The bio-availability of phosphorus from feed phosphates for broilers as influenced by bio-assay method, dietary Ca-level and feed form

ANIMAL FEED SCIENCE AND TECHNOLOGY Animal Feed Science Technology

69 (1997) 329-340

The bio-availability of phosphorus from feed phosphates for broilers as influenced by bio-assay method, dietary Ca-level and feed form G. De Groote *, G. Huyghebaert Gooernment Reseurch Station for Small Stock Husbandry, Burg. Van Gansberghelaan 92, 9820 Merelbeke, Belgium Accepted

17 December

1996

Abstract The apparent retention of P from feed phosphates was investigated in two balance trials (experiments 1 and 3) using a nearly P-free basal diet and fed respectively as pellets and crumbles. Experiment 2 was a 3-factorial comparative bio-assay growth trial, involving 2688 male broiler chicks, in which a low-P mash diet (0.7 g/kg non-phytin P) was supplemented to P levels of 1.3, 1.9, 2.5, 3.1 and 3.7 g/kg from commercial monobasic calcium phosphate monohydrate (MCPh) . H,O, dihydrated dibasic calcium phosphate (DCPh) CaHPO, .2H,O and anhydrous Ca(H2P0,), dibasic calcium phosphate (DCPa) CaHPO,. Asymptotic regression models were fitted to the response parameter toe ash-% to derive relative P bio-availability values for the feed phosphates. The effect of two Ca levels (9.1 vs. 10.5 g/kg in experiment 1 and 8.5 vs. 10 g/kg in experiment 2) on P utilization was also studied. The apparent P retention with the pelleted diet was on average 78.1, 74.2 and 63.6% respectively for MCPh, DCPh and DCPa and significantly different (LSD = 3.3%). The effect of Ca was not significant. With the crumbled diet the apparent P retention appeared to be distinctly higher, being 85.5 f 1.9 and 82.3 + 1.6 for MCPh and DCPh. The results suggest that apparent P retention is markedly influenced by diet form (hardness of the pellets) and that the results of the latter balance trial are more useful for the formulation of crumbled or mash diets. The relative bio-availability of P from MCPh, DCPh and DCPa, obtained in the comparative trial was respectively 100, 93 and 85. This ranking was comparable with the relative ranking based on the apparent P retention in the feed phosphates in the balance trials. The results from the three experiments suggest the use of relative P utilization values in diet formulation for broilers. 0 1997 Elsevier Science B.V. Keywords: Phosphorus;

* Corresponding

Bio-availability;

Broiler; Bio-assay

method; Ca-level; Feed form

author.

0377.8401/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved Pm SO377-8401(97)00029-l

330

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Feed Science Technology 69 (1997) 329-340

1. Introduction

Concern about phosphorus (P) pollution of soil and water increases the need to minimize P-excretion in animal feeding. Accurate knowledge of the bio-availability of P in various feed phosphates has become therefore more important. In the past such data have been considered as only needed to formulate diets, which meet the P-requirements of poultry and to prevent P-deficiencies. Nowadays bio-availability values serve at the same time to avoid feeding of excess P that will increase P-excretion into the manure and eventually into the environment. Different possibilities exist to measure in vivo the bio-availability of P. In growing chicks, P (and Ca) are essentially transferred to the skeleton (80%) and the tissues (20%). The measurement of the carcass P-retention is therefore the appropriate method to determine the net P-utilisation (NPU) in the chick diet or in different P sources. The use of long lasting balance trials, in which metabolic faecal-and endogenous urinary P-losses are to be measured, are complicated and not suitable as a quick and accurate assay (Edwards and Gillis, 1959; Gueguen, 1961). Also, direct measurement of P-retention by carcass analysis is rather cumbersome. Hurwitz (1964) found that there exist a fairly constant ratio between carcass- and tibia-P, indicating that tibia-P may serve as a good estimate of carcass-P. He demonstrated, that the linear part of the response of tibia-P (slope ratio) on the total P intake, measured the NPU and the relative availability of P. Gillis et al. (1954) were the first to quantify the availability of P for the chick by using as response parameter the percentage of tibia ash. As with tibia-P, the relationship of the tibia ash percentage to the dietary-P intake is linear, when levels sufficiently below the P requirement are applied. The same is true for the toe ash-% which can be determined more easily and which has been used as an equal sensitive criterion by other investigators (Grau and Zweigart, 1953; Fritz et al., 1969; Yoshida and Hoshii, 1977). This is not surprising since the composition and the percentage of P in bone ash is rather constant. Hoshii and Yoshida (1977) proved that P availabilities measured by slope-ratio assay of toe ash agree with those obtained from carcass-P retention, suggesting that the change in toe ash represents the change in the NPU. In most of the studies where comparative utilization data of P are reported for the chick and the broiler the assay technique utilised has been similar to that described by Gillis et al. (19541, using tibia ash percentage or toe ash percentage as the response criterion and taking into account influences of Ca level and Ca/P ratio (for a review see Harms and Damron, 1977). Slope ratio of the dose-response lines appears to be the most appropriate method, although non-linearity and different intercepts can raise interpretation problems of the results (Yoshida, 1976; Huyghebaert et al., 1980). The slightly modified procedure of Bums and Baker (1976) apparently eliminates these difficulties. More recently non-linear bio-assays (asymptotic or sigmoidal curves) of body weight and toe- or tibia ash, using the full range of P-supplementation, have been routinely applied to estimate bio-availability values of P (De Groote et al., 1991; Huyghebaert, 1995; Ketels and De Groote, 1988; Potchanacom and Potter, 1987; Potter, 1988; Potter et al., 1995; Ravindran et al., 1995).

