Phytate phosphorus utilization and intestinal phytase activity in laying hens

Abstract

The first aim of this study was to examine the effects of strain and age on phytate phosphorus (PP) utilization and intestinal phytase activity (IPA) in two strains of laying hens, Hy-line Brown (HB) and Hy-line White W-36 (HW) at 32, 52 and 72 weeks of age. A digestion trial was conducted using the indicator method and birds were sampled to measure IPA. The second aim was to examine the effect of feed grade exogenous phytase enzyme on total (TP), water soluble (WSP) and phytate phosphorus (PP) excretion in the HW strain fed varying levels of phosphorus. Hens were fed three concentrations of available P (AP): 0.2, 0.3 and 0.4%. Each level of P was supplemented with three levels of commercial feed grade exogenous phytase enzyme (0, 300, and 600 FTU/kg) and the amount of TP, WSP and PP in excreta per 100 g of feed consumed was calculated. The HB retained more PP as compared to HW. Intestinal phytase activity showed a significant (P<0.01) age effect with the highest activity occurring at 32 weeks. There were significant differences in the amount of TP and SP excreted between birds receiving the 3 levels of phosphorus with 300 units phytase (P<0.01). The results of this study showed that layers are capable of utilizing PP, and that utilization is regulated by IPA and varies with age. Exogenous phytase improved PP utilization but it increased the amount of TP and WSP in excreta.

Key Words:

• Strain
• Phytate phosphorus
• Intestinal phytase activity
• Laying hens
• Phosphorus excretion.

Introduction

Most ingredients used in poultry feeds are of plant origin. Phytate P in plants occurs as a mixed calcium-magnesium-potassium salt of phytic acid (Anderson, 1912). The chickens’ ability to utilize PP is a subject of debate. Phytate P utilization from corn-soybean type diets is influenced by dietary factors (Ballam et al., 1984; Mohammed et al., 1991; Marounek et al., 2008). Phytate P utilization in laying hens is increased when dietary Ca is lowered (Scheideler and Sell, 1987; Applegate et al., 2000).

The effect of age on the ability of chickens to utilize PP has not been clearly defined. Maddaiah et al. (1964) showed that mature hens utilized phytate more efficiently than chicks as measured by phytate retention. In laying hens fed corn-soybean diet, phytate P retention was 24% in 20-week and 53% in 47-week old hens (Marounek et al., 2008).

A possible explanation for the increased utilization of PP with age is the suggestion that more endogenous phytase is present in the gastrointestinal tract of older birds (Ravindran et al., 1995; Marounek et al., 2010).

Despite the contradictory observations and uncertainties concerning PP utilization with age, when birds are fed corn-soybean meal diets, the recommended levels of Ca and P were reduced when compared to earlier recommendations (NRC, 1994). An intake of 300 mg AP per hen/day is recommended to reduce P excretion by 20% (Summer, 1995). However, the results of a recent study with wheat-based diets without feed grade phytase indicated that dietary P concentration of 0.16-0.39% non-phytate P for 12 weeks had little effect on egg and shell production (Boorman and Gunaratne, 2001). Continued feeding of the 0.16% non-phytate P diet for 61 weeks did not influence performance, but plasma phosphorus and bone measurements indicated marginal P depletion (Boorman and Gunaratne, 2001). These results clearly showed that earlier, as well as the current (NRC, 1994) recommendations overestimate P requirements of layers. The equivocal reports of PP utilization by layers fed corn-soybean meal diets, the role of intestinal phytase, and the need to take into account the contribution of phytate, all provided the rationale to examine the effect of age on PP utilization.

Phytase supplementation of layer diets has generally been limited to 250-300 FTU/kg (Van der Kils et al., 1994; Gordan and Roland, 1997). Supplementation of 250 FTU of phytase/kg diet in laying hens was equivalent to 0.8 g of P from mono-calcium phosphate. Higher levels of phytase resulted in a lower equivalence per unit of phytase due to the significant quadratic response in P absorption (Van der Klis et al., 1994). Um and Paik (1999) reported that there were no significant differences between 0.2 to 0.5% NPP diets and supplementation of phytase gave no further improvement in performance.

Two experiments were conducted with layers to (a) examine the effects of age and strain on PP retention and IPA in laying hens, and (b) to evaluate the response of laying hens to graded levels of exogenous phytase supplementation.

