DOI: 10.1111/jpn.12341
ORIGINAL ARTICLE
Effect of super dosing of phytase on growth performance, ileal digestibility and bone characteristics in broilers fed corn–soya-based diets M. Manobhavan, A. V. Elangovan, M. Sridhar, D. Shet, S. Ajith, D. T. Pal and N. K. S. Gowda National Institute of Animal Nutrition and Physiology, Bangalore, India
Summary A feeding trial was designed to assess the effect of super dosing of phytase in corn–soya-based diets of broiler chicken. One hundred and sixty-eight day-old broilers were selected and randomly allocated to four dietary treatment groups, with 6 replicates having 7 chicks per treatment group. Two-phased diets were used. The starter and finisher diet was fed from 0 to 3 weeks and 4 to 5 weeks of age respectively. The dietary treatments were consisted of normal phosphorus (NP) group without any phytase enzyme (4.5 g/kg available/non-phytin phosphorus (P) during starter and 4.0 g/kg during finisher phase), three low-phosphorus (LP) groups (3.2 g/kg available/non-phytin P during starter and 2.8 g/kg during finisher phase) supplemented with phytase at 500, 2500, 5000 FTU/kg diet, respectively, to full fill their phosphorus requirements. The results showed that super doses of phytase (at 2500 FTU and 5000 FTU/kg) on low-phosphorus diet improved feed intake, body weight gain, ileal digestibility (serine, aspartic acid, calcium, phosphorus), blood P levels and bone minerals such as calcium (Ca), P, magnesium (Mg) and zinc (Zn) content. It could be concluded that super doses of phytase in low-phosphorus diet were beneficial than the normal standard dose (at 500 FTU/kg) of phytase in diet of broiler chicken. Keywords bone, broiler, nutrient utilization, phosphorus, phytase Correspondence A. V. Elangovan, National Institute of Animal Nutrition and Physiology, Bangalore, India. Tel: +91-9341380858; Fax: +91-80-25711420; Emails: [email protected]; [email protected] Received: 11 December 2014; accepted: 31 March 2015
Introduction Phosphorus (P) is the most expensive essential mineral for poultry. In plant-based diets, two-third of P is bound to phytate and not available to poultry. The presence of phytate can result in reduced efficiency of nutrient utilization increasing the cost of feeding and lead to environmental pollution. The best way to improve phosphorus utilization is with judicious use of natural resources by phytase enzyme supplementation in the diet of monogastrics. Phytase is used extensively and the generally recommended dose of phytase in poultry is 500 FTU/ kg diet (Selle and Ravindran, 2007; Cowieson et al., 2009; Pirgozliev et al., 2012; Lalpanmawia et al., 2014). Shirley and Edwards (2003) observed a quadratic increase in phytate P disappearance of almost 95% with increasing phytase dose to 12 000 FTU/ kg in corn-based diets. Further, the use of higher doses of phytase has been gaining interest not only due to more P release but also leaving less residual phytate and generation of myo-inositol with
vitamin-like/lipotropic effects (Shirley and Edwards, 2003). The use of super doses of phytase (>1000 FTU/kg of diet) has been shown to improve nutrient availability in poultry diets (Cowieson et al., 2006). The high doses of phytase may result in more liberated phosphate or restoration of P/Ca proportionate release with less residual phytate in the gut (Cowieson et al., 2011). Phytate forms complexes with multivalent cations to produce insoluble salts, this Ca–phytate complex is the weakest, and it has been reported to be the most beneficial by dietary phytase supplementation (Angel and Applegate, 2001). Chung et al. (2013) reported that the impact of phytase on the availability of minerals other than P is not definite. It is well established that available P requirement is 0.45 and 0.40% during starter and finisher phase. Hence, low-P (available P, 0.30%) group without phytase supplementation was not taken as reference or negative control. Moreover, our study was to compare the advantage of conventionally fed 500 FTU over super dosing under practical farming condition.