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Feed Science Technology 69 (1997) 329-340

331

Bio-assays based on growth, tibia- or toe ash percentage and bone strength do not enable to measure true- or apparent-P availability, but furnish comparative or relative values of P utilisation, by including in the assay a standard or reference phosphate to which arbitrary a value of 100% availability is assigned. Hitherto the majority of the data on the bio-availability of P from mineral, animal and vegetable feedstuffs are relative values, derived from comparative type bio-assays (for a review see e.g. De Groote, 1983; McGillivray, 1978; Sauveur, 1983, 1989). Attempts to measure true- or apparent-P retention by excreta collection in balance trials with chicks were rather unsuccessful (Edwards and Gillis, 1959; Sibbald, 1982). More recently Simons and Versteegh (1990) and Van der Klis and Versteegh (1993) developed successfully a balance assay based on excreta collection and using a nearly P-free basal diet (5 g/kg Ca> to which the test phosphates are added at a low level of approximately 2 g/kg available P. At this level there is no urinary P-excretion. Reliable, absolute values for utilizable P in feedstuffs from mineral, animal and plant origin have been obtained with this method. They form the basis for the preliminary system of P-evaluation in the Dutch feedstuffs table (CVB, 1994). It is well established that the Ca level and the Ca/P ratio affect P-retention and skeletal deposition and influence the responses of tibia ash percentage and toe ash percentage (Harms and Damron, 1977; Huyghebaert et al., 1981a,b). The inhibitory effect of Ca on P-absorption in turkeys was also clearly demonstrated (Hurwitz et al.. 1978). Very little information is available concerning the effect of particle size of feed phosphates and of meat meals and also of the diet feed form (mash, pellets, crumbles) on P-utilisation. Hotta et al. (1979) and De Groote et al. (1991) found that fine grinding of meat and bone meals improved P bio-availability markedly. In applying the method of Simons and Versteegh (1990) it was observed in the first experiment that the feed pellets of the diet were very hard and some incompletely digested. In the second experiment the pellets were broken and fed as crumbles. The purpose of the three experiments described in this report was to study the effect on the utilization of P in a number of commercial feed phosphates of: (1) two bio-assay methods, namely the comparative type using toe ash percentage as the response parameter and the balance type based on excreta collection, of (2) two Ca levels and of (3) the feed form (pellets vs. crumbles).

2. Materials

and methods

2.1. Diets and feed phosphates The composition of the experimental diets used in the two balance trials (experiments I and 3) is given in Table 1. The basal diets were nearly free of P ( < 0.3 g/kg P) and the test phosphates were added at levels corresponding to 3 g/kg P. The tested feed phosphates were analyzed for P and Ca content and their in vitro P-solubility in 2% citric acid and Petermann solution was also determined (Table 2).

