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Availability for growing pigs of minerals and protein of a high phytate barley-rapeseed meal diet treated with Aspergillus niger phytase or soaked with whey
ANIMAL FEED SCIENCE AND TECHNOLOGY
ELSEVIER
Animal Feed Science Technology 56 (199.5)83-98
Availability for growing pigs of minerals and protein of a high phytate barley-rapeseed meal diet treated with Aspergillus niger phytase or soaked with whey J.M. Nisi Department of Animal Science,
*, E.H. Helander, K.H. Partanen Uniuersity
of Helsinki, PO Box 28, FIN-00014 Helsinki , Finland
Received 17 August 1994; accepted 13 April 1995
Abstract The improvement of phytin-phosphorus utilization in barley-rapeseed meal (800 and 200 g kg-‘) diet due to Aspergillus niger phytase (EC 3.1.3.8) supplement was assayed in a 8 X 6 cyclic change over designed digestibility and balance trial with eight growing pigs of 28-70 kg live weight. The 2 X 2 X 2 factorially arranged diets were either fortified with dicalcium phosphate to supply total phosphorus (P) 6.7 and available P (aP) 2.8 g kg-’ or without inorganic P supplement, P 5.3, aP 1.4 g kg-‘; were fed either as soaked with whey at 40 “C for 3 h or without soaking; and half the diets were supplemented with phytase 1000 PU g-’ (Finase@FPSOO). Each diet contained calcium (Cal 8.0 g kg-‘. Other nutrients were at similar levels in each diet. Soaking of the meal with whey significantly improved ash and organic matter digestibilities (P < 0.001). The digestibilities of ash (P < 0.001) and ether extract (P < 0.05) were enhanced (2 and 3% units) by phytase treatment. No effect on nitrogen (N) utilization was found due to phytase addition, .but soaking significantly improved N retention in relation to N intake. Both the soaking of the diet and addition of microbial phytase significantly improved (P < 0.01, P < 0.001) the apparent absorption of P (3 and 9% units). The retained P in diets with supplementary phytase was significantly higher (P < 0.01) than that without, 3.8 vs. 2.8 g day-‘. The soaking had an enhancing effect on P retention. P retention in relation to intake was greater (P < 0.001) in diets with added phytase than without (36 vs. 31%). From the diet with inorganic P supplementation a significantly lower retention value, 29% (P < O.OOl), was achieved compared with the P-unsupplemented diets. Phytase supplementation did not affect Ca absorption but significantly increased Ca retention due to lower urinary excretion. The soaking had no effect on Ca absorption or retention. Utilization of magnesium and zinc was not affected by the treatments. The results
* Corresponding author 0377-8401/95/$09.50
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84
J.M. N&i et al. /Animal Feed Science Technology 56 (1995) 83-98
indicate that microbial phytase and soaking with whey improved the utilization barley-rapeseed meal diet and reduced the amount of P excretion in faces. Keywords:
of phytate P in
Phytase; Aspergillus niger; Phosphorus; Balance trials; Minerals; Digestibility; Availability
1. Introduction Phytic acid, myo-inositol hexa cis phosphate, is the main storage form of phosphorus (P) in cereals and oilseeds. The presence of high levels of phytic acid in rapeseed meal (RSM), 30 g kg - ’, indicates that more than half of P is in the phytate molecules of low availability. The adverse effects of phytic acid have been attributed to its ability to interact with essential minerals and proteins leading to the formation of insoluble complexes which are nutritionally unavailable (Erdman, 1979). To enable dephosphorylation of the dietary phytates, the pigs require intrinsic (EC 3.1.3.26) or supplementary microbial phytase (EC 3.1.3.8), the enzymes that cleave the orthophosphate groups from the phytate molecule. The temperatures used for oil extraction of rapeseeds inactivate the natural phytases and render RSM devoid of phytase. These circumstances suggest a poor availability of P to pigs, and a potential inhibition of the absorption of other minerals, and reduced protein utilization. The use of microbial phytase in diets as a means of releasing the P from phytate, thereby reducing the need for supplementing inorganic P, has recently received considerable attention. The application of microbial phytase in pig diets has been further studied because of environmental concern regarding phosphates in animal waste. New efficacy data for phytase preparation derived from either Aspergillus ficuum or A. niger have appeared recently in the literature (N&i, 1990; Simons et al., 1990; Jongbloed et al., 1992, Cromwell et al., 1993; Lei et al., 1993; NPsi and Helander, 1994). However, these studies have mainly been performed on maize and soybean meal (SBM) diets while RSM in addition to high phytate content, contains glucosinolates and other antinutritive factors. For this reason efficacy of phytase treatment could be different from that of maize and SBM diets. In addition, it has been indicated that when the feed is soaked for some time before feeding, a hydrolysis of phytate partly occurs. Kemme and Jongbloed (1993) noticed that soaking the diet with microbial phytase had an additional enhancing effect on P and Ca digestibility. In contrast, N&i and Helander (1994) found no response of mineral availability to the soaking of phytase-treated pelleted barley-SBM diets. Wet feeding of pigs has recently increased sharply and it is technically possible to soak feed before feeding in order to enhance nutrient availability due to enzymatic pretreatment. A low pH in fermented or preserved whey suggests improved phytase activity in liquid feeding due to an optimal pH for hydrolysing phytates (Simons et al., 1990). Dietary lactose supply has shown an augmenting effect on mineral absorption (Heijnen et al., 1993). The objective of this study was to evaluate the possibility of improving the usage of P from a barley-RSM diet by a phytase supplement produced by A. niger. In addition, the aim was to determine the effect of soaking the diet with whey in order to enhance of both intrinsic and supplementary phytase activity. Attention was also paid to the
J.M. Niisi et al. /Animal
reduction of the P content phosphates in soils.
2. Material
Feed Science Technology
of pig excrement
56 (1995) 83-98
in order to avoid
85
the accumulation
01
and methods
The experiment was conducted as an 8 X 6 cyclic changeover design with eight growing barrows (Large White), initial weight of 27.8 (SE 3.05) kg and final weight of 69.8 (SE 3.60) kg. The treatments were arranged factorially (2 X 2 X 2) to determine the effects of dietary P level, phytase supplementation and soaking the diet with whey on availability of P and other minerals. Nutrient digestibilities and nitrogen balance of the animals were also determined. The diets containing 160 g kg-’ crude protein were composed of ground barley (800 g kg-‘) and RSM (200 g kg-‘) supplemented with minerals except P, trace elements and vitamins to meet the requirements proposed by Salo et al. (1990). The diets fed as soaked were mixed in warm water containing 60 g kg-’ dried whey as a replacement for barley (Table 1). Phytase supplementation was made with Finase@FP500 which was produced by a mutant strain of A. niger (Alko Ltd. Biotechnology, Rajamlki Finland), at the level of 2 g kg-’ to supply 1000 phytase units (PU) g-’ diet. The RSM used was hydrothermally processed double zero low glucosinolate (total glucosinolates 3.9 pmol g-’ ) spring variety Kulta of Brassica campestris. The dried whey used in soaking was edible grade spray dried sweet whey (Kuivamaito Oy, Lapinlahti).
Table
I
Chemical composition of the feed ingredients used in diets for growing pigs Component Dry matter (g kgAsh (g kg-’
’)
DM)
Barley
Rape seed meal
Dried whey
870
886
971
22
74
55
Crude protein (g kg-
’ DM)
125
374
69
Ether extract (g kg-’
DM)
34
60
56
135
6 _
NDF (g kg - ’ DM)
200
271
ADF (g kg- ’ DM)
52
197
Crude fibre (g kg-
P(g kg-’
’ DM)
DM)
Ca (g kg-’
DM)
Mg (g kg-’
DM)
Zn (mg kg-
’ DM)
12.8
4.0
0.7
8.0
3.3
1.1
5.8
0.9
60
69
The basal diet was composed of barley (800 g kg-‘) 4, 6 and 8) contained 60 g kg-’ (nos. I-4)
and RSM (200 g kg-’ 1. The diets fed as soaked (nos. 2,
were enriched with dicalcium phosphate dihydrate to provide P content of 6.7 g total P kg-
total P 5.3 and available P element supplementation,
I .4
g kg-‘.
1.5 g kg-’
were without P supplementation containing
Addition of sodium chloride to the diets was 5.0 g kg-’
and trace
diet, which provided 19 mg Fe, 68 mg Zn, 20 mg Mn, 20 mg Cu. 0.19
0.09 mg Se per kg diet. The vitamin addition, 0.5 ml kg-’
kg diet: A 5000 IU, D,
’ and
Calcium content of the diets was adjusted with calcium
carbonate to supply 8.0 g kg -’ in all of the diets. Diet nos. 5-8
I and
2
dried whey as a replacement for barley. The diets with P supplementation
were estimated to supply available P of 2.8 g kg-‘.
mg
_
3.4
500 IU, E 15 mg, K,
I
pantothenic acid 5 mg and nicotinic acid 10 mg.
diet, provided the following vitamins per
mg, B, 2 mg, B, 2 mg, B,,
0.0075
mg. biotin 0.025 mg,
86
J.M. N&i et al. /Animal Feed Science Technology 56 (1995) 83-98
Each of the diets fed to the pigs were either soaked in 1 1 kg-’ (v/w) of warm whey at 40 “C for 3 h before feeding or given in the form of dry feed mixed with water immediately prior to feeding. Water was available between the meals. The daily diet allowance was 1400 g during the first period, and was increased by 200 g per period to supply 95 g DM kg-’ W”.75 on average. The animals were placed individually in metabolism cages throughout the entire trial. Each period was comprised of 5 days of adjustment and 5 days of total faeces and urine collection. The chemical analyses of feeds and faeces were performed according to the standard procedures (AOAC, 1984). Phytic acid contents were analyzed by extracting and quantitatively assaying by the ICP-AES method of Plaami and Kumpulainen (1991). Phosphorus was determined after dry ashing calorimetrically by the vanadomolybdate procedure of Tayssky and Shorr (1953) and other minerals were measured in the diet ingredients, faeces and urine with a Perkin-Elmer 5100 PC atomic-absorption spectrophotometer. Phytase activity (U) of barley and RSM was measured as free phosphate liberated from phytate when incubating the sample in a 0.1 M sodium acetate buffer, pH 5.0, at 35 “C for 30 min. A phytase unit (U) is defined as the amount of enzyme that liberates 1 pmol of inorganic phosphorus from sodium-phytate in one minute. FinaseOFP500 contained decleared phytase activity of 500 000 PU g-‘, the units expressed as nmol (Alko B-021, Alko Ltd., Biotechnology). The data were subjected to a least squares analysis of variance (Snedecor and Cochran, 1989) using the model yjk = /_L+ Ai + Pj + Tk + eijk where A, P and T are the effects of animal, period and treatment, respectively. The degrees of freedom for treatment effects were further partitioned into single degrees of freedom by making orthogonal contrasts.
