium feedstuffs. The feeding of large Ca particles may result in slow release of Ca since larger limestone particles remain longer in the gizzard, leading to availability of Ca for intestinal absorption during the period of shell formation. Due to the continuous availability of Ca for gut absorption by feeding larger particle size limestone (calcium source), there may be a reduction in the bone mobilization of Ca along with its counter ion P, especially during shell calcification, thereby reducing P excretion.
Coon and Leske (1999) reported that commercial layers fed increasing levels of NPP along with large particle size limestone compared to small particle limestone excrete less total phosphorus per g P consumed. In order to increase the availability of Ca for shell calcification from the diet rather than from the bone mobilization, hens should have adequate metabolic calcium during the time of egg shell calcification. Laying birds can be fed at the exact time of eggshell calcification (generally during the dark period) with a midnight snack or by feeding a calcium source of optimum size and solubility that will allow calcium to be retained in the gastrointestinal tract throughout the day and night, thus providing calcium for eggshell calcification. Feeding larger particle size limestone has been shown to increase the retention of limestone particles for a longer time period in the gizzard (Zhang and Coon, 1997) and increased the overall calcium retention for layers by improving calcium deposition in eggs.
The amount of calcium in a broiler diet has been reported to affect the use of phytase (Tamim and Angel, 2003). The solubility of phytic acid in feed ingredients has been reported to be affected by pH (Selle et al., 2000; Champagne, 1988). Selle et al. (2000) have suggested that most phytate mineral complexes are soluble at low pH’s (less than 3.5) with maximum insolubility occurring between 4 and 7. Champagne (1988) has reported that calcium phytate complexes precipitate at pH’s between 4 and 6 which is the approximate pH of the intestine where the calcium ions should be absorbed. Taylor (1965) has suggested that the primary factor affecting phytate phosphorus utilization is the Ca ion concentration in the small intestine where insoluble Ca-phytate complexes form. A precipitated phytate mineral complex would not be accessible for hydrolysis or absorption in the intestine. The impact of feeding different particle sizes of calcium carbonate on weight gain and feed conversions of broilers has not been studied extensively. Guinotte et al. (1991) reported that broilers did not show any benefits in weight gain, feed conversions, and tibia bone ash when fed coarse particle calcium carbonate when comparing several sources. Guinotte et al. (1991) suggested that the optimum size was finely ground limestone with a particle size less than 150mm. McNaughton (1981) showed that a medium size particle (USBS 20-60) produced the best weight gain and feed conversions and needed less available phosphorus for optimum bone ash compared to smaller particle size calcium carbonate (USBS 12 to 20) and larger particle size calcium carbonate (USBS 100 to 200). The solubility of calcium in the gastrointestinal tract may have a direct affect on forming phytate mineral complexes. Research has neglected to evaluate the effects of calcium particle size and solubility on phytate hydrolysis with an exogenous phytase in broilers. Broilers may gain more from feeding phytase by feeding larger particle calcium carbonate with lower solubility to minimize the solubility of the calcium carbonate in the crop and in anterior portion of the gastrointestinal tract. A low solubility form of calcium carbonate may allow the phytase enzyme more accessibility to the phytate phosphorus in the gut and provide a higher phytate phosphorus disappearance in the broiler.
Research to evaluate how phosphorus is being excreted for laying birds or broilers fed with different Ca and P nutritional feeding programs has been limited because of the technical difficulty in separating urine from the faeces. Colostomy studies were conducted to separate urine from the faeces at the University of Arkansas by surgically severing the colon (colostomy) just anterior to the rectum and exteriorizing it.