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Further, it may be difficult to completely replace inorganic P source from diet as the phytase supplementation also depends on the phytate content of the diet. This study was conducted to test the effect of super dosing of phytase on growth performance, ileal digestibility of nutrients and bone characteristics in broiler chicken. Materials and methods Experimental design
The animal care and use protocol (No. 5/2012) was reviewed and approved by the Institutional Animal Ethics committee of National Institute of Animal Nutrition and Physiology, Bangalore, India. The experiment was conducted in a completely randomized design. A total of one hundred sixty-eight, unsexed, day-old Cobb broiler chicks, vaccinated against Newcastle disease were obtained from commercial hatchery (Venkateswara hatchery). The chicks with a mean body weight of 45.8 g 1.46 (SD) were distributed into four treatments; each treatment had six replicates with seven chicks in each replicate. The chicks were housed in battery cages (72 cm wide 9 48 cm long 9 38 cm high) as groups in random manner, fitted with heating arrangements, feeders, waterer and dropping trays, with 24 h light and proper air ventilation, and reared under standard managemental conditions. The temperature inside the cage was maintained at 33 °C on day 1 and gradually reduced 24–25 °C by the end of the third week and maintained. The feed and fresh drinking water were provided ad libitum during the entire experimental period. Experimental diets
The dietary treatments consisted of one normal phosphorus (NP) diet, without any phytase enzyme (0.45% available P during starter and 0.40% during finisher phase), and three low-phosphorus diets (0.32% available P during starter and 0.28% during finisher phase) supplemented with phytase (commercial phytase, Finase EC from AB Vista, Pune, India, 5000 FTU/g) at 500, 2500 and 5000 FTU/kg diet designated as LP 500, LP 2500 and LP 5000 respectively. Ingredient and nutrient composition of experimental diets are given in Table 1. The diets were formulated based on Nutrient Requirement of Poultry published by ICAR (2013) using Excel-based feed formulation. The phytate phosphorus content of maize and soya bean meal was estimated by the method described by Haug and Lantzsch (1983). The Ca and P in feed 94
were analysed by inductively coupled plasma-optical emission spectroscopy (ICP-OES) using a Perkin Elmer instrument. The amino acid content of feed and digesta was analysed at Amino Lab, Evonik Industries (Nordic European Centre, Singapore). Measurements and laboratory analysis
Body weight gain and feed intake (FI) were recorded at weekly intervals. The feed conversion ratio (FCR) was calculated. Prior to sacrificing, blood samples of birds (two birds/replicate) were collected from jugular vein into a sterile centrifuge tube containing heparin (20 IU/ml). The blood was centrifuged at 1550 g for 10 min, and plasma was separated and stored at 20◦C for estimation of Ca and phosphorous by commercial diagnostic kits (Span diagnostics, Gujarat, India). At the end of 35 days, four birds were selected randomly per pen and sacrificed by cervical Table 1 Ingredient and nutrient composition of experimental diets during starter (0–3 week) and finisher (4–5 week) phase Starter (0–3 week)
Finisher (4–5 week)
NP
LP
NP
LP
584.2 360 20 16 10 0 3.5 2.5 1.3 2.5
612 325 25 10 15 5 3.5 1 1 2.5
612.5 325 25 17 7.5 5 3.5 1 1 2.5
12.6 223 13.7 4.9 10.7 6.30 3.12 3.18
12.8 193 11.3 4.3 10.1 6.96 2.98 3.97
12.8 199 11.3 4.3 10.3 5.68 2.98 2.69
Ingredient composition (g/kg) Corn 583.4 Soya bean meal (CP, 46.1%) 359.3 Sunflower oil 20 Limestone 10 Di calcium phosphate 17.5 Celite 0 Salt 3.5 Lysine 2.5 Methionine 1.3 Vitamin mineral premix* 2.5 Determined nutrient composition (g/kg) ME (MJ/kg)† 12.6 Crude protein 222 Lysine 13.7 Methionine 4.9 Calcium 10.9 Total phosphorus 7.57 Phytate phosphorus 3.12 Available phosphorus† 4.45
NP, normal phosphorus; LP, low phosphorus. *Trace mineral premix, 1 g/kg; Vit. Premix, 1 g/kg and choline 0.5 g/kg. Trace mineral premix supplied mg/kg diet: Mg, 300; Mn, 55; I, 0.4; Fe, 56; Zn, 30; Cu, 4. The vitamin premix supplied per kg diet: Vit. A, 8250 IU; Vit. D3, 1200 ICU; Vit. K, 1 mg; Vit. E, 40 IU; Vit. B1, 2 mg; Vit. B2 4 mg; Vit. B12, 10 mcg; niacin, 60 mg; pantothenic acid, 10 mg; choline, 500 mg. †Calculated.
