Agricultural and Food Science in Finland, Vol. 10 (2001): 19–26. Vol. 10 (2001): 19–26. 19 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 19–26. © Agricultural and Food Science in Finland Manuscript received November 2000 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 19–26. Effect of two microbial phytase preparations on phosphorus utilisation in broilers fed maize-soybean meal based diets Jarmo Valaja, Sini Perttilä, Kirsi Partanen, Tuomo Kiiskinen Agricultural Research Centre of Finland, Animal Production Research, Animal Nutrition, FIN-31600 Jokioinen, Finland, e-mail: jarmo.valaja@mtt.fi Jari Piironen Röhm Enzyme Finland Ltd, FIN-05200 Rajamäki, Finland The present study was carried out to determine the effect of two microbial phytases, Aspergillus niger (FINASE® FP-500, 291 PU (phytase units)/g) and Trichoderma reesei phytase (FINASE® P, 5880 PU/g) on phosphorus (P) and calcium (Ca) utilisation and ileal P and Ca digestibility in broiler chickens fed diets based on maize and soybean meal. A total of 96 Ross broiler chickens housed four birds to a cage were used. Four dietary treatments consisted of a positive control supplemented with dicalcium phosphate (17 g/kg), a negative control without inorganic P, basal diet without inorganic P supplemented with Aspergillus niger phytase (2.6 g/kg) and basal diet without inorganic P supple- mented with Trichoderma reesei phytase (0.13 g/kg). Both phytases provided 750 PU/kg feed. P retention per unit intake was lowest and P excretion highest in birds fed the positive control diet with inorganic P (P<0.001). Both phytases clearly decreased P excretion (P<0.05) and increased P utilisa- tion (P<0.001) compared to the negative control treatment. No differences were found between As- pergillus and Trichoderma phytases in P retention. Phytase supplementations also increased apparent ileal digestibility of P compared to the negative control treatment (P<0.05). Phytase supplementation also increased Ca retention per unit intake (P<0.001). In conclusion, both microbial phytases were equally effective in improving utilisation of P in broiler chickens fed maize-soybean meal diets. Key words: Aspergillus niger, enzymes, digestibility, minerals, poultry, Trichoderma reesei Introduction In cereals and vegetable protein sources between half to three quarters of phosphorus (P) is bound to phytic acid (Eeckhout and De Paepe 1994) and has therefore, only limited availability in poultry (Nelson et al. 1968, Simons et al. 1990, Kiiski- nen et al. 1994). Phytic acid does not only limit the utilisation of plant P, but also complexes with various cations, e.g. calcium and protein, reduc- ing their availability (Ravindran et al. 1999). mailto:jarmo.valaja@mtt.fi 20 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Valaja, J. et al. Effect of two microbial phytases on P utilisation in broilers Because of growing concern about environ- mental pollution, attempts have been made to reduce P output from broiler production. It has been demonstrated that microbial phytase is ef- fective in releasing a significant proportion of the phytate P present in maize and soybean meal (Nelson et al. 1968, Simons et al. 1990). Micro- bial phytase has enhanced P utilisation of maize- soybean meal diets in broilers and reduced the amount of P in excreta (Simons et al. 1990, Broz et al. 1994, Kornegay et al. 1996). Phytase from Aspergillus niger and Trichoder- ma reesei has been equally effective in improv- ing the digestibility of P in maize and soybean meal diet in pigs (Näsi et al. 1999). Aspergillus niger phytase has been shown to improve P uti- lisation in broilers (Broz et al. 1994, Kiiskinen et al. 1994), but so far little is known about the efficacy of Trichoderma reesei phytase in broil- er diets. Therefore, the current study was car- ried out to determine