The objectives of the present studies were:
1) to evaluate the effect of graded levels of microbial phytase (Phyzyme™ XP 5000G) with low and high dietary phytate P levels in comparison to their respective positive controls on litter water-soluble P;
2) to determine the relationship of dietary Ca and NPP for P retention, plasma inorganic P, urinary P and digestive tract P excretion using colostomized and non-colostomized broilers fed diets supplemented with different levels of NPP and Ca;
3) to evaluate the effects of two different Ca particle sizes on Ca and P retention and percent tibia ash in broiler breeder hens; and
4) to evaluate the impact of feeding different particle sizes of calcium carbonate on broiler performance and tibia ash, and to determine the effect of different particle sizes of calcium carbonate on phytate P hydrolysis in vitro at pHs of 2.5 and 6.5.
Effect of an added microbial phytase on broiler litter soluble phosphorus
An experiment was conducted to evaluate the effect of graded levels of microbial phytase (Phyzyme™ XP 5000G) with low and high dietary phytate P levels in comparison to their respective positive controls on litter water-soluble P.
One thousand five hundred and thirty six male day-old broiler chicks were assigned to 48 pens. Forty-eight pens were randomly allotted to 12 diets (4 pens/dietary treatment with 32 chicks per pen). Birds were reared under standard rearing practices on the floor. The chicks were fed experimental starter diets for 21 days.
The dietary treatments were:
1) Low-phytate basal;
2) Low-phytate basal plus phytase at 250 U/kg diet;
3) Low-phytate basal plus phytase at 500 U/kg diet;
4) Low-phytate basal plus phytase at 750 U/kg diet;
5) Low-phytate basal plus phytase at 1000 U/kg diet;
6) Low-phytate positive control;
7) High-phytate basal diet;
8) High-phytate basal plus phytase at 250 U/kg diet;
9) High-phytate basal plus phytase at 500 U/kg diet;
10) High-phytate basal plus phytase at 750 U/kg diet;
11) High-phytate basal plus phytase at 1000 U/kg diet;
12) High-phytate positive control diet.
The broilers were switched to experimental grower diets after three weeks and maintained on the grower diets until six weeks of age. The treatment groups fed either low or high phytate basal diets and the Units of phytase were the same in grower diets as during the starter period. The collection of litter and analysis of water-soluble litter P was based on the procedure of Self-Davis and Moore (2000). Total P was analyzed using ICP.
The data, except Positive Controls, were subjected to ANOVA using GLM procedure (SAS 1999). Data in each group (low-phytate and high-phytate) was compared to respective Positive Control group using Dunnet’s method. The means of two positive controls were compared using two-group t-test.
The increase in retainable P consumed/kg feed consumed increased the soluble P in the litter (Figures 1 and 2 and Table 1). In low phytate diets, the soluble P in the litter surpassed the positive control with 750 and 1000 units of phytase/kg diet, while in the high phytate fed groups, the litter soluble P/kg litter surpassed the high phytate positive control with 250 units of phytase/kg diet. There was no significant (P > 0.05) main effect of phytate P, and no significant interaction of phytate P and phytase on both total and soluble litter P. However, phytase had a significant effect (P < 0.05) on both total and soluble litter P. In the present study, the soluble litter P per kg litter for positive controls is well below the regression line (Figures 1 and 2) in both low and high phytate P fed groups based on the amount of retainable P consumed per kg feed consumed. The increase in phytase beyond 500 units/kg diet in both the low and high phytate P diets resulted in more litter soluble P, and this increase could be attributed to increased phytate P hydrolysis (Figures 1 and 2). The increase in dietary phytase supplementation beyond 500 units/kg diet did not produce an additional improvement in % tibia ash and broiler performance (Coon and Manangi, 2004) compared to the respective positive controls for both low and high phytate P groups. The increased available phosphorus due to increased phytase addition was not retained, but was excreted in the faeces.
Effect of different levels of dietary P and Ca on P excretion and relationship between plasma inorganic P and urinary excretion of P in broilers
Two feeding experiments were conducted to evaluate the relationship between plasma inorganic P levels in relation to P retention using normal birds and urinary P excretion using colostomized birds when the birds were supplemented with different levels of NPP and Ca.