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dislocation for evaluation of ileal digestibility and bone minerals. After sacrificing the birds, the contents of the ileum from Meckel’s diverticulum to ileo-caecal junction were pooled within a pen, collected and frozen immediately (20 °C). The digesta samples were freeze-dried, ground and stored in an air tight container for estimation of crude protein (AOAC, 1990), amino acids (Amino Lab, Evonik Industries), acid insoluble ash (AIA) (AOAC, 1990) and minerals (ICP). The P measured in the ileal digesta was used for calculating the total P in the excreta/faeces. The apparent ileal digestibility coefficient (AIDC) of the nutrients was calculated from the equation: AIDC ¼1 ½ðileal nutrient=ileal AIAÞ = ðdiet nutrient=diet AIAÞ: The left tibia bones (two birds per replicate) were collected after removing the muscles and adhering structures. The length and width of the tibia bone were measured with vernier callipers. Each tibia was defatted for 16 h in petroleum spirit (boiling point 60– 80 °C) using Soxhlet apparatus, dried and weighed before ashing. The ash samples were digested with dilute hydrochloric acid (1:2), and the mineral extract was used for estimation of minerals by inductively coupled plasma-optical emission spectroscopy (ICPOES) using a Perkin Elmer instrument (Sun et al., 2000). Statistical analysis
The data were subjected to one-way analysis of variance (ANOVA) for completely randomized design and tested for significance between the dietary treatments means employing Tukey’s HSD post hoc test (SPSS, 2010 Version 18.0).
Results Growth performance
Chicks fed the LP 2500 and LP 5000 ate more and gained more (p < 0.001) than chicks fed the LP 500 or NP during starter, finisher and overall phase (Table 2). There was no difference in body weight gains of chicks fed NP and LP 500 during whole experimental period. The feed intake followed the similar pattern as that of body weight gain. The feed conversion ratio was nonsignificant (p > 0.05) during the experimental period. Apparent ileal digestibility
There was no (p > 0.05) effect of diets on AIDC of crude protein and amino acids except serine and aspartic acid (Table 3). Broilers fed LP 2500 had higher AIDC for serine (p < 0.05) and aspartic acid (p < 0.05). Ca digestibility was higher (p < 0.01) in broilers fed LP 500 or LP 5000 compared to broilers fed NP, but this was not different than broilers fed LP 2500 (Table 4). P digestibility was higher (p < 0.001) in broilers fed LP 2500 in comparison with broilers fed NP. The AIDC of Mg, Mn and Zn were non-significant (p > 0.05) among the different treatment groups. Plasma minerals
Plasma Ca content was higher (p < 0.001) in broilers fed the NP or LP 500 compared with broilers fed LP 2500 or LP 5000 (Table 5). Plasma P content was higher (p < 0.001) in broilers fed LP 5000 compared with broilers fed all other diets (Table 5). Bone morphometry and mineralization
The bone weight was higher (p < 0.01) in broilers fed LP 2500 compared with broilers fed all other diets
Table 2 Growth performance of broiler chicken
Body weight gain (g/bird)
Feed conversion ratio, Feed intake (g/bird)/Body weight gain (g/bird)
Feed intake (g/bird)
Groups
0–3 week
4–5 week
0–5 week
0–3 week
4–5 week
0–5 week
0–3 week
4–5 week
0–5 week
NP LP 500 LP 2500 LP 5000 SEM Significance (p value)
717b 725b 798a 825a 11.0 0.001
952b 917b 1007a 1013a 11.8 0.003
1669b 1642b 1805a 1838a 20.6 0.001
984b 1024b 1104a 1177a 17.8 0.001
1713b 1610b 1803a 1850a 24.1 0.001
2697b 2634b 2907a 3027a 37.7 0.001
1.37 1.41 1.38 1.43 0.010 0.178
1.80 1.76 1.79 1.83 0.011 0.326
1.62 1.61 1.61 1.65 0.008 0.327
SEM: standard error of means; NP: normal phosphorus; LP 500: low phosphorus + phytase at 500 FTU/kg; LP 2500: low phosphorus + phytase at 2500 FTU/kg; LP 5000: low phosphorus + phytase at 5000 FTU/kg. Means in the same column bearing different superscript differ significantly.