the effects of two microbi- al phytases, Aspergillus niger (FINASE® FP- 500) and Trichoderma reesei (FINASE® P) on P and calcium (Ca) utilisation in broilers fed maize-soybean meal based diets. Material and methods Animals and experimental procedures A digestibility and balance experiment was con- ducted with 96 Ross broiler chickens between 21 and 37 days old. One-day old broiler chicks were sexed, marked with numbered wing marks and placed in 3-floor battery wire cages (size 47.5 x 56 x 40 cm), four birds (two males and two females) in each cage. Four cages were treat- ed as one experimental block, within which one cage was randomly allocated each treatment. Treatments were evaluated with six replicate cages. The experiment was conducted as a ran- domised block design with four experimental treatments: 1. maize-soybean meal diet supple- mented with dicalcium phosphate (positive con- trol), 2. maize-soybean meal diet without inor- ganic P supplementation (negative control), 3. maize-soybean meal diet without inorganic P supplemented with Aspergillus niger phytase (750 PU (phytase units)/kg feed) and 4. maize- soybean meal diet with inorganic P supplement- ed with Trichoderma reesei phytase (750 PU/kg feed). The duration of the trial was 17 days. Birds were fed for the first three weeks a standard start- er diet (200 g crude protein/kg diet) and experi- mental diets were introduced at age of 21 days. After a 7-day adaptation period, a total excreta collection was conducted during four days. Ex- creta of birds were collected from trays under cages once a day and placed into a freezer (–20°C) prior to analysis. A second adaptation period lasted five days. Before the collection of digesta at the age of 37 days, birds were fasted for 24 hours. After fasting birds had free access to feed for four hours one block at a time. Sam- ples of ileal content were taken from the birds after stunning and neck dislocation one block at a time, with a time interval between blocks of 45 minutes. The small intestine was removed and ileal digesta were sampled from distal half be- tween the Meckels diverticulum and ileo-caeco- colic junction. Samples of four birds from the same cage were pooled. Digesta samples were placed into a freezer (–20°C) immediately after collection. Animals were weighed at the begin- ning and at the end of the trial and at the begin- ning of the collection period and the second ad- aptation period. Feed intake of each cage was recorded during the collection period and after fasting. Temperature was maintained at +22°C and light was controlled according to Ross-broil- er breeder instructions (20-h light (5–10 lux) and 4 h dark cycle). Feed and water were available ad libitum. Experimental diets A maize-soybean meal based diet was formulat- ed to meet the nutrient requirements of broilers except for P (Tuori et al. 1995) (Table 1). A pos- 21 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 19–26. itive control diet was supplemented with dical- cium phosphate. Diets were formulated to con- tain equal amount of Ca. Phytase preparations were produced by two fungi of Aspergillus ni- ger (FINASE® FP-500, Röhm Enzyme Ltd, Ra- j a m ä k i , F i n l a n d ) a n d Tr i c h o d e r m a re e s e i (FINASE® P, Röhm Enzyme Ltd, Rajamäki, Fin- land). Phytases were derived from a mutant strain of Aspergillus niger or from a transgenic organ- ism of Trichoderma reesei (Piddington et al. 1993). Phytase activities were 291 PU/g and 5880 PU/g in Aspergillus niger and Trichoder- ma reesei phytases, respectively. A phytase unit (PU) is defined as the amount of enzyme activi- ty which liberates, under standard conditions (pH 5.0, 37°C), 1 nmol of inorganic phosphate from sodium phytate in one minute. Enzyme prepara- tions were premixed with barley starch and add- Table 1. Formulation and analysed