Materials and methods

Experiment #1

A total of 80 30-week old commercial laying hens, 40 Hy-line Brown (HB) and 40 Hy-line White W-36 (HW), were used. Hens of each strain were selected from individually caged hens according to egg production and were caged individually in an open-sided poultry house. Birds received a corn-soybean meal basal diet formulated to contain 3.7% Ca and 0.58% TP from 30 to 72 weeks of age (Table 1).

Table 1 Dietary ingredients and chemical composition of the experimental layer diets.

Diet
	Exp. 1	Exp.2°	Exp. 2°	Exp. 2°
	diet 1	diet 1	diet 2	diet 3
Ingredients
Yellow corn, g/kg	650.9	637.1	634.5	625.4
Soybean meal, g/kg	200.0	206.5	206.0	208.0
Poultry fat, g/kg	38.2	30.0	30.0	33.7
Dicalcium phosphate, g/kg	-	6.0	12.5	19.0
Defluorinated phosphate, g/kg	15.0	-	-	-
Ground limestone, g/kg	82.5	109.4	106.0	102.9
Salt, g/kg	3.5	3.0	3.0	3.0
Vitamin premix#, g/kg	2.5	2.5	2.5	2.5
Trace mineral mix§, g/kg	0.5	0.5	0.5	0.5
DL-Methionine, g/kg	0.8	2.0	2.0	2.0
Lysine-HCL, g/kg	3.0	3.0	3.0	3.0
Calculated analysis
Metabolizable energy, kcal/kg	3000	2892	2882	2887
Total phosphorus, g/kg	5.8	4.3	5.3	6.3
Available phosphorus, g/kg	2.0	3.0	4.0
Calcium, g/kg	36.8	40.0	40.0	40.0
Determined analysis
Total phosphorus, g/kg	7.0	4.5	5.7	7.2

° Nine dietary combinations were fed to the hens in Experiment 2; diets consisted of 3 concentrations of AP (0.2, 0.3, and 0.4%) and each level of P was supplemented with three levels of commercial feed grade phytase enzyme (0, 300, and 600 FTU/kg).

# Vitamin premix provided: Mn, 88 mg; Cu, 6.6 mg; Fe, 8.5 mg; Zn, 88 mg; Se, 0.30 mg; vitamin A, 6600 U; cholecalciferol, 2805 U; vitamin E, 10 U; vitamin K, 2.0 mg per kg diet.

§ Mineral premix provided: riboflavin, 4.4 mg; pantothenic acid, 6.6 mg; niacin 24.2, mg; choline, 110 mg; vitamin B12, 8.8 mg; ethoxyquin, 1.1 mg per kg of diet.

To determine PP and TP retention, 0.3% chromic oxide was added to the basal diet of 12 birds per strain and fed for a 10-day adaptation period before collecting the excreta for three days at 32, 52 and 72 weeks of age. The same birds were used in all three collection periods. On Day 10 of each collection period, dropping trays were cleaned and lined with aluminum foil for excreta collection. Each day’s collection was dried at 80°C to inhibit phytate hydrolysis by microorganisms. Fecal samples from the same bird were pooled and ground to pass a 0.5 mm screen, and stored in sealed plastic containers until they could be assayed. The diet was sampled and treated in the same manner. Extreme care was taken in collecting the fecal samples to ensure they were not contaminated with feed or feathers. All values from chemical analysis performed on the diet and excreta samples were made on a dry matter basis. Sub-samples of dried excreta and diet were analyzed for percentage of TP, PP (using a method modified from that of Latta and Eskin, 1980) and chromium (using a method modified from that of Bolin et al., 1952). The data were used to calculate PP and TP retentions (Edwards and Gillis, 1959).

An additional 12 birds per strain per period were sampled for intestinal phytase activity (Iqbal et al., 1994; Biehl and Baker, 1997). The duodenal loop mucosa from three birds per strain was pooled. Four pooled samples per strain were used for the assay.

Experiment #2

A total of 108 84-week old Hy-Line white (W-36) hens were selected and housed individually according to previous egg production data. Diets were formulated to meet or exceed NRC (1994) nutrient requirement except for P (Table 1). The hens were fed nine dietary combinations; diets consisted of 3 concentrations of AP (0.2, 0.3 and 0.4%) and each level of AP was supplemented with 3 levels of commercial feed grade phytase enzyme (Ronozyme P CT, Lot # HB950031, 2500 FYT/g) at 0, 300 and 600 FYT/kg.