For Experiment 1, Cobb 500 male broilers, 40 days of age, of a uniform body weight were placed in individual metabolic cages and offered corn soybean test diets (Table 2) consisting of 8 levels of NPP combined with a fixed level of calcium (0.5%). Ground limestone and calcium from reagent grade calcium phosphate, dibasic, monohydrate were used as additional calcium sources to the basal diet. Reagent grade calcium phosphate, dibasic, monohydrate (Product C129-12, Fisher Scientific, Fairlawn, NJ) was added to basal diet as additional non-phytate phosphorus source. Ten broiler chicks were fed each of the 8 test diets for the 80 experimental units. Two-percent acid insoluble ash (Celite) was added to the feed and used as a marker. Rearing of chicks and collection and processing of excreta samples were similar to the procedure as described by Coon and Manangi (2004). On day 5 of the experiment, blood samples were collected from 5 birds for each treatment group. Cobb-500 broiler chicks (3-4 wk old) of uniform body weights were colostomized and fed the eight experimental diets in order to collect excreta and urine samples separately.
In Experiment 2, Cobb 500 male broilers, 50 days of age, were fed eight test diets containing 0.9% Ca with 8 levels of reagent grade calcium phosphate, dibasic, monohydrate balanced with limestone. Experiment 2 was identical to Experiment 1 with the exception of dietary calcium levels.
In both experiments, diets and excreta were analyzed for total phosphorus and calcium by inductively coupled plasma emission spectroscopic method as described by Leske and Coon (2002). Plasma inorganic phosphorus (iP) was measured using an inorganic phosphorus reagent kit (Bayer Healthcare LLC, Tarrytown, NY). Acid insoluble ash was determined in experimental diets and excreta samples, using the dry ash and hydrochloric acid digestion technique of Scott and Balnave (1991).
Diet and excreta phytate phosphorus was measured as inositol hexa-phosphate by using ion-exchange chromatography as described by Bos et al. (1991). Moisture in both feed and excreta were determined by using standard AOAC procedures (AOAC, 1990). Retention values of total phosphorus, phytate phosphorus, and NPP were determined using the diet and excreta phosphorus, and acid insoluble ash concentrations with a marker digestibility equation reported by Scott and Balnave (1991). The collected urine samples from the colostomized birds were centrifuged at 2500 rpm for 15 minutes. The precipitate was freeze dried. Both the supernatant and the precipitate were analyzed for Ca and P quantification using ICP.
Data in both the experiments were subjected to analysis of variance (SAS Institute, 1999) to determine statistical significance, and differences among means were separated using the LSD procedure. The segmented/two-line models to estimate the break points and the slope(s) were developed using mathematical equations (Leske and Coon, 2002).
The two-line model adopted herein was:
Y = ( α- β1γ + β1X) (X ≤ γ) + (α-β2γ + β2X) (X >γ),
where α = Y value and γ = X value at intersection of two lines and β1 is slope of 1st line and β2 is slope of 2nd line.
Experiment 1: 0.50% Ca Diets
The excreta total P (mg) per g dry matter based on segmented/broken line analysis (Figure 3) indicates that the excretion of total P remained constant up to 0.21% (break point) dietary NPP (0.38% total P). Dietary increases in NPP above 0.21% caused an increase in excreta total P. The increasing levels of NPP in the diets above the physiological threshold (0.21%) were excreted at a different rate than before the P threshold.
The effects of increasing levels of dietary NPP on plasma inorganic P (iP) are presented in Figure 4. The broken line model (Figure 4) indicates that the concentration of plasma iP increased as dietary NPP levels were increased up to 0.26% (break point). The iP become constant with dietary NPP levels above 0.26%. The plasma iP increased from 4.81 mg/dL for broilers fed 0.08% dietary NPP and 0.25% total P and reached a plateau level of 8.13mg/dL with 0.26% dietary NPP (Figure 4).