chemical composition of experimental diets. Treatment 1 2 3 4 Dicalcium phosphate + – – – Phytase – – Aspergillus Trichoderma Ingredients, g/kg Soybean meal 350.0 350.0 350.0 350.0 Maize 601.7 607.7 605.1 607.6 Rapeseed oil 16.0 16.0 16.0 16.0 Limestone 6.0 17.0 17.0 17.0 Dicalcium phosphate 17.0 – – – Sodium chloride 3.0 3.0 3.0 3.0 Vitamin premix1 2.0 2.0 2.0 2.0 Trace element premix2 2.0 2.0 2.0 2.0 Cr-mordanted straw 1.5 1.5 1.5 1.5 Methionine 0.8 0.8 0.8 0.8 Phytase product3 – – 2.6 0.13 Analysed composition G/kg dry matter Dry matter, g/kg 899.7 900.5 900.2 900.9 Organic matter 947.4 952.9 953.4 952.6 Crude protein 228.2 227.3 228.0 229.2 Ether extract 69.7 68.1 67.7 68.9 Crude fibre 35.9 36.8 35.7 36.8 Ash 52.6 47.1 46.6 47.4 Lysine4 11.5 11.5 11.5 11.5 Methionine+cystine4 8.4 8.4 8.4 8.4 Calcium 8.3 8.0 8.3 8.2 Total phosphorus 7.7 4.8 4.6 4.6 Phytate P4 2.8 2.9 2.9 2.9 Ca:P-ratio 1.1 1.7 1.8 1.8 1 The vitamin premix supplied the following per kg diet: 1.4 mg calcium, 5.9 mg phosphorus, 12500 IU vitamin A, 3100 IU vitamin D 3 , 40.0 mg vitamin E, 36.0 mg tocopherol, 5.0 mg phylloquinone, 3.0 mg thiamin, 6.0 mg riboflavin, 4.0 mg pyridoxine, 0.03 mg cyanocobalamin, 0.30 mg biotin, 1.0 mg folic acid, 40.0 mg niacin, 15.0 mg pantothenic acid and 0.61 mg antioxidants. 2 The trace mineral premix supplied the following per kg diet: 0.64 g calcium, 29.2 mg iron, 5.1 mg copper, 50.3 mg magnesium, 65.1 mg zinc, 0.5 mg iodine and 0.2 mg selenium. 3 Provided 750 PU/kg diet. 4 Calculated from table values. 22 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Valaja, J. et al. Effect of two microbial phytases on P utilisation in broilers ed at 0.26 and 0.013% inclusion rates to treat- ments 3 and 4, respectively. Chromium (Cr)- mordanted straw (1.5 g/kg) was used as an indi- gestible marker. Diets were mixed and cold- pressed (temperature +55–60°C) as 4 mm pel- lets (Amandus Kahl Laborpresse L175) in the feed mill of the Agricultural Research Cen- tre. Laboratory analysis Feed, excreta and ileal digesta samples were freeze-dried and ground through a 1-mm mesh before analysis. Proximate analysis was per- formed according to standard methods (AOAC 1990). Nitrogen (N) from excreta was deter- mined in fresh samples using the Kjeldahl-tech- nique and that from freeze-dried feeds and ileal digesta samples by the Dumas method using a Leco FP 428 nitrogen analyzer (Leco Corp., St. Joseph, Missouri, USA). Calcium and Cr were determined by atomic absorption spectrometry and total P was assayed after dry ashing, using the colorimetric vanadate-molybdate based pro- cedure of Tayssky and Shorr (1953). Apparent ileal digestibilities were calculated using Cr as an indigestible marker. Determination of P, Ca and nitrogen retention was calculated based on total excreta collection. Statistical analysis Experimental data were subjected to the analy- sis of variance using the GLM procedure of SAS (SAS 1990) according to the following model: y ijkl =µ + b j + d k + e ijkl where y ijkl is the depend- ent variable, µ is the overall mean, b j is block effect, d k is diet effect and e ijkl is a normally dis- tributed random variable. Three single degree of freedom orthogonal contrasts were formed to test differences between dietary treatments: C1: Ba- sal diet with dicalcium phosphate (positive con- trol) vs. other treatments; C2: Basal diet with- out additional phosphate (negative control) vs. diets with microbial phytase supplementation; C3: Aspergillus niger phytase vs. Trichoderma reesei phytase. Results and discussion Two observations were removed from the total excreta collection period due to the presence of blood stains in excreta. Otherwise the birds suc- cessfully completed the experiment. Average body weight of the birds was 720 (SE 18.1) and 1499 (SE 41.3) g on days 21 and 37, respective- ly. During