3. Results and discussion The chemical composition of the experimental feed ingredients is shown in Table 1. Barley and rapeseed meal (RSM) contained phytic acid at concentrations of 5.9 and 30.3 g kg-’ DM, respectively, and phytate-P contributed 49% and 67% of the total P in barley and RSM. Phosphorus content of dicalcium phosphate was 153.3 g kg-‘, being considerably lower than the usual value 180 g kg-‘. The phytate-P in relation to total P were calculated to be 45% in diets 1-4, and 57% in diets 5-8, respectively. Dicalcium phosphate contributed 22% and 19% of the total P supply in diets l-2 and 3-4. All the P in the P-unsupplemented diets (5-8) came from the barley and RSM, and phytate-P supply was 3.0 g kg-’ diet. In the soaked diets, P of whey contributed only 4% of total P supply. The availability of P in spray dried whole whey has been found to be similar to that in inorganic P sources (Coffey and Cromwell, 1993) In barley the assayed phytase activity was found to be 200 U kg-’ while no phytase activity was detected in RSM. Jongbloed et al. (1991) reported higher phytase activities for barley 350-630 U kg-‘, while Pointillart (1988) and N&i and Helander (1994) reported findings similar to those of the present study. Rapeseeds undergo heating
J.M. Niisi et al. /Animal
Feed Science Technology
56 (1995) 83-98
87
during toasting and solvent extraction which totally destroys phytase activity in RSM. Also, Pointillart (1988) has reported nil for phytase activity in RSM. Measured pH values of diets 2, 4, 6 and 8 were 5.53, 5.57, 5.61 and 5.57 after soaking for 3 h with whey at 40 “C, while the corresponding values were 5.41, 5.51, 5.68 and 5.58, respectively, immediately after the addition of water. Thus, no pH changes were observed during the soaking. The enzyme preparation FinaseeFP500 contains acid phosphatase in addition to phytase. Cromwell et al. (1993) found the optimal pH of phytase to be 5.0 and that of the acid phosphatase 2.5. The sum of activities of the two phytin degrading enzymes was fairly constant, between pH 2.0 and 5.5. Preserved whey usually has a pH value close to 4 due to acid application and lactic acid fermentation, which creates quite an optimal environment for phytate decomposition by phytase. In this study, due to technical problems, dried whey dissolved in water was used instead of liquid whey. The optimal pH values for intrinsic wheat and barley phytase activities have been reported to be between 5.0 and 5.2 (Scheuermann et al., 1988). According to Simons et al. (19901, Aspergiffusficuum phytase showed maximal activities at pH 2.5 and 5.5. The conditions for both intrinsic and microbial phytase activity should be quite optimal. Under similar conditions for 3-4 h, from 60% to 80% of phytates have been found to get hydrolyzed (Lantzsch, 1990; Simons et al., 1990>, or when soaked for a longer period (8-14 h) with water (Kemme and Jongbloed, 1993). In addition, phytic acid of canola meal has been degraded by A. @urn using a solid state technique for growth up to 0.4 during 48 h and completely during 144 h (Nair et al., 1991). The ash digestibility was significantly improved by phytase supplementation (P < O.OOl), 54.9% compared with the unsupplemented 52.0% (Table 2). Obviously, this increase is mainly due to the hydrolysis of plant phytates which can be seen as enhanced Table 2 Nutrient digestibility coefficients (%) of the experimental diets fed to growing pigs Diets
I
2
3
4
s
6
7
8
P supplement
+
+
+
+
-
-
-
-
Whey soaking
_
+
-
+
-
+
-
t
Phytase
-
-
t
+
-
-
+
+
SEM
Cl
c2
C3
NS ***
Dry matter
79.0
80.4
79.7
80.7
80.1
80.9
80.0
81.3
0.39
*
***
Ash
49.3
52.4
51.6
55.4
52.1
54.3
54.4
58.1
0.96
***
***
Ether extract
52.6
S2.6
54.3
55.2
54.4
53.7
55.0
55.8
I .03
NS
NS
NS 0 *
CCH
83.0
84.7
83.5
84.6
83.9
84.9
835
84.9
0.32
NS
* * *
NS
NDF
44.7
47.0
45.7
45.5
47.4
47.0
44.9
45.7
1.10
NS
NS
NS
ADF
18.2
22.2
19.5
19.6
22.2
22.1
18.6
21.1
I .66
NS
NS
Hemicellulose
61.2
62.9
61.8
61.9
62.9
62.8
61.1
61.4
0.82
NS
NS
NS NS
Organic matter
80.8
82.2
81.5
82.3
81.8
82.5
81.6
82.7
0.37
NS
* * *
Crude protein
77.7
77.5
78.8
78.2
78.7
77.9
79.1
79.0
0.68
NS
NS
D
I=P
supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase
+ soaking DS = no P supplement D6 = no P supplement t soaking D7 = no P supplement + phytase D8 = no P supplement + phytase + soaking. CCH = crude carbohydrates, NDF =
neutral detergenttibre and ADF
= acid
detergent fibre. SEM = standard error of mean. Cl = Phosphorus supplementation vs. no phosphorus supplementation. C2 = Soaking
vs. no soaking. C3 = Phytase addition vs. no phytase addition. No statistically
significant interactions were found between the treatments.
88
J.M. Niisi et al. /Animal Feed Science Technology 56 (1995) 83-98
digestion of P (Table 4). Ash digestibility was found to be lower (P < 0.001) in diets with P supplementation than in those without. Soaking also significantly improved the ash digestibility (55.0 vs. 51.8% P < 0.001). In addition, phytase treatment tended (P < 0. I> to improve crude protein digestibility and significantly enhanced that of ether extract (P < 0.05, Table 2). The improved digestibilities are obvious because Finasea FP500 also contains protein-, starch- and pectin-degrading enzymes. Moreover, phytic acid is able to inhibit cr-amylases, trypsin, tyrosinase and pepsin (Nair et al., 1991; Caldwell, 1992), and consequently the degradation of phytates by phytase treatment could enhance digestibility of nutrients. Soaking had a significant effect on organic matter and carbohydrate digestibilities (P < 0.001). The differences were rather due to differences in carbohydrate composition than to the treatment effect which is supported by the observation that no effect was noticed in neutral detergent fibre (NDF) nor acid detergent fibre (ADF) digestibility. On the other hand, giving the meal in wet form in comparison with dry form has been found to improve the ratio of feed consumption to gain in live weight and carcass weight (Patterson, 1989). In this study, a 3 h time period and a temperature of 40°C was supposed to be optimal for the hydrolysis of some components by cereal intrinsic enzymes and some side activities of phytase preparation in order to enhance nutrient availability. It is, however, possible that the digestive capacity of a growing pig is not limiting and only a small response could be achieved as found in the study by Graham et al. (1989) in which supplemental enzymes were added to the barley diet. Contrary to those findings, Nasi and Helander (1994) did not find any digestibility response of organic components to soaking or phytase supplementation which is also supported by the results that phytase added to maize-SBM or tapioca, hominy feed-SBM diets does not cause any response to the digestibility of DM (Simons et al., 1990; Jongbloed et al., 1992). On the other hand, the present results are supported by Mroz et al. (1991), Mroz et al. (1994) and Eeckhout and De Paepe (1992), who reported supplemental microbial phytase to enhance significantly DM and N apparent digestibilities. Reported improvements in growth and FCR in piglets and growing pigs given phytase (Simons et al., 1990; Beers and Jongbloed, 1992; Pallauf et al., 1992a; Cromwell et al., 1993; Lei et al., 1993; Mroz et al., 1994) may indicate a general benefit to phytate destruction, possibly due to its involvement in other aspects of digestion (Campbell and Bedford, 1992). No effect on nitrogen digestibility or N retention was noticed due to the dietary treatments (Table 3). N retention in relation to intake or absorption was significantly higher (P < 0.01) and urinary N excretion lower (P < 0.001) in pigs fed on soaked diets, but they were also supplied with a little less nitrogen (P < 0.001). Phytase supplementation tended to improve N retention (g kg-’ W’.“, P < 0.066) but, on the contrary, increased urinary N and urea N excretion (P < 0.001). Improved N retention in pigs fed diets with supplementary phytase has been found by Mroz et al. (1991) Ketaren et al. (1993) and Mroz et al. (1994). Other investigations have demonstrated that there is often a strong binding between phytic acid and protein (Mega, 1982; Zhu et al., 1990), and inhibition in the proteolytic enzymes such as pepsin and trypsin. However, in this study phytase treatment had only a small enhancing effect on protein digestibility, whereas P digestibility was improved considerably due to phytase. Phytate reduction in rapeseed flour had no significant effect on protein digestibility and amino acid absorp-
741
636
686
17.3 37.2 47.2 0.96 14.2 0.79 55.1
78.7 19.2
46.3 9.8 36.6
3
739
18.5 40.4 51.7 1.02 13.7 0.75 59.5
78.1 17.3
687
18.6 40.1 51.0 1.05 12.7 0.7 1 58.6
78.7 17.9
46.4 9.8 36.5
5
diets fed to growing
45.5 9.7 35.8
4
pigs
693
18.6 41.1 52.9 1.06 13.0 0.73 60.6
77.9 16.8
45.3 9.8 35.4
6
734
18.0 38.7 49.0 1.00 13.5 0.75 56.8
79.0 18.8
46.3 9.5 36.8
7
684
18.8 41.2 52.3 1.04 13.3 0.73 60.0
78.9 17.3
45.6 9.5 36.1
8
33.1
0.5 1 1.07 1.10 0.030 0.52 0.026 I .oo
0.68 0.35
0.16 0.32 0.33
SEM
NS
NS NS NS NS NS NS NS
NS NS
NS NS NS
Cl
NS
NS * ** NS NS NS ***
NS *+*
*** NS ***
C2
NS
,* * * **
NS NS
NS *a*
0 NS *
c3
Dl = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement + soaking D7 = no P supplement + phytase D8 = no P supplement + phytase + soaking. SEM = standard error of mean. C 1 = Phosphorus supplementation vs. no phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. No statistically significant interactions were found between the treatments.