332

G. De Groote, G. Huyghebaert/Animal

Table 1 Composition

Feed Science Technology 69 (1997) 329-340

of the basal diets used in balance trials 1 and 3 with growing

Ingredients

Experiment

Corn starch Cellulose Whey protein isolate (BIPRO) Lard Saccharose L-arginine WC, NaHCO, NaCl Vitamin + mineral premix” CaCO, (variablejb P source (variablejb Starch:cellulose (l:ljb Total, kg

1

broilers Experiment

3

Ca = 9.1 g/kg

Ca = 10.5 g/kg

Ca = 9 g/kg

290 242 209 12 90 5 6 2 3 10 9.8/5/5 13.2/16.7/14.3 - /1.3/3.7 1

292 236 209 120 90 5 6 2 3 10 13.8/9.4/9.4 13.2/16.7/14.3 -/1.3/3.7 1

289.6 241.5 208.6 119.7 89.8 5 5.9 1.9 3.0 10 11.2/5.7/5.7/6/5.9 13/16.7/16.5/15.5/17.2 0.8/2.6/2.8/3.5/2.1

9.1 3.3 125.6 209.0 20.9 12.6 14.4

10.5 3.3 125.6 209.0 21.0 12.6 14.3

9.0 3.4 12.56 209 20.9 12.6 14.3

Analysis Ca, g/kg P, g/kg MEn, MJ/kg (calculated) CP, g/kg (calculated) Lysine, g/kg (calculated) S-amino acids, g/kg (calculated) C18:2, g/kg (calculated)

aDiets contained monensin (100 mg/kg) and avoparcin (10 mg/kg). bRespectively for MCPh (monobasic calcium phosphate monohydrate), DCPh (dihydrated dibasic phosphate), DCPa (anhydrous dibasic calcium phosphate) (experiment 1) and MCPh (monobasic phosphate monohydrate), DCPh (dihydrated dibasic calcium phosphate), DCPh-#1 (dihydrated calcium phosphate), DCPh-#2 (dihydrated dibasic calcium phosphate) and bone dicalcium phosphate ment 3) diets.

MCPh, DCPh, DCPa and bone-DCP are commercial P sources, DCPh-#2 were produced under a slightly modified manufacturing

2.2. Balance

trials

(experiments

while DCPh-#l process.

calcium calcium dibasic (experi-

and

1 and 3)

Because of the very fine particle size, all experimental diets (Table 1) were pelleted (0 = 2.5 mm). However, with the purified diet the pellets were very hard and were therefore broken (with a 2-roller mill) in experiment 3, in order to improve solubility and digestibility. Each experimental diet was fed to six replicates of four broilers per replicate (22-25 days of age). Each balance trial was carried out according to the European Reference Method (Bourdillon et al., 1990), consisting of a 7-day period of adaptation to the respective

G. De Groote,

G. Huyghehaert/Animal

Feed Science Technology

69 (1997) 329-340

333

Table 2 Analytical

composition

of the different feed phosphates P

Experiments

(g/k)

Ca

P-solubility in Petermann

228.5 180.5 213.0

174 246 287

_ 96.2 95.4

_ _ _

230.8 180.0 180.5 192.4 174.7

164.0 243.3 244.1 256.1 240.1

99.2 94.7 94.2 97.7 97.3

99.1 99.4 98.9 99.1 98.7

solution (So)

P-solubility in citric acid (%;)

I and 2

MCPh” DCPhh DCPa’ Experiment

(g/kg)

3

MCPh” DCPhh DCPh-# lh DCPh-#2’ Bone-dicalcium

phosphate

“Monobasic calcium phosphate monohydrate. hDihydrated dibasic calcium phosphate. ‘Anhydrous dibasic calcium phosphate.

experimental diets and a 4-day main balance period with restricted feeding and total excreta collection. Samples of feeds and freeze-dried excreta were analyzed for Ca and P, according to AOAC methods: calorimetry for P (vanadium-molybdate method) and atomic spectrometry for Ca. The apparent retention of both Ca and P in the experimental diets was calculated from their excreta/feed (E/F) ratios as well as from their corresponding Caand P-contents. The retention results were not corrected for endogenous secretions and metabolic losses.