the total excreta collection period, birds on the positive control diet ate more feed than birds fed other treatments (P<0.05) (Table 2). This is likely to be due to the dilution of the metabolisable energy content of the diet as a re- sult of dicalcium phosphate supplementation. Dry matter intake during the 4 hour period be- fore ileal digestibility assay was 76, 53, 49 and 54 g for treatments 1, 2, 3 and 4, respectively. Consistent with the total excreta collection pe- riod, dry matter intake of birds fed the diet con- taining dicalcium phosphate was the highest (P<0.001). Phosphorus intake and excretion was high- e s t f o r b i r d s f e d t h e p o s i t i v e c o n t r o l d i e t (P<0.001) (Table 2). Phosphorus retention per unit intake clearly increased from 38.5% to 51.1% with phytase supplementation (P<0.001). Phytase supplementation also decreased P excre- tion of broilers compared to the negative con- trol diet without inorganic P (P<0.05). This is in agreement with previous results of Kornegay et al. (1996), Yi et al. (1996) and Broz et al. (1994) which demonstrated that Aspergillus phytase lin- early increased P retention of maize-soybean meal diets (from 44.3 to 62.6%). Kiiskinen et al. (1994) also observed that Aspegillus niger phytase increased the retention of P from 50.9% to 60.0% in diets based on wheat, barley and soybean meal. Leske and Coon (1999) deter- mined P retention responses of different feed- stuffs to phytase addition and observed that P retention of soybean meal and maize was in- 23 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 19–26. creased from 27.0 to 58.0% and 34.8 to 40.9%, respectively. It would appear that the effect of phytase supplementation is partly feed ingredi- ent dependent, being higher in feeds of high phytate P and low intrinsic phytase content, such as maize and soybean meal. Utilisation of phytate P is also influenced by the dietary Ca:total P- ratio as increases in this ratio has linearly de- creased the effect of phytase on P retention (Schöner et al. 1993, Sebastian et al. 1996a, Qian et al. 1997). According to Sebastian et al. (1996a) optimal responses to microbial phytase are achieved at low dietary Ca concentrations (0.6%). In the present study, dietary Ca content was above the recommended optimal levels, which may have influenced the magnitude of observed responses. However, the Ca contents were similar between treatments and had no in- fluence on the relative differences between phytase supplements. Microbial phytase supplementations also in- creased Ca and ash retention per unit intake (P<0.001) and decreased Ca excretion (P<0.05) compared to the negative control, a finding in agreement with the studies of Schöner et al. (1993), Kiiskinen et al. (1994), Yi et al. (1996) and Kornegay et al. (1996). Improvement in Ca utilisation was expected, since phytase liberates Ca from the Ca-phytate complex and the availa- bility of P increases with availability of Ca (Se- bastian et al. 1996b). Nitrogen retention per unit intake also slight- ly increased with phytase supplementation (P<0.05). Similarly, N retention of maize-soy- bean meal diet has also been shown to be im- proved by increasing phytase addition in previ- ous studies (Schöner et al. 1993, Sebastian et al. 1996b, Yi et al. 1996). Phytase supplementation may also release phytate-bound protein and ami- no acids for absorption and utilisation and there- by improve N retention (Ravindran et al. 1999). Both Trichoderma and Aspergillus phytases improved (P<0.05) apparent ileal digestibility of P in maize-soybean meal diets compared to the negative control treatment (Table 3). There is limited information available concerning the ef- Table 2. Effect of microbial phytases on nutrient retention of nutrients of broilers fed maize-soybean meal based diets. Treatment 