’)
18.8 40.3 51.8 I .05 13.0 0.73 59.5
N retained (g day- ’) _ of intake (96) - of absorption (%) N (g kg-o.75 day- ‘) Urinary urea N (g day _ ’) Urea N (g kg-o.75 day- ‘) Biological value (%)
Daily gain (g day-
71.5 16.6
77.7 17.3
- of intake (o/o) Nurine(gday-‘1
18.4 40.7 52.6 1.03 12.0 0.67 60.4
45.1 10.1 35.0
46.2 10.1 36. I
N intake (g day-’ > N in faeces (g day- ’) N absorption (g day- ’)
2
in pigs fed on experimental
I
Diet
Table 3 Nitrogen balance and protein utilization
90
.I.M. N&i et al. /Animal Feed Science Technology 56 (1995) 83-98
tion in rats (Thompson, 1990). In addition to rich phytates, RSM contains other antinutritive (ANF) compounds in spite of glucosinolates which were at a minimal level, 3.9 pm01 g-‘. These dietary ANF did not appear to respond to phytase or soaking in this study. The average daily gain of the pigs was 700 g without any difference due to treatments. Unsupplemented low P diets have led to reduced performance, but Cromwell et al. (1993) reported that phytase supplementation of the low P diets restored the growth rate and FCR to levels that approached those of pigs fed the adequate P control diet. Daily phosphorus intakes of pigs receiving the P supplemented diets were 11.3 g on average. Pigs fed diets without added inorganic phosphates consumed 8.8 g P day-’ (P < 0.001) (Table 4). Dicalcium phosphate supplied P at 1.5 g kg-’ diet, contributing 20% of the total P supply in the control diets. No supplemental phosphorus was added in diets 5-8; all of the P supply was of plant origin supplying phytate-P at 3.0 g kg-’ diet. The proportions of phytate-P to total P were calculated to be 45% and 57% in diets l-4 and 5-8, respectively. The apparent digestibility of P in the diets with P supplement averaged 41% and did not differ significantly from those of P-unsupplemented diets, 39%, P > 0.05). The inorganic P addition which was 20% of total P supply was without any response, probably due to high phytic acid of RSM that interfered with absorption. In spite of the fact that soaking the diet for 3 h before feeding significantly enhanced P digestibility, it did not affect on any parameter of P usage measured. In contrast to this, the results of Kemme and Jongbloed (1993) indicated that soaking has no effect on P absorption. Phytase supplementation of the diet resulted in a significant improvement in phosphorus digestibility (P < 0.001) compared with that of the unsupplemented diet (45 vs. 36%). There was a significant interaction between P level and phytase supplementation. Added phytase in the non-soaked diet enhanced P absorption by 11% units whereas soaking the phytase treated diet additionally improved the absorption up to 15% units. The effect of soaking alone was as low as 4% units. The responses of P absorption to phytase addition in the barley-RSM diet generally were much lower than those found on maize-SBM or barley-SBM diets fed to growing pigs (Nisi, 1990; Simons et al., 1990; Jongbloed et al., 1992; Kemme and Jongbloed, 1993, Mroz et al., 1994, Nasi and Helander, 1994). The few absorption or availability values, which have been published for RSM, are rather low, 1 l-24% (Coffey and Cromwell, 1993, Larsen and Sandstrom, 1993). The high phytic acid contents 30 g kg-’ and other ANF of RSM may be involved in the low effect of treatments compared to other feed ingredients. The daily gain of pigs fed on canola diet was also unaffected by P additions (Coffey and Cromwell, 1993). Furthermore, Eeckhout and De Paepe (1992) proposed that the effect of microbial phytase is partly additive to the effect of vegetable phytase, and the low response may be associated with the low intrinsic phytase activity of the RSM diet. The present responses to phytase and soaking are supported by findings of Kemme and Jongbloed (1993), which showed that the added microbial phytase in the non-soaked diet significantly enhanced digestibility of P and Ca by 18 and 6% units, respectively. Soaking the diet without phytase had no significant effect on dP or dCa. Soaking the diet with phytase tended to have an enhancing effect on dP and dCa as compared with non-soaking and phytase (8 and 6% units). Han et al. (1987) reported that approximately
11.1 6.9 4.2 38.1 1.0 3.2 29.4 76.8 0.18
11.6 6.5 5.1 43.9 1.8 3.3 28.6 63.8 0.18
3
and retention
11.2 6.0 5.2 46.7 1.8 3.4 31.0 66.7 0.19 8.7 5.9 2.8 32.4 0.05 2.8 31.8 97.8 0.16
5 8.8 5.6 3.2 35.9 0.12 3.1 34.5 96.8 0.17
6
in pigs fed on experimental 4 8.8 5.2 3.6 42.3 0.16 3.4 40.2 95.3 0.20
7 8.9 4.7 4.2 46.9 0.39 3.8 42.8 91.8 0.21
8
diets fed to growing
pigs
0.14 0.19 0.19 1.4 0.102 0.19 I .6 1.6 0.010
SEM
** +** * *
NS 0
NS NS
*** ***
NS
***
***
NS
NS
*** *** 0 ***
NS
c3 *** *** ***
c2 NS ** * **
***
Cl
*** ** **
*
NS
0
NS
*
NS NS
NS NS NS ***
Cl’C3
NS NS NS
*
Cl’C2
Dl = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement +soaking D7 = no P supplementtphytase D8 = no P supplement+ phytase+soaking. SEM = standard error of mean. Cl = Phosphorus supplementation VS. no phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. Cl * C2 = interaction of phosphorus supplementation and soaking. C I * C3 = interaction of phosphorus supplementation and phytase and phytase addition.