2.3. Growth trial (experiment

21

A flock of male broiler chicks (Ross) was purchased at one-day-old from a local hatchery. During the first week they were fed a normal starter diet (including Ca = 10 g/kg and Pav = 5 g/kg). At 7 days of age, 10% of the birds were removed, thereby improving weight uniformity. Then the broilers (2688) were assigned at random to 96 litter floor pens. They were reared for a 4-week experimental period (7-35 days of age) under normal environmental conditions for lighting, temperature, and ventilation. The respective all-mash (non-pelleted, IZ= 32) diets (Table 3) and fresh water were freely available. There were 96 pens of 28 male chicks, allowing three complete replications per treatment, in a 3-factorial design (6 X 3 X 2). The low Ca- and P-basal diet was least-cost formulated in which the remainder fraction was used to supplement with graded levels of Ca and P (Table 3). At 7 and 35 days of age, live body weights were measured on a pen basis and total weight gain was calculated. Feed intake per pen for the experimental period was recorded in order to calculate mean daily feed intake and feed efficiency. At 35 days of age 5 birds were selected (with a body weight close to the pen average) and slaughtered.

G. De Groote, G. Huyghebaert/Animal

334 Table 3 Composition

of the basal diet used in growth-trial

Ingredients

Feed Science Technology 69 (1997) 329-340

with broilers (experiment

2)

kg

Sorghum Soybean meal (48) Animal rendered fat Soybean oil DL-methionine NaCl Vitamin + mineral premixa Remainderb Total, kg

54.10 33.45 6.50 1.54 0.16 0.35 1.00 2.90 100

Analysis 1.2 3.9 0.7 12.47 215 11.5 8.4

Ca, g/kg P, g/kg P, non-phytin, g/kg (calculated) MEn-broilers, MJ/kg (calculated) Protein, g/kg (calculated) Lysine, g/kg (calculated) s-amino acids, g/kg (calculated)

“Diets were supplemented with 100 mg monensin and 10 mg avoparcin (per kilogram of complete diet). bRemainder contains variable amounts of sorghum, CaCO,, MCPh, DCPh and DCPa. Ca was supplemented up to a total dietary level of 8.5 and 10 g/kg, respectively. These Ca levels were combined with five supplementations of each P source: 0.6, 1.2, 1.8, 2.4 and 3 g/kg mineral P.

Ten toes (nail including two metatarsi) were cut, dried at 50°C for 16 h in a ventilated oven (dry matter weight) and ashed at 600°C for 16 h (ash weight). Afterwards the percentage ash was calculated. 2.4,

Statistical

evaluation

All results were subjected to a computerized analysis of variance with significant treatment differences identified by LSD-multiple range test (Statgraphics Version 5, 1991). The relative bio-availability value of the P sources was calculated by means of multiple exponential regression modelling (at each dietary Ca level): i-3

Toeash%=a-b.e-

cCjXj i=

1

with Xi = the daily P-intake (basal non-phytin + supplemental), mg/bird.

3. Results and discussion The results of experiment 1 are summarized in Table 4. The apparent retention of P in the three feed phosphates was significantly different. The effect of the Ca level was

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Feed Science Technology 69 (19971329-340

Table 4 The apparent retention in broilers of P and Ca in commercial by dietary Ca level (pelleted diets) (experiment

P-retention, % MCPh’ DCPh’ DCPa’ Mean Significance P source Ca level Pxca &retention, R MCPh’-diet DCPh’-diet DCPa3-diet Mean Si~nijicance P source Ca level caxp

335

sources of MCPh, DCPh and DCPa as influenced

1)

Ca = 9.1 g/kg

Ca = 10.5 g/kg

Mean

77.7+3.7(=100) 74.8 f 3.2 (96) 65.4+ 3.4 (84) 72.6

78.4k6.0 (= 100) 73.5 +4.5 (94) 6 I .9 f 2.5 (79) 71.3

78.1” (= 100) 74.2 (95) 63.6’ (82) LSD = 3.3 (P = 0.05)

74.0 f 4.8 70.1 k3.8 61.0+3.6 68.4

75.9” (= 100) 72.0’ (95) 64.7’ (85) LSD = 3. I (P: 0.05)

*** N.S. N.S. 77.8 + 2.3 74.0 + 2.9 68.4 + 3.3 73.4

** ***

I

N.S.