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Table 3 Apparent ileal digestibility coefficient (AIDC) of crude protein and amino acids at 5 week of age Groups Amino acids
NP
LP 500
LP 2500
LP 5000
SEM
Significance (p value)
Crude protein Cystine Serine Proline Alanine Aspartic acid Glutamic acid Methionine Lysine Threonine Arginine Isoleucine Leucine Valine Histidine Phenylalanine Glycine
0.824 0.804 0.867ab 0.911 0.853 0.88ab 0.913 0.914 0.913 0.814 0.902 0.876 0.893 0.857 0.886 0.845 0.819
0.813 0.783 0.852b 0.899 0.837 0.865b 0.896 0.911 0.907 0.808 0.886 0.865 0.875 0.847 0.861 0.837 0.813
0.823 0.823 0.89a 0.915 0.861 0.901a 0.928 0.932 0.931 0.831 0.906 0.887 0.903 0.866 0.887 0.845 0.825
0.815 0.818 0.877ab 0.914 0.831 0.888ab 0.916 0.916 0.91 0.817 0.905 0.879 0.893 0.856 0.879 0.844 0.814
0.0049 0.0061 0.0053 0.0032 0.0055 0.0048 0.0048 0.0035 0.0036 0.0041 0.0035 0.0038 0.0044 0.0035 0.0044 0.0033 0.0033
0.8747 0.0670 0.0324 0.2589 0.2084 0.0193 0.1046 0.1524 0.0619 0.2623 0.1636 0.2576 0.1483 0.3905 0.1247 0.8417 0.6617
SEM: standard error of means; NP: normal phosphorus; LP 500: low phosphorus + phytase at 500 FTU/kg; LP 2500: low phosphorus + phytase at 2500 FTU/kg; LP 5000: low phosphorus + phytase at 5000 FTU/kg. Means in the same row bearing different superscript differ significantly.
Table 4 Apparent ileal digestibility coefficient (AIDC) of minerals at 5 week of age Groups Minerals
NP
LP 500
LP 2500
LP 5000
SEM
Significance (p value)
Ca P Mg Mn Zn
0.574b 0.464c 0.272 0.471 0.455
0.635a 0.518b 0.275 0.511 0.467
0.602ab 0.557a 0.281 0.542 0.516
0.626a 0.533ab 0.245 0.511 0.457
0.0073 0.0079 0.0242 0.0177 0.0195
0.006 0.001 0.961 0.599 0.082
Means in the same row bearing different superscript differ significantly.
(Table 6). The bone length, proximal epiphysis width, diaphysis and distal epiphysis width were higher (p < 0.001) in broilers fed LP 2500 and LP 5000 compared to broilers fed NP and LP 500. The ash, Ca, P, Mg and Zn content of bone were higher (p < 0.001) in broilers fed LP 2500 and LP 5000 compared to broilers fed NP and LP 500 (Table 7). The bone Fe content was higher (p < 0.05) in LP 500. The Mn content of bone showed no significant (p > 0.05) difference among the treatment groups. Phosphorus intake and excretion
The total P intake was higher (p < 0.001) in broilers fed NP followed by LP 5000, LP 2500 and lower in LP 96
Table 5 Plasma Calcium and Phosphorus content of broiler chicken at 5 week of age
Groups
Calcium (mg/dl)
Phosphorus (mg/dl)
NP LP 500 LP 2500 LP 5000 SEM Significance (p value)
10.3a 11.2a 9.09b 8.94b 0.268 0.001
4.39b 4.06b 4.88b 5.35a 0.236 0.001
Means in the same column bearing different superscript differ significantly.
500. The P excretion g/kg feed intake differed significantly (p < 0.001). It was higher in broilers fed NP followed by LP 500, LP 5000, and least excretion was noticed in broilers fed LP 2500 (Table 8). Discussion It is well documented that the phytase supplementation in low-P diet increases growth performance in broiler chicken (Selle and Ravindran, 2007). Shirley and Edwards (2003) observed that broilers consuming a P-deficient corn–soya bean meal diet can achieve maximum performance with phytase supplementation of 12 000 U/kg in diet and also suggested that
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Table 6 Bone morphometry (left tibia) of broiler chicken at 5 week of age
Groups NP LP 500 LP 2500 LP 5000 SEM Significance (p value)
Bone weight (g/kg live weight 3.19ab 3.08b 3.36a 3.33ab 0.029 0.01
Bone length (cm)
Bone width (cm) proximal epiphysis
Bone width (cm) diaphysis
Bone width (cm) distal epiphysis
8.53b 8.35b 8.89a 9.00a 0.053 0.001
2.06b 2.06b 2.26a 2.27a 0.019 0.001
0.830b 0.833b 0.876a 0.892a 0.009 0.021
1.65b 1.67b 1.80a 1.78a 0.014 0.001
Means in the same column bearing different superscript differ significantly.