11.5 7.3 4.2 37.1 1.1 3.2 28.2 76.0 0.18
2
absorption
P intake (g day-’ ) P in faeces (g day - ’) P absorption (g day- ’) _ of intake (%) Pinurine(gday-‘) P retention (g day- ’) -of intake (%I -of absorption (o/o) P (g kg -“.75 day- ‘)
apparent
1
excretion,
Diet
Table 4 Phosphorus
2 z
2
2
92
J. M. Niisi et nl. /Animal Feed Science Technology 56 (I 995) 83-98
38% of phytic acid in SBM and 43% in cotton seed meal was hydrolysed by pretreating the substrates with phytase produced by A.jicuum. Nisi and Helander (1994) in contrast to those above found that soaking pelleted phytase treated diets in warm water for 3 h had no effect. Lactose inclusion in the pig diet increased preceacal digestibility of P but total tract digestibility of P was unaffected (Ahlborn, 1993). Retention of P was enhanced due to lactose supplement in rats (Heijnen et al., 1993). Fermentation of lactose lowered the pH in the ileal lumen of rats and improved the solubility of mineral components thus stimulating the absorption (Heijnen et al., 1993). Phytase inclusion in a diet without P supplementation decreased by one third the amount of P excreted in faeces compared to the value without phytase addition to the diet. Phosphorus excretion in urine was manyfold in P supplemented diets compared with others (P < 0.001). The phytase supplemented diet produced a significantly higher (P < 0.001) urine P excretion compared to that produced without phytase addition. Signs of P deficiency appearing as hypophosphaturia and hypercalciuria (Pointillart, 1991) were found in pigs fed a P unsupplemented diet without phytase. The treatment with phytase alleviated the symptoms of deficiency. The total excretion of P was only one third lower in a phytase supplemented diet (5.5 g day-‘) compared with an inorganic P supplemented diet, in which the faecal and urinary excretion was 8.1 g day-‘. In previous experiments with maize-SBM and barley-SBM diets treated with phytase, the reductions have been much higher (Nisi, 1990; N’asi and Helander, 1994). The retention of P in pigs fed diets with inorganic P did not differ from that in pigs without P supplementation; however inorganic P supply from dicalcium phosphate was 1.5 g kg-’ diet and P deficiency was obvious in pigs fed an unsupplemented diet without phytase. Soaking the diets had no effect on the retention of P in spite of the increased absorption of P. Phytase supplement improved P retention significantly (P < 0.011, and the enhancement was on average 1.0 g day-’ (2.8 g vs. 3.8 g> corresponding to an increase of 36%. The retention of phosphorus was 29% in relation to P intake in the diets with inorganic P and 37% in diets without inorganic P supplementation. The observed availability of P in barley and RSM diets is in accordance with limited existing literature (Pointillart, 1988; Coffey and Cromwell, 1993; Larsen and Sandstriim, 1993; NPsi and Helander, 1994). Microbial phytase supplementation improved retention of phosphorus to the level of 36% and the difference was significant (P < 0.01) compared to the untreated non P-supplemented diet (31%). Retention of the absorbed P was very high (92%) in diets supplemented with phytase, but lower (6% units) relative to the untreated diets (P < 0.01). Calcium digestibility was lower in a P-supplemented diet (P < 0.01) compared to unsupplemented, but urinary Ca excretion was higher (P < 0.001). Soaking reduced Ca excretion in faeces, but was without any significant effect on Ca retention or utilization. Phytase treatment did not affected Ca digestibility but decreased urinary Ca excretion (P < 0.001). Pigs fed the diet with phytase had significantly (P < 0.01, P < 0.001) improved absolute Ca retention and in relation to Ca intake or absorption (Table 5). Nisi and Helander (1994) found only a marginal improvement in Ca absorption or retention due to phytase addition in a barley-SBM diet, whereas Ca digestibility was increased when phytase was added to a diet composed of tapioca and hominy feed or maize-SBM (Simons et al., 1990; Kemme and Jongbloed, 1993; Mroz et al., 1994) Also improve-
’)
day
’)
’)
0.33
0.32
0.33 0.34
6.2 41.3 92.3
5.9 38.1 92.4
5.7 38.2 93.5
5.9 38.4 93.3
44.7 0.5
41.2 0.5
40.9 0.4
41.2 0.4
15.0
4
8.3 6.7
15.5
3
0.32
5.7 35.1 77.2
16.1 a.1 1.4 45.5 1.7
5
0.3
5.3 35.0 76.8
1
1.6
15.4 8.4 7.0 45.6
6
in pigs fed on experimental
9.1 6.4
8.8 6.1
14.9
2
and retention
9.1 6.3
15.4
1
absorption
0.38
6.8 42.1 88.4
16.1 8.4 1.6 47.5 0.9
7
0.016
0.30 1.7 1.0
6.6 42.4 89.0 0.36
0.05 0.30 0.30 1.8 0.10
SEM
pigs
15.5 8.1 7.4 47.1 0.8
8
diets fed to growing
NS
NS NS NS
NS NS **r NS
* NS NS NS
***
c2
0 *** ** *+*
***
Cl
**
NS NS NS *** ** ** ***
*
c3
NS
NS NS NS NS NS NS NS
*
CI’C2
*
NS NS NS NS *** * * ***
CI’C3
DI = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement vs. no + soaking D7 = no P supplement + phytase D8 = no P supplement + phytase + soaking, SEM = standard error of mean. C I = Phosphorus supplementation phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. Cl * C2 = interaction of phosphorus supplementation and soaking. Cl * C3 = interaction of phosphorus supplementation and phytase addition.
Ca (g kg-‘.”
Ca retention (g g-’ ) -of intake (%I -of absorption (%)
-of intake (%I Ca in urine (g day-
’) ’)
apparent
Ca in faeces (g day Ca absorption (g day
Ca intake (g day
Diet
Table 5 Calcium excretion,
a \
9
4.2 2.9 1.3 30.3 0.5 0.75 18.0 59.5 43
1 4.2 3.0 1.2 29.3 0.5 0.74 17.9 59.3 42
3
and retention
4.1 2.8 1.2 29.8 0.5 0.73 18.2 61.2 42
2
apparent absorption
intake (g day- ’) in faeces (g day- ’) absorption (g day- ’) intake (%I in urine (g day- ’) retention (g day- ’) intake (8) absorption (%) (mg kg-‘.” day-‘)
excretion,
4.2 2.8 1.4 32.2 0.5 0.87 20.2 62.5 47
4 4.0 2.6 1.4 34.1 0.6 0.76 19.2 57.3 44
5 4.0 2.6 1.4 33.9 0.6 0.71 18.4 54.5 42
6
in pigs fed on experimental
4.1 2.7 1.4 34.9 0.6 0.85 20.9 60.0 47
7 4.0 2.5 1.5 36.6 0.6 0.84 20.9 57.2 47
8
diets fed to growing Cl * * * *** 0 ** ** NS NS NS NS
SEM 0.04 0.08 0.09 1.8 0.04 0.078 1.6 3.3 4
pigs
0 NS NS NS NS NS NS NS NS
c2
* NS NS NS NS NS NS NS NS
c3
Cl *c3 NS NS NS NS NS NS NS NS NS
Cl *c2 NS NS NS NS NS NS NS NS NS
Dl = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement tsoaking D7 = no P supplement +phytase D8 = no P supplement +phytase+ soaking. SEM = standard error of mean. Cl = Phosphorus supplementation vs. no phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. Cl * C2 = interaction of phosphorus supplementation and soaking. C I * C3 = interaction of phosphorus supplementation and phytase addition.
Mg Mg Mg -of Mg Mg -of -of Mg
Diet
Table 6 Magnesium
240 194 46 19.1 10 37 15.2 15.6 2.1
I
absorption
intake (mg day ’) in faeces (mg day- ’) absorption (mg day- ’) intake (%) in urine (mg day-‘) retention (mg day _ ’) intake (%) absorption (%o) (mg kg-‘.” day-‘)
apparent
232 182 51 21.7 10 41 17.6 76.1 2.3
2
and retention
241 188 53 22.1 11 42 17.3 55.1 2.3
3 234 177 57 23.9 11 46 19.4 81.8 2.5
4 240 170 69 28.2 10 59 23.7 83.7 3.2
5
in pigs fed on experimental
232 170 62 26.6 13 49 20.9 79.0 2.8
6 240 181 58 24.1 14 44 17.9 74.0 2.4
7
diets fed to growing
234 177 57 24.1 12 45 18.7 16.2 2.4
8
pigs
0.9 4.9 4.8 1.87 1.2 5.0 1.93 9.69 0.26
SEM NS ** ** ** * * * NS *
Cl *** 0 NS NS NS NS NS NS NS
C2 0 NS NS NS 0 NS NS NS NS
c3
NS NS NS NS NS NS NS NS NS
CI*C2
NS 0 * * NS * * NS *
Cl *c3
Dl = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement + soaking D7 = no P supplement + phytase D8 = no P supplement + phytase + soaking. SEM = standard error of mean. C I = Phosphorus supplementation vs. no phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. C 1 * C2 = interaction of phosphorus supplementation and soaking. C 1 * C3 = interaction of phosphorus supplementation and phytase addition.
Zn Zn Zn -of Zn Zn -of -of Zn
Diet
Table 7 Zinc excretion,
96
J.M. Niisi et al. /Animal Feed Science Technology 56 (1995) 83-98
ments in Ca digestibility has been found in piglet fed maize diets with phytase (Pallauf et al., 1992a; Hoppe et al., 1993). Ca content in RSM is rather high compared to cereals, 7.0 vs 0.35 g kg-‘, and it contributed 20% of the total Ca supply in this study. The availability of Ca in RSM is reduced due to high phytic acid following rather low digestibility as reported by Larsen and Sandstrom (19931, and also, both Ca and P utilization have been decreased with time in pigs fed a phytase rich diet containing rapeseed (Pointillart, 1988). Another reason for the low Ca availability can be the lower P retention in diets without P and phytase additions. If the intake of Ca is adequate but that of P inadequate, the retention of Ca falls (Jongbloed, 1987). In the present study, the calculated Ca/P ratios were 1.33:1 and 1.78:l (P < 0.001) for diets l-4 and diets 5-8, respectively, while the optimum Ca/P ratio is from 1.2 to 1.3:1 (ARC, 1981). However, Mroz et al. (1994) suggested that Ca:total P ratios are not relevant indicators in practical diet formulation for pigs, but Ca:digestible P ratios should be used, and the ratios 2.8-3.3 were most optimal. P addition reduced the ratio Ca/dP from 4.72 to 3.27 (P < O.OOl), soaking from 4.17 to 3.8 1 (P < 0.001) and phytase addition from 4.46 to 3.52 (P < 0.001). As a result of the phytase treatment, the ratio approached quite an optimal level which is also seen in the improved utilization of P. It is well known that phytic acid has a high affinity to bi- and trivalent cations (Wise, 1983). Digestibility of magnesium was lower (P < 0.01) in a P-supplemented diet while urinary excretion was reduced (P < 0.01, Table 61, but there were also differences in Mg supply. Phytase treatment or soaking did not affect Mg absorption or utilization. In the barley-SBM diet, phytase supplement improved Mg digestibility and retention (N&i and Helander, 1994) in agreement with the results found in piglets when phytase was supplemented (Pallauf et al., 1992b). P supplementation increased faecal excretion of zinc but decreased urinary Zn excretion significantly. Soaking or phytase supplementation did not have any effect on the retention or utilization of Zn (Table 7). Phytic acid readily forms complexes with several essential minerals, such as calcium, iron, zinc and manganese, impairing their utilization by the animal (Nelson et al., 1971; Pallauf et al., 1992b). The results of the present study demonstrate that the addition of a microbial phytase or soaking with the whey barley-RSM diets enhance the absorption of vegetable phosphorus and essential minerals in the diet as Ca and Mg. An increasing proportion of the pigs’ requirement for P could be met by P in barley and RSM when phosphorus could be converted to an available form. Phosphorus content in pig manure is reduced following the improved plant-P utilization resulting from dietary phytase supplementation.
Acknotiledgements The authors are grateful to Ms. Mari Korkeaoja, Ms. Anneli Pakarinen and Mr. Jari Miettinen for technical assistance. The financial support to this study was received from Academy of Finland.