1 2 3 4 SEM Significance Dicalcium phosphate + – – – C1 C2 C3 Phytase – – Aspergillus Trichoderma n 6 5 6 5 Dry matter intake, g/day 103.8 89.7 93.7 89.2 4.95 * ns ns Dry matter excretion, g/day 29.2 25.5 25.5 24.2 1.51 * ns ns N intake, g/day 3.79 3.26 3.42 3.27 0.180 * ns ns N excretion, g/day 1.69 1.55 1.57 1.44 0.085 ns ns ns N retention per intake, % 55.5 52.8 53.9 55.9 0.69 ns * o Ash intake, g/day 0.54 0.42 0.44 0.42 0.023 *** ns ns Ash excretion, g/day 0.41 0.35 0.32 0.31 0.018 ** ns ns Ash retention per intake, % 25.7 17.5 27.3 26.9 1.01 ns *** ns Ca intake, g/day 0.86 0.72 0.77 0.73 0.040 * ns ns Ca excretion, g/day 0.51 0.55 0.44 0.43 0.028 ns * ns Ca retention per intake, % 41.0 24.0 42.6 40.8 1.31 ** *** ns P intake, g/day 0.80 0.42 0.43 0.41 0.026 *** ns ns P excretion, g/day 0.55 0.26 0.21 0.20 0.016 *** * ns P retention per intake, % 31.4 38.5 51.2 51.1 1.19 *** *** ns Significance: ns non-significant, o P<0.10, * P<0.05, ** P<0.01 and *** P<0.001. Contrasts: C1: Basal diet supplemented with dicalciumphosphate (1) vs. other treatments (2, 3 and 4), C2: basal diet without inorganic P (2) vs. phytase supplementations (3 and 4), C3: Aspergillus phytase (3) vs. Trichoderma phytase (4). SEM=standard error of means. 24 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Valaja, J. et al. Effect of two microbial phytases on P utilisation in broilers fects of microbial phytase on apparent ileal P digestibility. In the study of Ravindran et al. (2000) phytase supplementation increased appar- ent ileal P digestibility of wheat-sorgum-soybean meal diet up to 30.3%-units, the improvement being clearly greater than that in the current study. However, the apparent ileal P digestibili- ty of control diets was lower than that in our study (42.2 vs. 56.3%), which probably arised due to differences in basal diet ingredients. In pigs, phytase supplementation effectively en- hanced apparent ileal P digestibility in maize and soybean meal based diets (Jongbloed et al. 1992, Mroz et al. 1994). Apparent ileal digestibility of Ca was lowest for the positive control diet that was supplemented with inorganic P (P<0.001). In contrast to nutrient retention, phytase supple- mentations decreased apparent ileal digestibili- ty of Ca relative to the negative control treat- ment (P<0.01). Apparent ileal digestibility of dry matter ( P < 0 . 0 0 1 ) , o r g a n i c m a t t e r ( P < 0 . 0 0 1 ) , N (P<0.001) and ash (P<0.01) were lower for the positive control diet compared to other treat- ments. Phytase supplementation had no effect on the apparent ileal digestibility of organic mat- ter, N or ash. The current findings are consistent with previous studies demonstrating that phytase has no effect (Sebastian et al. 1997) or elicits only minor improvements (Namkung and Lee- son 1999) on apparent ileal N digestibility of maize-soybean meal based diets. In contrast, Ravindran et al. (1999) observed that phytase supplementation improved apparent ileal digest- ibility of N by 3.1 and 4.8%-units in maize and soybean meal, respectively, and the apparent ileal N and amino acid digestibility responses to phytase appeared to be negatively related to the inherent N digestibility of dietary ingredient. Furthermore, phytase additions improved appar- ent ileal N digestibility of wheat-sorgum-soy- bean meal diets between 1.6 and 4.7%-units (Ravindran et al. 2000). Both Aspergillus niger and Trichoderma ree- sei phytases were equally effective in improv- ing P and Ca utilisation and decreasing P excre- tion (Table 2). No differences in apparent ileal digestibity of P were indentified between the two phytase sources (Table 3). Apparent ileal digest- ibility of Ca (P<0.001), N (P<0.05) and ash (P<0.05) were lower for the Trichoderma than the Aspergillus phytase supplemented diet. How- ever, N retention per unit