* _ ’ P < 0.001: means followed by different

letters are significantly

different from each other (P < 0.05)

’Monobasic calcium phosphate monohydrate. ’Dihydrated dibasic calcium phosphate. ‘Anhydrous dibasic calcium phosphate.

not significant, although P-retention was on average slightly lower at 10.5 g/kg Ca. The Ca-effect appears to be small, compared to what could be expected from the results of Hurwitz et al. (1978) with turkeys. The obtained values were within the range published by Simons and Versteegh (19901, which used the same method but with a considerably lower Ca level of 5 g/kg. There was no significant Ca X P interaction. Mean P-retention was 5% higher in MCPh and 19% lower in the anhydrous form of DCP, relative to DCPh. Since the amounts of CaCO, were variable in the six experimental diets, the apparent Ca-retention of the feed phosphates could not be calculated from our data. Dilworth et al. (1964) presented evidence of differences in the Ca-availability of feed-grade phosphates compared to CaCO,, based on tibia ash percentage. Their data suggest a positive correlation between the availability of P and Ca in feed phosphates. Our data showing significant change in Ca-retention in the diets due to different feed phosphates can be interpreted as showing the same relationship, certainly with respect to the DCPh and DCPa where the CaCO, supplements were identical. The higher Ca level decreased significantly dietary Ca-retention in the three diets. It may be assumed that this is not due to the higher CaCO, levels, since Ca-availability in CaCO, appears to be higher than in feed phosphates (Dilworth et al., 1964). In this experiment it was observed that due to the very hard pellets, some apparently incomplete digested pellets were present in the excreta. For this reason experiment 1 was

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Feed Science Technology 69 (1997) 329-340

Table 5 The apparent retention in broilers of P in commercial sources of MCPh, DCPh, bone dicalcium in two experimental hydrated dicalcium phosphates (crumbled diets-Ca = 9 g/kg) (experiment

phosphate 3)

P sources

Retention of P (o/o)

*SE.

Relative

MCPh’ DCPh’ DCPh-# 1’ DCPh-#22 Bone dicalcium

85.5a 82.3 bc 81.0cd 83.6ab 79.8d LSD (P = 0.05) = 2.4

f 1.9 f1.6 & 2.2 +1.4 t2.0

(= 100)

phosphate

Means followed by different letters are significantly ‘Monobasic calcium phosphate monohydrate. 2Dihydrated dibasic calcium phosphate.

and

(96) (95) (98) (93)

different from each other (P < 0.05).

repeated with the same low-P purified diet (Table l), fed as crumbles and testing five feed phosphates (experiment 3). The MCPh and DCPh were from the same producer and comparable in chemical analysis (Table 2) although from a different batch. The results of experiment 3 are summarized in Table 5. Compared to experiment 1 the P-retention with the crumbled diet was 78% points higher for both feed phosphates at the same Ca level of 9 g/kg. The relative, significant difference of 4% between the MCPh and DCPh remained however the same in both experiments and appeared to be unaffected by diet form. These results support the view that absolute P-utilisation data are very specific for a given set of experimental conditions and therefore some limits on their practical application should be taken into consideration (McGillivray, 1978). The widespread use of apparent P retention values (CVB, 1994) in formulating commercial feeds, f.i. differing in physical form (pellets, crumbles, mash), may therefore be questioned and our observations argue for the application of relative P utilization values in diet formulation. The definite confirmation of this hypothesis awaits the results of a study in which a direct comparison of pellets and crumbles (mash) is made in the same experiment. The retention of P in bone-DCP was significantly (P = 0.05) and relatively 3% lower than in DCPh-commercial. This agrees with earlier results (De Groote et al., 1991; Huyghebaert and De Groote, 1982) where, based on tibia- and toe ash percentage, the relative P-availability of another commercial bone-DCP was found to be 3-7% lower than in DCPh. The results of the comparative bio-assay, using toe ash percentage as the response parameter in a growth trial (7-35 days), are summarized in a factorial manner in Table 6. As expected P-supplementation improved all parameters significantly (P < 0.001) in a curvilinear manner. The higher Ca level (10 vs. 8.5 g/kg) affected adversely and significantly broiler performance, except toe ash percentage, which was only slightly depressed. The last observation corresponds with the small, non-significant negative effect of the higher Ca level (10.5 vs. 9.1 g/kg) on P retention observed in experiment 1. The depressive effect of Ca on broiler performance appeared very clearly at P levels below 2.5-3 g/kg, but was non-existing above that level (significant Ca X P level interactions).