Hens were fed these diets for a 2-week acclimation period, then dropping trays were lined with aluminum foil for total excreta collection for two consecutive days. Excreta collected and feed samples were subjected to the same preparation as described previously in Experiment 1. Subsamples of the excreta and feed samples were analyzed for percentage of TP, PP and WSP.

Statistical analysis

Analysis of Experiment 1 was based on repeated measures design. The pen was used as the experimental unit. All calculations were performed using SAS (1996). A model was defined for the experiment that included the following sources of variation: hen strain; age; age × strain. An analysis of variance (ANOVA) was performed to test for significance for each model term. Analysis of Experiment 2 was based on a 3×3 factorial arrangement of AP and phytase treatments in a randomized complete block design. The pen was used as the experimental unit. A model was defined that included the main effects of AP, phytase level and an interaction of AP × phytase. Significant model term differences in the analysis of variance were further tested using pair wise comparisons. A probability of P<0.05 or less was considered significant, and when appropriate the means±SEM are presented.

Results

Values of PP retention measured at 32, 52 and 72 weeks of age for both strains are presented in Table 2. Phytate P utilization was influenced by strain (P<0.02) and age (P<0.001) but a strain × age interaction was not observed (Figure 1). Hy-Line Brown strain retained 15.4% more PP (P<0.01) compared to strain HW (38.3 vs 33.2%±1.07, respectively). At 32 weeks of age birds retained a significantly (P<0.01) higher amount of PP compared to 52 weeks of age and a numerically (P>0.05) higher amount of PP compared to 72 weeks of age (39.5, 31.3 and 36.5%±1.18, respectively). The Hy-Line Brown birds retained more PP than HW at 32 weeks of age (42.1 vs 36.9%±1.67, respectively) and it declined 0.079% and 0.074% per week of age in strains HB and HW, respectively.

Table 2 Effects of age and strain on phytate phosphorus and phosphorus retentions and phytase activity in laying hens.

Phytate phosphorus retention, %	Phosphorus retention, %	Phytase activity, nmol/mg/min
Age, weeks
32	39.5^a	39.9^a	36.3^a
52	31.3^b	31.5^b	25.9^b
72	36.5^a	44.5^a	30.0^b
SEM	1.18	2.21	1.43
Strain
HB	38.3^a	36.3	32.6
HW	33.2^b	41.1	29.1
SEM	1.06	1.63	1.17
Statistical probabilities
Age	0.001	0.001	0.001
Strain	0.002	ns	ns
Age × strain	ns	0.05	ns

a,b Means within columns followed by different superscripts are significantly different (P<0.05); ns, not significant.

Figure 1 Effect of age on phytate phosphorus retention.

Total phosphorus retention measured at 32, 52 and 72 weeks of age for the 2 strains are presented in Table 2. A significant strain × age interaction was detected for TP retention (P<0.05) indicating differences in the slopes of the two strains (Figure 2). At 32 and 52 weeks of age, HW retained higher TP compared to HB (43.7, 37.2 vs 36.3, 25.9±3.1, respectively). At 72 weeks of age, HW retained lower TP (42.3 vs 46.8±3.1, respectively) resulting in a significant strain × age interaction. Retention of TP increased by 0.263% per week for HB while it decreased by 0.036% for HW birds. At 32 weeks of age, birds retained significantly higher amounts (P<0.001) of TP compared to 52 weeks of age and numerically lower amounts (P>0.05) of TP compared to 72 weeks of age (40.0, 31.6 and 44.5%±2.2, respectively). Strain showed no significant effect on TP retention values (P>0.05).

Figure 2 Effect of age on total phosphorus retention.

No significant strain × age interaction was observed for IPA but it was influenced by age of the birds (P<0.001). Higher activity was observed at 32 weeks of age compared to 52 and 72 weeks of age (36.3, 25.9 and 30.0±1.4, respectively) (Table 2).