J.&f. Niisi et al. /Animal
Feed Science Technology
56 (1995) 83-98
95
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ELSEVIER
Animal Feed Science Technology 56 (199.5)83-98
Availability for growing pigs of minerals and protein of a high phytate barley-rapeseed meal diet treated with Aspergillus niger phytase or soaked with whey J.M. Nisi Department of Animal Science,
*, E.H. Helander, K.H. Partanen Uniuersity
of Helsinki, PO Box 28, FIN-00014 Helsinki , Finland
Received 17 August 1994; accepted 13 April 1995
Abstract The improvement of phytin-phosphorus utilization in barley-rapeseed meal (800 and 200 g kg-‘) diet due to Aspergillus niger phytase (EC 3.1.3.8) supplement was assayed in a 8 X 6 cyclic change over designed digestibility and balance trial with eight growing pigs of 28-70 kg live weight. The 2 X 2 X 2 factorially arranged diets were either fortified with dicalcium phosphate to supply total phosphorus (P) 6.7 and available P (aP) 2.8 g kg-’ or without inorganic P supplement, P 5.3, aP 1.4 g kg-‘; were fed either as soaked with whey at 40 “C for 3 h or without soaking; and half the diets were supplemented with phytase 1000 PU g-’ (Finase@FPSOO). Each diet contained calcium (Cal 8.0 g kg-‘. Other nutrients were at similar levels in each diet. Soaking of the meal with whey significantly improved ash and organic matter digestibilities (P < 0.001). The digestibilities of ash (P < 0.001) and ether extract (P < 0.05) were enhanced (2 and 3% units) by phytase treatment. No effect on nitrogen (N) utilization was found due to phytase addition, .but soaking significantly improved N retention in relation to N intake. Both the soaking of the diet and addition of microbial phytase significantly improved (P < 0.01, P < 0.001) the apparent absorption of P (3 and 9% units). The retained P in diets with supplementary phytase was significantly higher (P < 0.01) than that without, 3.8 vs. 2.8 g day-‘. The soaking had an enhancing effect on P retention. P retention in relation to intake was greater (P < 0.001) in diets with added phytase than without (36 vs. 31%). From the diet with inorganic P supplementation a significantly lower retention value, 29% (P < O.OOl), was achieved compared with the P-unsupplemented diets. Phytase supplementation did not affect Ca absorption but significantly increased Ca retention due to lower urinary excretion. The soaking had no effect on Ca absorption or retention. Utilization of magnesium and zinc was not affected by the treatments. The results
* Corresponding author 0377-8401/95/$09.50
0 1995 Elsevier Science B.V. All rights reserved
SSDI 0377.8401(95)00812-8
84
J.M. N&i et al. /Animal Feed Science Technology 56 (1995) 83-98
indicate that microbial phytase and soaking with whey improved the utilization barley-rapeseed meal diet and reduced the amount of P excretion in faces. Keywords:
of phytate P in
Phytase; Aspergillus niger; Phosphorus; Balance trials; Minerals; Digestibility; Availability
1. Introduction Phytic acid, myo-inositol hexa cis phosphate, is the main storage form of phosphorus (P) in cereals and oilseeds. The presence of high levels of phytic acid in rapeseed meal (RSM), 30 g kg - ’, indicates that more than half of P is in the phytate molecules of low availability. The adverse effects of phytic acid have been attributed to its ability to interact with essential minerals and proteins leading to the formation of insoluble complexes which are nutritionally unavailable (Erdman, 1979). To enable dephosphorylation of the dietary phytates, the pigs require intrinsic (EC 3.1.3.26) or supplementary microbial phytase (EC 3.1.3.8), the enzymes that cleave the orthophosphate groups from the phytate molecule. The temperatures used for oil extraction of rapeseeds inactivate the natural phytases and render RSM devoid of phytase. These circumstances suggest a poor availability of P to pigs, and a potential inhibition of the absorption of other minerals, and reduced protein utilization. The use of microbial phytase in diets as a means of releasing the P from phytate, thereby reducing the need for supplementing inorganic P, has recently received considerable attention. The application of microbial phytase in pig diets has been further studied because of environmental concern regarding phosphates in animal waste. New efficacy data for phytase preparation derived from either Aspergillus ficuum or A. niger have appeared recently in the literature (N&i, 1990; Simons et al., 1990; Jongbloed et al., 1992, Cromwell et al., 1993; Lei et al., 1993; NPsi and Helander, 1994). However, these studies have mainly been performed on maize and soybean meal (SBM) diets while RSM in addition to high phytate content, contains glucosinolates and other antinutritive factors. For this reason efficacy of phytase treatment could be different from that of maize and SBM diets. In addition, it has been indicated that when the feed is soaked for some time before feeding, a hydrolysis of phytate partly occurs. Kemme and Jongbloed (1993) noticed that soaking the diet with microbial phytase had an additional enhancing effect on P and Ca digestibility. In contrast, N&i and Helander (1994) found no response of mineral availability to the soaking of phytase-treated pelleted barley-SBM diets. Wet feeding of pigs has recently increased sharply and it is technically possible to soak feed before feeding in order to enhance nutrient availability due to enzymatic pretreatment. A low pH in fermented or preserved whey suggests improved phytase activity in liquid feeding due to an optimal pH for hydrolysing phytates (Simons et al., 1990). Dietary lactose supply has shown an augmenting effect on mineral absorption (Heijnen et al., 1993). The objective of this study was to evaluate the possibility of improving the usage of P from a barley-RSM diet by a phytase supplement produced by A. niger. In addition, the aim was to determine the effect of soaking the diet with whey in order to enhance of both intrinsic and supplementary phytase activity. Attention was also paid to the
J.M. Niisi et al. /Animal
reduction of the P content phosphates in soils.
2. Material
Feed Science Technology
of pig excrement
56 (1995) 83-98
in order to avoid
85
the accumulation
01
and methods
The experiment was conducted as an 8 X 6 cyclic changeover design with eight growing barrows (Large White), initial weight of 27.8 (SE 3.05) kg and final weight of 69.8 (SE 3.60) kg. The treatments were arranged factorially (2 X 2 X 2) to determine the effects of dietary P level, phytase supplementation and soaking the diet with whey on availability of P and other minerals. Nutrient digestibilities and nitrogen balance of the animals were also determined. The diets containing 160 g kg-’ crude protein were composed of ground barley (800 g kg-‘) and RSM (200 g kg-‘) supplemented with minerals except P, trace elements and vitamins to meet the requirements proposed by Salo et al. (1990). The diets fed as soaked were mixed in warm water containing 60 g kg-’ dried whey as a replacement for barley (Table 1). Phytase supplementation was made with Finase@FP500 which was produced by a mutant strain of A. niger (Alko Ltd. Biotechnology, Rajamlki Finland), at the level of 2 g kg-’ to supply 1000 phytase units (PU) g-’ diet. The RSM used was hydrothermally processed double zero low glucosinolate (total glucosinolates 3.9 pmol g-’ ) spring variety Kulta of Brassica campestris. The dried whey used in soaking was edible grade spray dried sweet whey (Kuivamaito Oy, Lapinlahti).
Table
I
Chemical composition of the feed ingredients used in diets for growing pigs Component Dry matter (g kgAsh (g kg-’
’)
DM)
Barley
Rape seed meal
Dried whey
870
886
971
22
74
55
Crude protein (g kg-
’ DM)
125
374
69
Ether extract (g kg-’
DM)
34
60
56
135
6 _
NDF (g kg - ’ DM)
200
271
ADF (g kg- ’ DM)
52
197
Crude fibre (g kg-
P(g kg-’
’ DM)
DM)
Ca (g kg-’
DM)
Mg (g kg-’
DM)
Zn (mg kg-
’ DM)
12.8
4.0
0.7
8.0
3.3
1.1
5.8
0.9
60
69
The basal diet was composed of barley (800 g kg-‘) 4, 6 and 8) contained 60 g kg-’ (nos. I-4)
and RSM (200 g kg-’ 1. The diets fed as soaked (nos. 2,
were enriched with dicalcium phosphate dihydrate to provide P content of 6.7 g total P kg-
total P 5.3 and available P element supplementation,
I .4
g kg-‘.
1.5 g kg-’
were without P supplementation containing
Addition of sodium chloride to the diets was 5.0 g kg-’
and trace
diet, which provided 19 mg Fe, 68 mg Zn, 20 mg Mn, 20 mg Cu. 0.19
0.09 mg Se per kg diet. The vitamin addition, 0.5 ml kg-’
kg diet: A 5000 IU, D,
’ and
Calcium content of the diets was adjusted with calcium
carbonate to supply 8.0 g kg -’ in all of the diets. Diet nos. 5-8
I and
2
dried whey as a replacement for barley. The diets with P supplementation
were estimated to supply available P of 2.8 g kg-‘.
mg
_
3.4
500 IU, E 15 mg, K,
I
pantothenic acid 5 mg and nicotinic acid 10 mg.
diet, provided the following vitamins per
mg, B, 2 mg, B, 2 mg, B,,
0.0075
mg. biotin 0.025 mg,
86
J.M. N&i et al. /Animal Feed Science Technology 56 (1995) 83-98
Each of the diets fed to the pigs were either soaked in 1 1 kg-’ (v/w) of warm whey at 40 “C for 3 h before feeding or given in the form of dry feed mixed with water immediately prior to feeding. Water was available between the meals. The daily diet allowance was 1400 g during the first period, and was increased by 200 g per period to supply 95 g DM kg-’ W”.75 on average. The animals were placed individually in metabolism cages throughout the entire trial. Each period was comprised of 5 days of adjustment and 5 days of total faeces and urine collection. The chemical analyses of feeds and faeces were performed according to the standard procedures (AOAC, 1984). Phytic acid contents were analyzed by extracting and quantitatively assaying by the ICP-AES method of Plaami and Kumpulainen (1991). Phosphorus was determined after dry ashing calorimetrically by the vanadomolybdate procedure of Tayssky and Shorr (1953) and other minerals were measured in the diet ingredients, faeces and urine with a Perkin-Elmer 5100 PC atomic-absorption spectrophotometer. Phytase activity (U) of barley and RSM was measured as free phosphate liberated from phytate when incubating the sample in a 0.1 M sodium acetate buffer, pH 5.0, at 35 “C for 30 min. A phytase unit (U) is defined as the amount of enzyme that liberates 1 pmol of inorganic phosphorus from sodium-phytate in one minute. FinaseOFP500 contained decleared phytase activity of 500 000 PU g-‘, the units expressed as nmol (Alko B-021, Alko Ltd., Biotechnology). The data were subjected to a least squares analysis of variance (Snedecor and Cochran, 1989) using the model yjk = /_L+ Ai + Pj + Tk + eijk where A, P and T are the effects of animal, period and treatment, respectively. The degrees of freedom for treatment effects were further partitioned into single degrees of freedom by making orthogonal contrasts.