G. De Groote, G. Huyghebaert/Animal Table 6 The effect of P level, P source (experiment

and Ca content

Feed Science Technology 69 (1997) 329-340

on performance

and toe ash-%

331

of broilers

(7-35

days)

2) Weight

Feed intake

gain, g (7-35 d)

(g/d)

Feed/growth ratio

Toe ash-% (35 days)

(%c)

(52.2) 72.9* 88.4’ 94.0b 97.4” 97.2” 2.1

(2.25) 1.86d 1.78‘ 1.70h 1.68”b 1.66” 0.036

(3.77) 4.61* 5.25’ 5.85b 6.14” 6.25” 0.14

(34.5) 9.5h 4.0” 4.6” 3.4” 4.6” 2.7

91” 89b 9oab

1.74” 1.73” I .74” 0.028

5.73” 5.61b 5.52b 0.1 I

4.8” 5.0” 5.8” 2.1

1.72” 0.023

5.65” 5.59” 0.09

4.1” 6.3b 1.7

***

**

Non-phytin P lerel. g/kg 0.7 ’ (658) 1.3 1 102d 1.9 1405c 1547b 2.5 3.1 1627” 1634” 3.7 34 LSD (P < 0.05) P Source MCPh’ 1478” DCPh’ 1450b 1461ab DCPa4 26 LSD (P < 0.05) Ca let’el, g/kg 8.5 1505d 10.0 1422b LSD (P < 0.05) 21 Statistical significance5 *** P level P source N.S. **x Ca level 111 Ca X P level Ca X P source N.S. P level X P source *

1.7 91.7a 88.3b 1.4

** *A*

xx*

N.S. N.S.

x

1.76’

N.S. ** x

L

N.S. N.S.

*

Mortality

I

*

N.S. N.S. N.S. N.S.

N.S. I

I

N.S. N.S.

Means followed by different letters are significantly different from each other (P < 0.05). ‘Not included in the 3-factorial analysis of variance. ‘Monobasic calcium phosphate monohydrate. ‘Dihydrated dibasic calcium phosphate. ‘Anhydrous dibasic calcium phosphate. ‘Significantly different at * * *P < 0.001, * * 0.01, * 0.05, respectively.

The P source affected significantly (P < 0.01) the response parameter toe ash percentage, being highest for MCPh and lowest for DCPa. Also feed intake and growth were marginally influenced. Plots of toe ash percentage response (35 days) on non-phytin P-intake (mg/day, 7-35 days) with the three feed phosphates at the two Ca levels are shown in Fig. 1. Asymptotic curves of best fit are presented. From the regression coefficients of the equations relative bio-availability values of P for the three commercial feed phosphates were derived, using MCPh as the reference ( = 100). Averaged for the two Ca levels the relative P-availabilities were 100, 93 and 85, respectively, for MCPh, DCPh and DCPa. This ranking compared well with the relative ranking derived from the apparent P-retention values obtained in the digestibility trials (Tables 4 and 5).

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Feed Science Technology 69 (1997) 329-340

Ca = 8.5 g/kg

Ca-log/kg

~~/yyq ux)

P’L-p&7-

int., mzd

Y - 8.975 - 3.937 * e -(O.OOm * Xl + 0.00503 * x2 +0.00443 .X3) Rd. biiWail.,

% :

l&.0 MCPh (1)

Rd. bit-avail., %

:

1d0.0 MCPh (1)

95 .4 DCPh (2)

1 Eta

(3)

(1) monobasic calcium phosphate monohydrate (2) dihydrated dibasic calcium phosphate (3) anhydrous dibasic calcium phosphate Fig. 1. Response of toe ash percentage

of broilers on the average daily

P intake from three feed phosphatesat

two dietary Ca-levels (at 35 days of age).

From the limited information presented, it may be concluded that neither the two bio-assay methods, nor the two Ca levels changed in a significant way the relative utilization of P from the three feed phosphates and that all differences noted were within the experimental errors. On the other hand the results of our study suggest that apparent retention values of P obtained in a balance trial with a hard pelleted basal diet may be too low for application in the formulation of crumbled or mash broiler diets. Recently Ravindran et al. (1995) evaluated various response parameters in assessing the relative bio-availability of P in mineral sources. Toe ash and body-weight gain appeared to be slightly more sensitive than tibia ash in this study, They found also that relative P bio-availabilities as well as their relative ranking varied considerably according to the response criteria used. If this observation ‘is confirmed, it makes the evaluation of the bio-availa&ity of P in different sources more complex.

Acknowledgemebts This work was partially supported by a grant from EMC-Tessenderlo Chemie, Belgium. The authors wish to thank Pierre De Corte and Carine Saelens for their skilful technical assistance.

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