The addition of exogenous phytase to laying hen diets in Experiment 2 improved the degradation of PP. Phytase addition resulted in lower amounts of PP recovered in excreta (Table 3). The effect of the addition of the first 300 units of phytase was greater than that of the second 300 units. The first 300 units of phytase lowered PP in excreta per 100 g feed from 0.27 to 0.20 g, and the further addition of 300 units resulted in no further drop in PP (0.19 g). A comparison between the level of phosphorus in the diets indicates that the degradation of PP by phytase and measured by PP content in excreta is not dependent on the P content in the feed (P>0.05). Mean PP excreted were 0.23, 0.22 and 0.21 per 100 g feed intake for birds that received 0.2, 0.3 and 0.4% AP, respectively (Table 3). The phytase × phosphorus level term was not significant indicating that the slopes of the three lines do not differ from each other. A significant P × phytase interaction for the TP excreted per 100 g of feed intake indicated a significant difference in the slopes of the lines (Table 3). The addition of 300 or 600 units of phytase to the diet that contained 0.2% available phosphorus lowered the amount of TP in excreta (Figure 3). No further improvement in TP excretion was seen from the addition of 600 units of phytase. Birds that consumed the diet containing 0.4% AP without phytase excreted lower amounts of TP compared to the other two groups, resulting in a significant phosphorus × phytase interaction.

Table 3 Effects of available phosphorus and exogenous phytase on phytate, total and soluble phosphorus excretion in laying hens.

Available phosphorus, %	Exogenous phytase, FYT/kg	Excreta phytate phosphorus, g/100 g feed	Excreta total phosphorus, g/100 g feed	Excreta soluble phosphorus, mg/g excreta
0.2	0	0.28	0.35	30.4
0.3	300	0.30	0.26	34.1
0.4	600	0.26	0.41	37.1
0.2	0	0.21	0.26	26.9
0.3	300	0.19	0.31	36.7
0.4	600	0.20	0.43	56.9
0.2	0	0.20	0.26	26.9
0.3	300	0.19	0.32	51.4
0.4	600	0.18	0.43	79.5
SEM		0.009	0.02	6.6
Phosphorus
0.2		0.23^a	0.29^c	27.9^c
0.3		0.22^b	0.33^b	40.8^b
0.4		0.21^b	0.42^a	57.8^a
SEM		0.005	0.01	3.8
Phytase
0		0.27^a	0.37^a	33.9^b
300		0.20^b	0.33^b	40.2^b
600		0.19^b	0.34^ab	52.5^a
SEM		0.05	0.01	3.8
Statistical probabilities
Phosphorus		0.02	0.001	0.001
Phytase		0.001	0.05	0.005
Phosphorus × phytase		ns	ns	0.02

a-c Means within columns followed by different superscripts are significantly different (P<0.05);

ns, not significant.

Figure 3 Effect of available phosphorus level on total phosphorus excretion in laying hens.

There was no significant difference in TP excreted between birds that received 0.2, 0.3 or 0.4% AP without phytase enzyme (P>0.05), while there were significant differences in the amount of TP excreted between birds that received the 3 levels of AP with 300 units phytase (P<0.01). Birds that received the diet with 0.2% AP excreted the lowest amount and those that received 0.4% excreted the highest amount of TP per 100 g feed (Table 3). The difference between the 3 levels increased with the addition of 600 units of phytase. Birds that received 0.2 and 0.3% AP had negative slopes while those that received 0.4% had a positive slope (P<0.01) (Figure 4).

Figure 4 Effect of exogenous phytase level on total phosphorus excretion in laying hens.

Significant differences in the slopes of the 3-phytase levels indicated a significant phosphorus × phytase interaction on the WSP in excreta (Figure 5). At 0.2% AP, there were no significant differences in the amounts of WSP in excreta from birds that received 0, 300 and 600 units of phytase/kg diet (P>0.05). While at 0.3 and 0.4% AP levels the amount of WSP in excreta was higher for those receiving 300 or 600 units of phytase (P<0.01) than those that received no supplement. Increasing the amount of enzyme added had no influence on the amount of SP in excreta from birds receiving 0.2% AP. Increasing the enzyme from 0 to 600 units resulted in a significant increase in the WSP content of excreta (P<0.01).

Figure 5 Effect of available phosphorus level on soluble phosphorus excretion in laying hens.