The effects of NPP levels on total urinary excretion of P per day in colostomized birds are presented in Figure 5. The total urinary excretion of P per day in broilers remained low from 0.08% dietary NPP to 0.28% dietary NPP but additional levels of dietary NPP produced an increased excretion of total urinary P (Figure 5).
The effects of varied levels of dietary NPP on phytate P disappearance, and NPP and total P retentions are presented in Figures 6-8. The retention of total P increased to 53.87% as the levels of dietary total P increased up to 0.46% [(0.29% NPP) (point of intersection of two lines- Figure 8)], and as the levels of total P increased further beyond 0.46%, the retention of total P started declining. Similarly, for NPP retention, increased levels of dietary NPP increased the NPP retention to 78.36% with dietary NPP supplementation up to 0.29% (point of intersection of two lines- Figure 7), and further increases in NPP resulted in a decrease in NPP retention. As opposed to total P or NPP retention, the phytate P disappearance showed a decline from 35.1% with the lowest NPP (0.08%) to 14.93% with the supplementation up to 0.31% dietary NPP (break point- Figure 6), and remained at a plateau with further increases in NPP supplementation.
The percent dietary retainable phosphorus values are presented in Table 3. In normal birds, the increase in dietary NPP levels increased the retainable phosphorus values up to 0.33% dietary NPP, and further increase in dietary NPP did not produce further improvement in retainable P.
Experiment 2: 0.90% Ca Diets
The excreta total P (mg) per g dry matter based on segmented/broken line analysis (Figure 9) indicates that the excretion of total P remained constant up to 0.23% (break point) dietary NPP (0.40% total P). Dietary increases in NPP above 0.23% caused an increase in excreta total P. The increasing levels of NPP in the diets above the physiological threshold (0.23%) were excreted at a different rate than before the P threshold.
The effect of increasing levels of dietary NPP on plasma inorganic P (iP) are presented in Figure 10. The broken line model (Figure 10) indicates that the concentration of plasma iP increased as dietary NPP levels were increased up to 0.27% (break point). The iP become constant with dietary NPP levels above 0.27%. The plasma iP increased from 4.56 mg/dL for broilers fed 0.08% dietary NPP and 0.25% total P and reached a plateau level of 7.51 mg/dL with 0.27% dietary NPP (Figure 10).
The effects of NPP levels on total urinary excretion of P per day in colostomized broilers are presented in Figure 11. The total urinary excretion of P per day in broilers was a low baseline level from 0.08% dietary NPP to 0.21% dietary NPP but additional levels of dietary NPP produced an increased excretion of total urinary P (Figure 11).
The effects of varied levels of dietary NPP on phytate P disappearance and the retention of total P and NPP are presented in Figures 12-14. The retention of total P increased to 51.82% as the levels of dietary total P (NPP) increased up to 0.42% [(0.25% NPP) (point of intersection of two lines - Figure 12)]. Dietary increases in total P above 0.46% caused a decline in the retention of total P. The NPP retention reached a peak of 82.2% with 0.25% dietary NPP (point of intersection of two lines - Figure 13). Dietary NPP levels above 0.25% resulted in decreased NPP retention. Maximum phytate P disappearance or utilization occurred at the lowest dietary NPP level (Figure 14). When dietary NPP levels were increased the phytate P disappearance or utilization decreased until the dietary NPP reached 0.29%. The phytate P disappearance did not change with NPP levels above 0.29%.
The percent dietary retainable phosphorus values are presented in the Table 3. The retainable P values increased from 0.09 to 0.23% as the dietary NPP levels increased from 0.08 to 0.28% (0.25 to 0.45% total P) in broilers.
Results from both experiments indicate that there exists a physiological steady state for P in broilers based on the plasma inorganic P profile and urinary excretion of P.