3. Results and discussion The chemical composition of the experimental feed ingredients is shown in Table 1. Barley and rapeseed meal (RSM) contained phytic acid at concentrations of 5.9 and 30.3 g kg-’ DM, respectively, and phytate-P contributed 49% and 67% of the total P in barley and RSM. Phosphorus content of dicalcium phosphate was 153.3 g kg-‘, being considerably lower than the usual value 180 g kg-‘. The phytate-P in relation to total P were calculated to be 45% in diets 1-4, and 57% in diets 5-8, respectively. Dicalcium phosphate contributed 22% and 19% of the total P supply in diets l-2 and 3-4. All the P in the P-unsupplemented diets (5-8) came from the barley and RSM, and phytate-P supply was 3.0 g kg-’ diet. In the soaked diets, P of whey contributed only 4% of total P supply. The availability of P in spray dried whole whey has been found to be similar to that in inorganic P sources (Coffey and Cromwell, 1993) In barley the assayed phytase activity was found to be 200 U kg-’ while no phytase activity was detected in RSM. Jongbloed et al. (1991) reported higher phytase activities for barley 350-630 U kg-‘, while Pointillart (1988) and N&i and Helander (1994) reported findings similar to those of the present study. Rapeseeds undergo heating
J.M. Niisi et al. /Animal
Feed Science Technology
56 (1995) 83-98
87
during toasting and solvent extraction which totally destroys phytase activity in RSM. Also, Pointillart (1988) has reported nil for phytase activity in RSM. Measured pH values of diets 2, 4, 6 and 8 were 5.53, 5.57, 5.61 and 5.57 after soaking for 3 h with whey at 40 “C, while the corresponding values were 5.41, 5.51, 5.68 and 5.58, respectively, immediately after the addition of water. Thus, no pH changes were observed during the soaking. The enzyme preparation FinaseeFP500 contains acid phosphatase in addition to phytase. Cromwell et al. (1993) found the optimal pH of phytase to be 5.0 and that of the acid phosphatase 2.5. The sum of activities of the two phytin degrading enzymes was fairly constant, between pH 2.0 and 5.5. Preserved whey usually has a pH value close to 4 due to acid application and lactic acid fermentation, which creates quite an optimal environment for phytate decomposition by phytase. In this study, due to technical problems, dried whey dissolved in water was used instead of liquid whey. The optimal pH values for intrinsic wheat and barley phytase activities have been reported to be between 5.0 and 5.2 (Scheuermann et al., 1988). According to Simons et al. (19901, Aspergiffusficuum phytase showed maximal activities at pH 2.5 and 5.5. The conditions for both intrinsic and microbial phytase activity should be quite optimal. Under similar conditions for 3-4 h, from 60% to 80% of phytates have been found to get hydrolyzed (Lantzsch, 1990; Simons et al., 1990>, or when soaked for a longer period (8-14 h) with water (Kemme and Jongbloed, 1993). In addition, phytic acid of canola meal has been degraded by A. @urn using a solid state technique for growth up to 0.4 during 48 h and completely during 144 h (Nair et al., 1991). The ash digestibility was significantly improved by phytase supplementation (P < O.OOl), 54.9% compared with the unsupplemented 52.0% (Table 2). Obviously, this increase is mainly due to the hydrolysis of plant phytates which can be seen as enhanced Table 2 Nutrient digestibility coefficients (%) of the experimental diets fed to growing pigs Diets
I
2
3
4
s
6
7
8
P supplement
+
+
+
+
-
-
-
-
Whey soaking
_
+
-
+
-
+
-
t
Phytase
-
-
t
+
-
-
+
+
SEM
Cl
c2
C3
NS ***
Dry matter
79.0
80.4
79.7
80.7
80.1
80.9
80.0
81.3
0.39
*
***
Ash
49.3
52.4
51.6
55.4
52.1
54.3
54.4
58.1
0.96
***
***
Ether extract
52.6
S2.6
54.3
55.2
54.4
53.7
55.0
55.8
I .03
NS
NS
NS 0 *
CCH
83.0
84.7
83.5
84.6
83.9
84.9
835
84.9
0.32
NS
* * *
NS
NDF
44.7
47.0
45.7
45.5
47.4
47.0
44.9
45.7
1.10
NS
NS
NS
ADF
18.2
22.2
19.5
19.6
22.2
22.1
18.6
21.1
I .66
NS
NS
Hemicellulose
61.2
62.9
61.8
61.9
62.9
62.8
61.1
61.4
0.82
NS
NS
NS NS
Organic matter
80.8
82.2
81.5
82.3
81.8
82.5
81.6
82.7
0.37
NS
* * *
Crude protein
77.7
77.5
78.8
78.2
78.7
77.9
79.1
79.0
0.68
NS
NS
D
I=P
supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase
+ soaking DS = no P supplement D6 = no P supplement t soaking D7 = no P supplement + phytase D8 = no P supplement + phytase + soaking. CCH = crude carbohydrates, NDF =
neutral detergenttibre and ADF
= acid
detergent fibre. SEM = standard error of mean. Cl = Phosphorus supplementation vs. no phosphorus supplementation. C2 = Soaking
vs. no soaking. C3 = Phytase addition vs. no phytase addition. No statistically
significant interactions were found between the treatments.
88
J.M. Niisi et al. /Animal Feed Science Technology 56 (1995) 83-98
digestion of P (Table 4). Ash digestibility was found to be lower (P < 0.001) in diets with P supplementation than in those without. Soaking also significantly improved the ash digestibility (55.0 vs. 51.8% P < 0.001). In addition, phytase treatment tended (P < 0. I> to improve crude protein digestibility and significantly enhanced that of ether extract (P < 0.05, Table 2). The improved digestibilities are obvious because Finasea FP500 also contains protein-, starch- and pectin-degrading enzymes. Moreover, phytic acid is able to inhibit cr-amylases, trypsin, tyrosinase and pepsin (Nair et al., 1991; Caldwell, 1992), and consequently the degradation of phytates by phytase treatment could enhance digestibility of nutrients. Soaking had a significant effect on organic matter and carbohydrate digestibilities (P < 0.001). The differences were rather due to differences in carbohydrate composition than to the treatment effect which is supported by the observation that no effect was noticed in neutral detergent fibre (NDF) nor acid detergent fibre (ADF) digestibility. On the other hand, giving the meal in wet form in comparison with dry form has been found to improve the ratio of feed consumption to gain in live weight and carcass weight (Patterson, 1989). In this study, a 3 h time period and a temperature of 40°C was supposed to be optimal for the hydrolysis of some components by cereal intrinsic enzymes and some side activities of phytase preparation in order to enhance nutrient availability. It is, however, possible that the digestive capacity of a growing pig is not limiting and only a small response could be achieved as found in the study by Graham et al. (1989) in which supplemental enzymes were added to the barley diet. Contrary to those findings, Nasi and Helander (1994) did not find any digestibility response of organic components to soaking or phytase supplementation which is also supported by the results that phytase added to maize-SBM or tapioca, hominy feed-SBM diets does not cause any response to the digestibility of DM (Simons et al., 1990; Jongbloed et al., 1992). On the other hand, the present results are supported by Mroz et al. (1991), Mroz et al. (1994) and Eeckhout and De Paepe (1992), who reported supplemental microbial phytase to enhance significantly DM and N apparent digestibilities. Reported improvements in growth and FCR in piglets and growing pigs given phytase (Simons et al., 1990; Beers and Jongbloed, 1992; Pallauf et al., 1992a; Cromwell et al., 1993; Lei et al., 1993; Mroz et al., 1994) may indicate a general benefit to phytate destruction, possibly due to its involvement in other aspects of digestion (Campbell and Bedford, 1992). No effect on nitrogen digestibility or N retention was noticed due to the dietary treatments (Table 3). N retention in relation to intake or absorption was significantly higher (P < 0.01) and urinary N excretion lower (P < 0.001) in pigs fed on soaked diets, but they were also supplied with a little less nitrogen (P < 0.001). Phytase supplementation tended to improve N retention (g kg-’ W’.“, P < 0.066) but, on the contrary, increased urinary N and urea N excretion (P < 0.001). Improved N retention in pigs fed diets with supplementary phytase has been found by Mroz et al. (1991) Ketaren et al. (1993) and Mroz et al. (1994). Other investigations have demonstrated that there is often a strong binding between phytic acid and protein (Mega, 1982; Zhu et al., 1990), and inhibition in the proteolytic enzymes such as pepsin and trypsin. However, in this study phytase treatment had only a small enhancing effect on protein digestibility, whereas P digestibility was improved considerably due to phytase. Phytate reduction in rapeseed flour had no significant effect on protein digestibility and amino acid absorp-
741
636
686
17.3 37.2 47.2 0.96 14.2 0.79 55.1
78.7 19.2
46.3 9.8 36.6
3
739
18.5 40.4 51.7 1.02 13.7 0.75 59.5
78.1 17.3
687
18.6 40.1 51.0 1.05 12.7 0.7 1 58.6
78.7 17.9
46.4 9.8 36.5
5
diets fed to growing
45.5 9.7 35.8
4
pigs
693
18.6 41.1 52.9 1.06 13.0 0.73 60.6
77.9 16.8
45.3 9.8 35.4
6
734
18.0 38.7 49.0 1.00 13.5 0.75 56.8
79.0 18.8
46.3 9.5 36.8
7
684
18.8 41.2 52.3 1.04 13.3 0.73 60.0
78.9 17.3
45.6 9.5 36.1
8
33.1
0.5 1 1.07 1.10 0.030 0.52 0.026 I .oo
0.68 0.35
0.16 0.32 0.33
SEM
NS
NS NS NS NS NS NS NS
NS NS
NS NS NS
Cl
NS
NS * ** NS NS NS ***
NS *+*
*** NS ***
C2
NS
,* * * **
NS NS
NS *a*
0 NS *
c3
Dl = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement + soaking D7 = no P supplement + phytase D8 = no P supplement + phytase + soaking. SEM = standard error of mean. C 1 = Phosphorus supplementation vs. no phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. No statistically significant interactions were found between the treatments.