intake tended (P<0.10) to be higher for diets supplemented with Tri- choderma than Aspergillus phytases. In other respect, no differences were observed between Table 3. Effect of microbial phytases supplementations on apparent ileal nutrient digestibility in broilers fed maize-soybean meal based diet. Treatment 1 2 3 4 SEM Significance Dicalcium phosphate + – – – C1 C2 C3 Phytase – – Aspergillus Trichoderma n 6 6 6 6 Dry matter 78.7 82.2 82.4 81.4 0.40 *** ns o Organic matter 80.1 83.3 83.5 82.6 0.40 *** ns ns Ash 52.8 59.2 60.2 56.5 1.11 ** ns * Nitrogen 75.8 79.5 79.9 78.6 0.45 *** ns * Calcium 45.2 59.7 57.5 46.6 1.74 *** ** *** Phosphorus 59.8 56.3 63.7 59.5 1.77 ns * ns Significance: ns non-significant, o P<0.10, * P<0.05, ** P<0.01 and *** P<0.001. Contrasts: C1: Basal diet supplemented with dicalciumphosphate (1) vs. other treatments (2, 3 and 4), C2: basal diet without inorganic P (2) vs. phytase supplementations (3 and 4), C3: Aspergillus phytase (3) vs. Trichoderma phytase (4). SEM=standard error of means. 25 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 19–26. the two phytases. Phytase supplementations from various sources have generally been equally ef- fective in enhancing P utilisation (Kiiskinen et al. 1994, Broz et al. 1994, Kornegay et al. 1996, Yi et al. 1996). Responses to phytase supple- ments are dose-dependent, since the release of P per unit phytase decreases as the amount of phytase per unit diet increases (Kornegay et al. 1996). Nelson et al. (1968) reported that differ- ent cultures of Aspergillus derived phytases had variable effects on P utilisation, with the high- est response with Aspergillus niger. However, absolute phytase activities of different supple- ments were not determined. Use of both As- pergillus niger and Trichoderma reesei phytase supplementations of maize-soybean meal based diets in pigs have been shown to improve simi- larly phytate-P availability (Näsi et al. 1999). 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SELOSTUS Fytaasientsyymilisäyksen vaikutus fosforin hyväksikäyttöön maissi-soijarouhepohjaisessa broilerrehussa Jarmo Valaja, Sini Perttilä, Kirsi Partanen, Tuomo Kiiskinen ja Jari Piironen Maatalouden tutkimuskeskus ja Röhm Enzyme Finland Oy Sulavuus- ja tasekokeessa selvitettiin kahden erilai- sen fytaasientsyymilisäyksen vaikutuksia maissi-soi- jarouherehuseosta saaneiden broilereiden fosforin hyväksikäyttöön ja eritykseen. Tutkittavat fytaasient- syymivalmisteet, FINASE® FP-500 ja FINASE® P, oli tuotettu kahdella erilaisella mikrobikannalla, Asper- gillus nigerillä tai Trichoderma reeseillä. Kokeessa oli 96 broilerpoikasta, joiden ikä kokeen alussa oli 21 ja lopussa 37 vuorokautta. Broilereita pidettiin kolmikerroksisessa häkkipatterissa neljän eläimen häkeissä. Rakeistetun koerehun pääasiallisina raaka- aineina olivat maissi ja soijarouhe. Koekäsittelyjä oli neljä: positiivinen vertailu, jossa dikalsiumfosfaatti- täydennys, negatiivinen vertailu ilman epäorgaanis- ta fosforitäydennystä sekä kaksi rehua ilman epäor- gaanista fosforia, joihin oli lisätty joko Aspergillus tai Trichoderma pohjaista fytaasientsyymiä. Molemmat fytaasientsyymilisäykset paransivat selvästi maissi-soijarouhe-rehun fosforin käyttökel- poisuutta ja vähensivät ulosteissa erittyvän fosforin määrää. Kasvifosforin hyväksikäyttö parani 38,5 %:sta 51,1%:iin verrattuna rehuun, joka ei sisältänyt epäorgaanista fosforia tai fytaasientsyymiä. Myös fosforin ohutsuolisulavuus parantui fytaasientsyymin vaikutuksesta. Sekä Aspergillus että Trichoderma fy- taasin vaikutukset fosforin hyväksikäyttöön olivat samanlaiset. Fytaasientsyymi paransi myös rehun kal- siumin hyväksikäyttöä, mutta vaikutukset muiden ra- vintoaineiden hyväksikäyttöön tai sulavuuteen olivat vähäisiä. Title Introduction Material and methods Results and discussion References SELOSTUS