Effect of Ca particle size on P excretion in layers and broiler breeder hens
Effect of Ca particle size on P excretion in layers
A sixteen-week experiment with Hyline-36 layers was conducted to determine the effect of feeding small and large calcium particles from limestone on the excretion of P at different dietary non¬phytate P levels with a fixed phytate P level. Coon and Leske (1999) fed the commercial layers two different basal diets consisting of corn-soy with a total of 0.128% non-phytate P, 0.22% phytate P, 3.5% Ca, 18% protein and 2900 kcal ME/kg. The two basal diets contained the same nutrients and ingredients with the exception of using different particle size limestone. A small particle limestone with 0.5-0.8 mm sieve size and a large particle limestone with 3.3-4.7mm sieve size were used for the two basal diets. The two basal diets were fed alone and then each basal was supplemented with three levels of NPP from monocalcium phosphate providing 0.228%, 0.328%, and 0.428% non-phytate P, respectively.
The layers fed large calcium particle size showed a reduction of 2.4, 1.7, and 4.1 mg P/g DM excreta compared to small calcium particle size fed groups in treatments 1, 2, and 3, respectively (Figure 15). In treatment 4, the P excretion increased by 1.2 mg/g excreta for hens fed with large calcium particles compared to hens fed the same NPP levels along with being fed small calcium particles. The reason the hens fed the large calcium particles excreted slightly more P with the high levels of dietary NPP may be attributed to less efficient use of P when feeding high levels of P.
Effect of Ca particle size on P excretion in broiler breeder hens
A six-week experiment was conducted to evaluate the effects of two different Ca particle sizes on Ca and P retention and percent tibia ash in broiler breeder hens. Three hundred broiler breeder pullets (Cobb 700), 23 wk of age, were offered a corn-soybean meal breeder diet (based on NRC, 1994 recommendation) in individual cages until 30 wk of age. During the 31st week, 150 hens were divided into two groups of 75 each and offered the test diets. The calcium added to the basal diets consisted of two different particle sizes of limestone. The smaller particle size limestone passed a 300 microns mesh screen, whereas the larger particle size limestone passed a 4.57 mm mesh screen. The nutrient retention study consisted of feeding ten broiler breeders the experimental diets with added acid insoluble ash (Celite). Two-percent Celite was added to the feed and used as a marker. The Celite mixed feed was fed to the hens that were acclimated to the diets for four days prior to one-day excreta collection period (42nd day). The excreta from individual birds were collected on trays, frozen (-20 °C) and freeze dried for further analysis. Eggs were collected for 3 days before day 42. Egg weights were recorded after each collection and were kept at room temperature for 3-4 h before the determination of specific gravity. Twenty-five birds on day 42 from each treatment group (normal birds) were euthanized by CO2 inhalation for tibiae collection. Tibiae (right and left) were taken from each bird and cleaned of all exterior tissue and frozen until analysis.
Diets and excreta samples were analyzed for total phosphorus and calcium by inductively coupled plasma emission spectroscopic method as mentioned by Leske and Coon (2002). Acid insoluble ash was determined in experimental diets and excreta samples, using dry ash and hydrochloric acid digestion technique of Scott and Balnave (1991). Moisture in both the feed and excreta were determined by standard AOAC procedures (AOAC, 1990). Diet and excreta phytate phosphorus were measured as inositol hexa-phosphate by using ion-exchange chromatography as described by Bos et al. (1991). Phytate phosphorus disappearance and retention values of total phosphorus and calcium were determined using the method of Scott and Balnave (1991). Tibiae were cut lengthwise and defatted in refluxing petroleum ether in Soxhlet apparatus for 48 h. The defatted tibiae were oven dried and ashed in ceramic crucibles for 16 h 600o C. Ash content was expressed as percent tibia ash on defatted dried basis. The specific gravity of each egg was determined by floatation using graded levels of salt with a specific gravity range from 1.06 to 1.10 at increments of 0.05 (Moreng and Avens, 1985).