’)
18.8 40.3 51.8 I .05 13.0 0.73 59.5
N retained (g day- ’) _ of intake (96) - of absorption (%) N (g kg-o.75 day- ‘) Urinary urea N (g day _ ’) Urea N (g kg-o.75 day- ‘) Biological value (%)
Daily gain (g day-
71.5 16.6
77.7 17.3
- of intake (o/o) Nurine(gday-‘1
18.4 40.7 52.6 1.03 12.0 0.67 60.4
45.1 10.1 35.0
46.2 10.1 36. I
N intake (g day-’ > N in faeces (g day- ’) N absorption (g day- ’)
2
in pigs fed on experimental
I
Diet
Table 3 Nitrogen balance and protein utilization
90
.I.M. N&i et al. /Animal Feed Science Technology 56 (1995) 83-98
tion in rats (Thompson, 1990). In addition to rich phytates, RSM contains other antinutritive (ANF) compounds in spite of glucosinolates which were at a minimal level, 3.9 pm01 g-‘. These dietary ANF did not appear to respond to phytase or soaking in this study. The average daily gain of the pigs was 700 g without any difference due to treatments. Unsupplemented low P diets have led to reduced performance, but Cromwell et al. (1993) reported that phytase supplementation of the low P diets restored the growth rate and FCR to levels that approached those of pigs fed the adequate P control diet. Daily phosphorus intakes of pigs receiving the P supplemented diets were 11.3 g on average. Pigs fed diets without added inorganic phosphates consumed 8.8 g P day-’ (P < 0.001) (Table 4). Dicalcium phosphate supplied P at 1.5 g kg-’ diet, contributing 20% of the total P supply in the control diets. No supplemental phosphorus was added in diets 5-8; all of the P supply was of plant origin supplying phytate-P at 3.0 g kg-’ diet. The proportions of phytate-P to total P were calculated to be 45% and 57% in diets l-4 and 5-8, respectively. The apparent digestibility of P in the diets with P supplement averaged 41% and did not differ significantly from those of P-unsupplemented diets, 39%, P > 0.05). The inorganic P addition which was 20% of total P supply was without any response, probably due to high phytic acid of RSM that interfered with absorption. In spite of the fact that soaking the diet for 3 h before feeding significantly enhanced P digestibility, it did not affect on any parameter of P usage measured. In contrast to this, the results of Kemme and Jongbloed (1993) indicated that soaking has no effect on P absorption. Phytase supplementation of the diet resulted in a significant improvement in phosphorus digestibility (P < 0.001) compared with that of the unsupplemented diet (45 vs. 36%). There was a significant interaction between P level and phytase supplementation. Added phytase in the non-soaked diet enhanced P absorption by 11% units whereas soaking the phytase treated diet additionally improved the absorption up to 15% units. The effect of soaking alone was as low as 4% units. The responses of P absorption to phytase addition in the barley-RSM diet generally were much lower than those found on maize-SBM or barley-SBM diets fed to growing pigs (Nisi, 1990; Simons et al., 1990; Jongbloed et al., 1992; Kemme and Jongbloed, 1993, Mroz et al., 1994, Nasi and Helander, 1994). The few absorption or availability values, which have been published for RSM, are rather low, 1 l-24% (Coffey and Cromwell, 1993, Larsen and Sandstrom, 1993). The high phytic acid contents 30 g kg-’ and other ANF of RSM may be involved in the low effect of treatments compared to other feed ingredients. The daily gain of pigs fed on canola diet was also unaffected by P additions (Coffey and Cromwell, 1993). Furthermore, Eeckhout and De Paepe (1992) proposed that the effect of microbial phytase is partly additive to the effect of vegetable phytase, and the low response may be associated with the low intrinsic phytase activity of the RSM diet. The present responses to phytase and soaking are supported by findings of Kemme and Jongbloed (1993), which showed that the added microbial phytase in the non-soaked diet significantly enhanced digestibility of P and Ca by 18 and 6% units, respectively. Soaking the diet without phytase had no significant effect on dP or dCa. Soaking the diet with phytase tended to have an enhancing effect on dP and dCa as compared with non-soaking and phytase (8 and 6% units). Han et al. (1987) reported that approximately
11.1 6.9 4.2 38.1 1.0 3.2 29.4 76.8 0.18
11.6 6.5 5.1 43.9 1.8 3.3 28.6 63.8 0.18
3
and retention
11.2 6.0 5.2 46.7 1.8 3.4 31.0 66.7 0.19 8.7 5.9 2.8 32.4 0.05 2.8 31.8 97.8 0.16
5 8.8 5.6 3.2 35.9 0.12 3.1 34.5 96.8 0.17
6
in pigs fed on experimental 4 8.8 5.2 3.6 42.3 0.16 3.4 40.2 95.3 0.20
7 8.9 4.7 4.2 46.9 0.39 3.8 42.8 91.8 0.21
8
diets fed to growing
pigs
0.14 0.19 0.19 1.4 0.102 0.19 I .6 1.6 0.010
SEM
** +** * *
NS 0
NS NS
*** ***
NS
***
***
NS
NS
*** *** 0 ***
NS
c3 *** *** ***
c2 NS ** * **
***
Cl
*** ** **
*
NS
0
NS
*
NS NS
NS NS NS ***
Cl’C3
NS NS NS
*
Cl’C2
Dl = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement +soaking D7 = no P supplementtphytase D8 = no P supplement+ phytase+soaking. SEM = standard error of mean. Cl = Phosphorus supplementation VS. no phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. Cl * C2 = interaction of phosphorus supplementation and soaking. C I * C3 = interaction of phosphorus supplementation and phytase and phytase addition.
11.5 7.3 4.2 37.1 1.1 3.2 28.2 76.0 0.18
2
absorption
P intake (g day-’ ) P in faeces (g day - ’) P absorption (g day- ’) _ of intake (%) Pinurine(gday-‘) P retention (g day- ’) -of intake (%I -of absorption (o/o) P (g kg -“.75 day- ‘)
apparent
1
excretion,
Diet
Table 4 Phosphorus
2 z
2
2
92
J. M. Niisi et nl. /Animal Feed Science Technology 56 (I 995) 83-98
38% of phytic acid in SBM and 43% in cotton seed meal was hydrolysed by pretreating the substrates with phytase produced by A.jicuum. Nisi and Helander (1994) in contrast to those above found that soaking pelleted phytase treated diets in warm water for 3 h had no effect. Lactose inclusion in the pig diet increased preceacal digestibility of P but total tract digestibility of P was unaffected (Ahlborn, 1993). Retention of P was enhanced due to lactose supplement in rats (Heijnen et al., 1993). Fermentation of lactose lowered the pH in the ileal lumen of rats and improved the solubility of mineral components thus stimulating the absorption (Heijnen et al., 1993). Phytase inclusion in a diet without P supplementation decreased by one third the amount of P excreted in faeces compared to the value without phytase addition to the diet. Phosphorus excretion in urine was manyfold in P supplemented diets compared with others (P < 0.001). The phytase supplemented diet produced a significantly higher (P < 0.001) urine P excretion compared to that produced without phytase addition. Signs of P deficiency appearing as hypophosphaturia and hypercalciuria (Pointillart, 1991) were found in pigs fed a P unsupplemented diet without phytase. The treatment with phytase alleviated the symptoms of deficiency. The total excretion of P was only one third lower in a phytase supplemented diet (5.5 g day-‘) compared with an inorganic P supplemented diet, in which the faecal and urinary excretion was 8.1 g day-‘. In previous experiments with maize-SBM and barley-SBM diets treated with phytase, the reductions have been much higher (Nisi, 1990; N’asi and Helander, 1994). The retention of P in pigs fed diets with inorganic P did not differ from that in pigs without P supplementation; however inorganic P supply from dicalcium phosphate was 1.5 g kg-’ diet and P deficiency was obvious in pigs fed an unsupplemented diet without phytase. Soaking the diets had no effect on the retention of P in spite of the increased absorption of P. Phytase supplement improved P retention significantly (P < 0.011, and the enhancement was on average 1.0 g day-’ (2.8 g vs. 3.8 g> corresponding to an increase of 36%. The retention of phosphorus was 29% in relation to P intake in the diets with inorganic P and 37% in diets without inorganic P supplementation. The observed availability of P in barley and RSM diets is in accordance with limited existing literature (Pointillart, 1988; Coffey and Cromwell, 1993; Larsen and Sandstriim, 1993; NPsi and Helander, 1994). Microbial phytase supplementation improved retention of phosphorus to the level of 36% and the difference was significant (P < 0.01) compared to the untreated non P-supplemented diet (31%). Retention of the absorbed P was very high (92%) in diets supplemented with phytase, but lower (6% units) relative to the untreated diets (P < 0.01). Calcium digestibility was lower in a P-supplemented diet (P < 0.01) compared to unsupplemented, but urinary Ca excretion was higher (P < 0.001). Soaking reduced Ca excretion in faeces, but was without any significant effect on Ca retention or utilization. Phytase treatment did not affected Ca digestibility but decreased urinary Ca excretion (P < 0.001). Pigs fed the diet with phytase had significantly (P < 0.01, P < 0.001) improved absolute Ca retention and in relation to Ca intake or absorption (Table 5). Nisi and Helander (1994) found only a marginal improvement in Ca absorption or retention due to phytase addition in a barley-SBM diet, whereas Ca digestibility was increased when phytase was added to a diet composed of tapioca and hominy feed or maize-SBM (Simons et al., 1990; Kemme and Jongbloed, 1993; Mroz et al., 1994) Also improve-
’)
day
’)
’)
0.33
0.32
0.33 0.34
6.2 41.3 92.3
5.9 38.1 92.4
5.7 38.2 93.5
5.9 38.4 93.3
44.7 0.5
41.2 0.5
40.9 0.4
41.2 0.4
15.0
4
8.3 6.7
15.5
3
0.32
5.7 35.1 77.2
16.1 a.1 1.4 45.5 1.7
5
0.3
5.3 35.0 76.8
1
1.6
15.4 8.4 7.0 45.6
6
in pigs fed on experimental
9.1 6.4
8.8 6.1
14.9
2
and retention
9.1 6.3
15.4
1
absorption
0.38
6.8 42.1 88.4
16.1 8.4 1.6 47.5 0.9
7
0.016
0.30 1.7 1.0
6.6 42.4 89.0 0.36
0.05 0.30 0.30 1.8 0.10
SEM
pigs
15.5 8.1 7.4 47.1 0.8
8
diets fed to growing
NS
NS NS NS
NS NS **r NS
* NS NS NS
***
c2
0 *** ** *+*
***
Cl
**
NS NS NS *** ** ** ***
*
c3
NS
NS NS NS NS NS NS NS
*
CI’C2
*
NS NS NS NS *** * * ***
CI’C3
DI = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement vs. no + soaking D7 = no P supplement + phytase D8 = no P supplement + phytase + soaking, SEM = standard error of mean. C I = Phosphorus supplementation phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. Cl * C2 = interaction of phosphorus supplementation and soaking. Cl * C3 = interaction of phosphorus supplementation and phytase addition.