phytase supplementation levels within the poultry industry need to be re-evaluated. Onyango et al. (2005) reported increase in body weight gain of 555 and 569 g/bird by supplementing phytase at 500 and 1000 FTU/kg, respectively, in low-P diet, which was comparable with normal P diet (568 g/bird) and significantly higher than low-P group without phytase (476 g/bird). Cowieson et al. (2006) reported improvements in body weight gain (15.47–17.59 g/b/ day) as phytase supplementation was increased in logarithmic doses from 150 to 24 000 FTU/kg in low-P diet and was higher than normal P diet (16.07 g/b/ day). Pirgozliev et al. (2008) reported increase in feed intake as the level of phytase increased from 250, 500 and 2500 FTU/kg which in turn increased body weight gain 39.6, 41.1 and 44.0 g/b/day accordingly. The same authors (Pirgozliev et al., 2011), on phytase supplementation of 12 500 in low-P diet, observed higher weight gain (33.1 g/b/day) than normal P (30.8 g/b/day) and low-P diet with phytase 500 FTU/ kg (28.1 g/b/day). Chung et al. (2013) reported that there was no effect (p > 0.05) on feed intake and weight gain of broiler chicken fed low-P diet with
Table 8 Total P intake and excretion of broiler chicken
Groups
NP
LP 500 LP 2500 LP 5000
SEM
Significance (P value)
Total P intake (g/bird) Total P in excreta/ faeces (g/bird) P excretion (g/kg feed intake)
19.4a
15.6c
0.304
0.001
10.4a
3.85a
17.2b
17.9b
7.52c
7.62c
8.37b
0.245
0.001
2.86b
2.62d
2.77c
0.101
0.001
Means in the same row bearing different superscript differ significantly.
phytase supplementation (at 1500 and 3000 FTU/kg). The growth-promoting effect in the present study can be partially attributed to the increase in feed intake and better utilization of phosphorus. Shirley and Edwards (2003) reported that graded dose of phytase (0–12 000 FTU) improved feed intake from 381 to 595 g/chick. Pirgozliev et al. (2008) stated that 14.5% increase in feed intake was observed with 500 FTU/kg phytase supplementation compared to low-P diet and the response was 22% more, when the phytase level was increased to 2500 FTU/kg. Pirgozliev et al. (2010) observed that the feed intake of control diet, 49.7 g/b/day, was comparable with low-P diet supplemented with graded level of phytase at 250, 500, 12 500 FTU/kg with 47.4, 46.6 and 48.8 g/b/day feed intake respectively. In the present study, the increased phytase dose improved feed intake as observed in many of the earlier studies. Cowieson et al. (2006) reported that chickens fed on low-P diets supplemented with either 2400 or 24 000 FTU had 14% better feed conversion ratio compared to birds fed a diet with adequate supplies of P. Pirgozliev et al. (2010) reported 9.4% better FCR
Table 7 Bone ash and mineral content of tibia bone at 5 week of age Groups Ash and minerals
NP
LP 500
LP 2500
LP 5000
SEM
Significance (p value)
Ash (%) Ca (%) Mg (%) P (%) Zn (ppm) Fe (ppm) Mn (ppm)
43.4c 19.2bc 0.403b 9.15c 147bc 422ab 40.9
43.37c 18.23c 0.394b 8.67d 133c 447a 40.5
46.45b 19.93ab 0.453a 9.67b 165ab 390b 31.3
48.96a 20.83a 0.473a 10.22a 178a 405ab 31.3
0.4247 0.1992 0.0069 0.1008 3.41 7.08 1.89
0.001 0.001 0.001 0.001 0.001 0.022 0.147
Means in the same row bearing different superscript differ significantly.