Discussion

It has been reported that the ability of laying hens to utilize PP may decline with age, averaging 46.7, 9.1 and 16.5% at 34, 50 and 72 weeks of age, respectively (Scheideler and Sell, 1987). Other investigators reported that PP utilization increases with age in chicks and laying hens (Maddaiah et al., 1964; Edwards et al., 1989; Marounek et al., 2008). In this study, the highest PP retention occurred at 32 weeks of age. This agrees with the fact that there is higher demand for P during the early stages of the laying cycle when egg production, egg weight and body weight are increasing. The high PP retention at 32 weeks was also associated with high IPA and, as a result, TP retention was significantly higher compared to the other two periods.

Feeding diets high in phosphorus have a tendency to lower the utilization of PP. Phytate P retention increased when low levels of NPP were fed to 3-week old broiler chicks (Ballam et al., 1984). In general, the data reported in this experiment did not show a consistent effect of dietary P in the range of 0.2 to 0.4% AP on PP retention.

It was reported that the addition of 250 units of phytase/kg feed of 24-week old laying pullets resulted in an improvement in PP degradation and a further addition of phytase resulted in slight improvement (Van der Klis and Versteegh, 1991). The results of this experiment show that phytase is effective in improving the availability of PP in corn-soybean meal diets and the effect of the first 300 units of phytase was greater than that of the second 300 units. Phytase supplementation also increased the amount of TP and SP even at the low level (300 units), which could have negative effects on the environment. An addition of 300 units of phytase to the diet that contained 0.2% AP resulted in a reduction of TP excreted but no further improvement was seen after increasing the amount of phytase in the diets to 600 units. Addition of phytase at either level to the diet with 0.4% AP resulted in more TP being excreted. These results indicated that environmental contamination by P can be reduced by lowering dietary P levels. Phosphorus in excreta consists of an undigested portion of bound phytate and NPP from ingredients, and undigested portions of P from mineral supplements and surplus amounts of bioavailable P in excess of the bird’s need. Phosphorus losses from soil are generally associated with SP, which is dissolved in run off. Organic P associated with manure has greater bioavailability and solubility, and most dissolved P is immediately available for biological uptake (Sharpley, 1999). The NRC (1994) requirement for available P in laying hens is 250 mg/day. Several authors (Temperton et al., 1965a,^b; Boorman and Gunaratne, 2001) have shown that the P requirements of laying hens can be met entirely from plant ingredients to sustain egg production and egg shell quality. However, examination of the diets used by these researchers showed that they included phytase rich ingredients such as wheat. Owings et al. (1977) showed that 0.19% NPP (0.09% must be in inorganic form) will sustain a high rate of egg production but to maintain livability, the requirement was at least 0.28%. Garlich et al. (1975) reported that 0.227% NPP in the diet did not maintain normal hen weight gains. Gordan and Roland (1997) reported that hens consuming 0.1% NPP without supplemental phytase had a decrease in egg production, compared to 0.2 to 0.5 NPP. But a 0.1% NPP diet supplemented with 300 units phytase corrected the adverse effect. There were no significant differences between 0.2 to 0.5% NPP diets, and supplementation of phytase to 0.2 to 0.5% NPP diets gave no further improvement in performance. Boling et al. (2000a,^b) obtained similar results in a study involving 0.15% NPP with phytase. Supplementation of 500 units of phytase/kg diet of 21-week old ISA Brown hens reduced the NPP level to 0.12% in layer diet without affecting laying performance (Um and Paik, 1999). The use of an NPP regimen of 0.25, 0.2 and 0.15% plus 300 units phytase/kg diet for the periods 20 to 35, 36 to 51, and 52 to 63 weeks of age, reduced the daily TP excretion by 55.6% over the control 0.45% NPP for the period of 20 to 63 weeks of age (Keshavarz, 2003). Equivalency of phytase to P is very important in diet formulation to determine how much inorganic P can be saved by supplementation with phytase.

Conclusions

Birds are able to utilize PP and this is regulated by intestinal phytase. Exogenous phytase improved the utilization of PP but at the same time, the amounts of TP and SP in excreta were increased by phytase and inorganic supplementation. One portion of P in excreta consists of surplus amounts of bioavailable P in excess of the bird’s need. The most significant factor in reducing P in excreta is dietary P level. These results clearly indicate that levels of P pollution can be reduced by lowering dietary inorganic P level and by the judicious use of exogenous phytase.

Acknowledgments:

this project was supported by the King Saud University, Deanship of Scientific Research, College of Food & Agriculture Sciences, Research Center.