Data from this experiment were subjected to “two group t- test” to determine the statistical significance (SAS Institute, 1999).
Although the P excretion difference was not significant (P=0.15) during the six-week breeder study, a numerical improvement of 2.09 % P retention and 3.69% Ca retention was obtained for breeders fed larger size limestone particles compared to breeders fed smaller particle size limestone (Table 4). There was a significant (P < 0.0001) improvement of 3.22% in tibia ash content in the group fed larger particle size limestone compared to hens fed smaller particle size limestone for a 6 wk (Table 4) period.
Average hen day egg production for a period of 6 wk showed no difference (P > 0.05) between the two groups of broiler breeder hens fed two different particle sizes of limestone. Feeding a larger particle size limestone for broiler breeder hens significantly (P < 0.05) increased the specific gravity of eggs as compared to smaller particle size limestone fed group.
In conclusion, feeding large particle size Ca compared to small particle size results in a reduction (P=0.1585) of 1.83 mg P/g DM excreta and an improvement (P<0.0001) of 3.22% in tibiae ash in broiler breeder hens based on a six week feeding study.
Effect of Ca particle size on broilers; in vitro and in vivo studies
Impact of feeding different particle sizes of calcium carbonate for broilers
A twenty-eight day floor pen experiment was carried out using 1680 Cobb 500 male day old chicks to evaluate the impact of feeding different particle sizes of calcium carbonate on broiler performance and tibia ash. The experiment consisted of 8 treatments with 6 replications per treatment to test 8 different CaCO3 particle sizes (Table 5). The eight treatments had 8 different CaCO3 particle sizes. The corn soybean basal contained 21.5% CP, 3025 kcal ME/kg, 0.78% Ca, 0.20% NPP with 500 FTU/kg of Danisco Phyzyme XP added. The average particle sizes of CaCO3 (along with % solubility) tested were 28 (74.4), 137 (56.4), 299 (47.0), 388 (53.0), 519 (46.7), 760 (45.3), 796 (42.2), and 1306 (43.4) microns.
Significantly (P < 0.05) increased weight gains were (Table 6) obtained in chicks fed CaCO3 particle sizes between 137 and 388 microns compared to the gains obtained by feeding either the smallest (28) or largest particle (1306) sizes. An increased mg of ash per tibia was also obtained for the chicks fed CaCO3 particle sizes ranging from 137-388 as compared to the smallest (28) or largest particle (1306) sizes.
Effect of Ca particle size and pH on phytate P hydrolysis
An in vitro phytate P hydrolysis by a 3-phytase at pH 2.5 and 6.5 using 8 different particles (Table 5) sizes of CaCO3 at 9g/kg diet was carried out to evaluate the effect of Ca particle size on phytate P hydrolysis at 15, 30, 60, and 120 min incubation at 37 C. The results indicate a significant (P < 0.05) interaction of Ca particle size and pH on phytate P hydrolysis with greater effect at pH 6.5 (Table 7). The main effect of particle size showed that the smallest particle size (28 microns) with more solubility (74.4%) had the lowest phytate P hydrolysis indicating the interference on the action of phytase on phytate hydrolysis due to Ca-phytate complex formation (Table 7).
In summary, both in vivo and in vitro studies indicate that a small calcium particle size (28 microns) limestone with a high solubility (>70.0%) limits phytase hydrolysis to provide available P for growth and bone ash formation.
Acknowledgments
The authors are indebted to ILC Resources, Danisco Animal Nutrition, and Mosaic Co. for providing financial support, test ingredients, and phytase for the broiler and broiler breeder feeding studies. The authors are also indebted to Cobb-Vantress for providing the broilers and broiler breeders utilized in the research.
References are available on request
From Proceedings of the “Midwest Poultry Federation Convention”, St. Paul, Minnesota, U.S.A.
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Craig N. COON
Megharaja K. MANANGI
Department of Poultry Science University of Arkansas Fayetteville, AR
U.S.A.