Ca (g kg-‘.”
Ca retention (g g-’ ) -of intake (%I -of absorption (%)
-of intake (%I Ca in urine (g day-
’) ’)
apparent
Ca in faeces (g day Ca absorption (g day
Ca intake (g day
Diet
Table 5 Calcium excretion,
a \
9
4.2 2.9 1.3 30.3 0.5 0.75 18.0 59.5 43
1 4.2 3.0 1.2 29.3 0.5 0.74 17.9 59.3 42
3
and retention
4.1 2.8 1.2 29.8 0.5 0.73 18.2 61.2 42
2
apparent absorption
intake (g day- ’) in faeces (g day- ’) absorption (g day- ’) intake (%I in urine (g day- ’) retention (g day- ’) intake (8) absorption (%) (mg kg-‘.” day-‘)
excretion,
4.2 2.8 1.4 32.2 0.5 0.87 20.2 62.5 47
4 4.0 2.6 1.4 34.1 0.6 0.76 19.2 57.3 44
5 4.0 2.6 1.4 33.9 0.6 0.71 18.4 54.5 42
6
in pigs fed on experimental
4.1 2.7 1.4 34.9 0.6 0.85 20.9 60.0 47
7 4.0 2.5 1.5 36.6 0.6 0.84 20.9 57.2 47
8
diets fed to growing Cl * * * *** 0 ** ** NS NS NS NS
SEM 0.04 0.08 0.09 1.8 0.04 0.078 1.6 3.3 4
pigs
0 NS NS NS NS NS NS NS NS
c2
* NS NS NS NS NS NS NS NS
c3
Cl *c3 NS NS NS NS NS NS NS NS NS
Cl *c2 NS NS NS NS NS NS NS NS NS
Dl = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement tsoaking D7 = no P supplement +phytase D8 = no P supplement +phytase+ soaking. SEM = standard error of mean. Cl = Phosphorus supplementation vs. no phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. Cl * C2 = interaction of phosphorus supplementation and soaking. C I * C3 = interaction of phosphorus supplementation and phytase addition.
Mg Mg Mg -of Mg Mg -of -of Mg
Diet
Table 6 Magnesium
240 194 46 19.1 10 37 15.2 15.6 2.1
I
absorption
intake (mg day ’) in faeces (mg day- ’) absorption (mg day- ’) intake (%) in urine (mg day-‘) retention (mg day _ ’) intake (%) absorption (%o) (mg kg-‘.” day-‘)
apparent
232 182 51 21.7 10 41 17.6 76.1 2.3
2
and retention
241 188 53 22.1 11 42 17.3 55.1 2.3
3 234 177 57 23.9 11 46 19.4 81.8 2.5
4 240 170 69 28.2 10 59 23.7 83.7 3.2
5
in pigs fed on experimental
232 170 62 26.6 13 49 20.9 79.0 2.8
6 240 181 58 24.1 14 44 17.9 74.0 2.4
7
diets fed to growing
234 177 57 24.1 12 45 18.7 16.2 2.4
8
pigs
0.9 4.9 4.8 1.87 1.2 5.0 1.93 9.69 0.26
SEM NS ** ** ** * * * NS *
Cl *** 0 NS NS NS NS NS NS NS
C2 0 NS NS NS 0 NS NS NS NS
c3
NS NS NS NS NS NS NS NS NS
CI*C2
NS 0 * * NS * * NS *
Cl *c3
Dl = P supplement D2 = P supplement + soaking D3 = P supplement + phytase D4 = P supplement + phytase + soaking D5 = no P supplement D6 = no P supplement + soaking D7 = no P supplement + phytase D8 = no P supplement + phytase + soaking. SEM = standard error of mean. C I = Phosphorus supplementation vs. no phosphorus supplementation. C2 = Soaking vs. no soaking. C3 = Phytase addition vs. no phytase addition. C 1 * C2 = interaction of phosphorus supplementation and soaking. C 1 * C3 = interaction of phosphorus supplementation and phytase addition.
Zn Zn Zn -of Zn Zn -of -of Zn
Diet
Table 7 Zinc excretion,
96
J.M. Niisi et al. /Animal Feed Science Technology 56 (1995) 83-98
ments in Ca digestibility has been found in piglet fed maize diets with phytase (Pallauf et al., 1992a; Hoppe et al., 1993). Ca content in RSM is rather high compared to cereals, 7.0 vs 0.35 g kg-‘, and it contributed 20% of the total Ca supply in this study. The availability of Ca in RSM is reduced due to high phytic acid following rather low digestibility as reported by Larsen and Sandstrom (19931, and also, both Ca and P utilization have been decreased with time in pigs fed a phytase rich diet containing rapeseed (Pointillart, 1988). Another reason for the low Ca availability can be the lower P retention in diets without P and phytase additions. If the intake of Ca is adequate but that of P inadequate, the retention of Ca falls (Jongbloed, 1987). In the present study, the calculated Ca/P ratios were 1.33:1 and 1.78:l (P < 0.001) for diets l-4 and diets 5-8, respectively, while the optimum Ca/P ratio is from 1.2 to 1.3:1 (ARC, 1981). However, Mroz et al. (1994) suggested that Ca:total P ratios are not relevant indicators in practical diet formulation for pigs, but Ca:digestible P ratios should be used, and the ratios 2.8-3.3 were most optimal. P addition reduced the ratio Ca/dP from 4.72 to 3.27 (P < O.OOl), soaking from 4.17 to 3.8 1 (P < 0.001) and phytase addition from 4.46 to 3.52 (P < 0.001). As a result of the phytase treatment, the ratio approached quite an optimal level which is also seen in the improved utilization of P. It is well known that phytic acid has a high affinity to bi- and trivalent cations (Wise, 1983). Digestibility of magnesium was lower (P < 0.01) in a P-supplemented diet while urinary excretion was reduced (P < 0.01, Table 61, but there were also differences in Mg supply. Phytase treatment or soaking did not affect Mg absorption or utilization. In the barley-SBM diet, phytase supplement improved Mg digestibility and retention (N&i and Helander, 1994) in agreement with the results found in piglets when phytase was supplemented (Pallauf et al., 1992b). P supplementation increased faecal excretion of zinc but decreased urinary Zn excretion significantly. Soaking or phytase supplementation did not have any effect on the retention or utilization of Zn (Table 7). Phytic acid readily forms complexes with several essential minerals, such as calcium, iron, zinc and manganese, impairing their utilization by the animal (Nelson et al., 1971; Pallauf et al., 1992b). The results of the present study demonstrate that the addition of a microbial phytase or soaking with the whey barley-RSM diets enhance the absorption of vegetable phosphorus and essential minerals in the diet as Ca and Mg. An increasing proportion of the pigs’ requirement for P could be met by P in barley and RSM when phosphorus could be converted to an available form. Phosphorus content in pig manure is reduced following the improved plant-P utilization resulting from dietary phytase supplementation.
Acknotiledgements The authors are grateful to Ms. Mari Korkeaoja, Ms. Anneli Pakarinen and Mr. Jari Miettinen for technical assistance. The financial support to this study was received from Academy of Finland.
J.&f. Niisi et al. /Animal
Feed Science Technology
56 (1995) 83-98
95
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