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compared to low-P diet with phytase supplementation at 500 FTU/kg and even 10.1% better FCR compared to normal P diet when phytase was supplemented at 12 500 FTU/kg in low-P diet. Rutherfurd et al. (2012) observed that there was no effect (p > 0.05) on the feed intake to weight gain ratio for the birds fed phytase (at 1000 and 2000 FTU/kg)-supplemented low-P diet compared with unsupplemented low-P diet such that performance was equal to that of the adequate P diet. The apparent ileal digestibility of CP was not altered by phytase supplementation (Woyengo et al., 2010; Rutherfurd et al., 2012; Lalpanmawia et al., 2014). However, Ravindran et al. (2006) reported improved ileal CP digestibility in broilers fed phytase of 500 FTU/kg from medium to high concentrations of phytate. Cowieson et al. (2006) reported that broilers fed with low-P diet with phytase showed improved (p < 0.001) apparent ileal digestibility of amino acids when compared to normal P diet. Pirgozliev et al. (2011) observed that higher dose (12 500 FTU) of phytase supplementation in low-P diet improved the total tract amino acid digestibility when compared to phytase supplemented at 500 FTU/kg. Rutherfurd et al. (2012) and Chung et al. (2013) reported that the apparent ileal digestibility of amino acids was improved with phytase supplementation in low-P diets. Sebastian et al. (1997) reported that microbial phytase supplementation had no effect (p < 0.065) on the apparent ileal digestibility (AID) of CP or any AA except methionine and phenylalanine in male broiler chicken, whereas, in females, adding phytase increased the AID of all AA except lysine, methionine, phenylalanine and proline. Effect of phytase on amino acids digestibility is varied (Selle et al., 2000; Adeola and Sands, 2003), and it is clear that when phytase influences amino acids digestibility coefficients, it does not do so to the same extent for all amino acids. This may be linked to differential interactions between amino groups and phytate or it may be associated with the ability of phytate to increase the loss of endogenous compounds, such as mucins, that are rich in certain amino acids (Mansoori and Acamovic, 1998; Cowieson et al., 2004). Improved P digestibility and Ca digestibility in phytase-supplemented diet were reported by several researchers (Ravindran et al., 2006; Lalpanmawia et al., 2014). This is probably related to the release of minerals from the phytate bond as well as prevention of bounding with phytate. Further, Chung et al. (2013) also observed that the impact of phytase on the availability of minerals other than P is not definite. 98
Ayasan and Okan (1999) reported a value of 15.47– 20.68 mg of Ca/100 ml and 5.33–7.71 mg of P/100 ml of blood on feeding dietary levels of different calcium and phosphorus in laying Japanese quails. Shirley and Edwards (2003) reported that supplementation of phytase of 0–12 000 FTU/kg in low-P diet increased blood P level and reduced blood Ca level. It may be observed higher doses of phytase in low-P diet increased plasma p values probably more P released from phytate. However, reduced blood Ca levels indicated more utilization of Ca. Qian et al. (1996) observed higher tibial length with phytase supplementation at 600 FTU/kg in comparison with low-P diet. The bone length was higher probably due to the higher live weight of the birds pertaining to super dosing groups. Kocabagli (2001) found that the tibio-tarsal weight/length index was found to be significantly higher in phytase-supplemented groups than in control group. Rutherfurd et al. (2012) and Guo et al. (2009) reported that tibial weight was significantly higher in phytase-supplemented group. Rousseau et al. (2012) reported that tibial diameter/mid-shaft width was improved by phytase supplementation of 500 FTU/kg in low-P diets but no improvement in tibial bone length. Shirley and Edwards (2003) observed that phytase supplementation (0–12 000 FTU) in low-P diets improved tibia ash from 26 to 41%, tibia ash weight from 0.200 to 0.601 g/tibia, total phosphorus retention from 51 to 80%. The increase in tibial ash is considered to be a good indicator of bone mineralization (Sebastian et al., 1996; Leeson et al., 2000). This improvement in ash percentage in tibia can be related to the increase in Ca and P retention. Cowieson et al. (2006) observed that phytase supplementation (0–24 000 FTU) in low-P diet improved (p < 0.001) apparent retention of P, K, Na, Mg, Ca, S, Cu, Fe and Mn, which was higher than normal P-fed group. Guo et al. (2009) reported that phytase supplementation of 500 and 1000 FTU/kg in diets containing 2.0 g non-phytate phosphorus/kg linearly or quadratically increased performance and tibia ash of broilers during the whole growing period. Rutherfurd et al. (2012) reported that phytase supplementation of 1000 and 2000 FTU/kg in low-P diet increased both tibial mineral concentration and density by 35% and 24%, respectively, compared to low-P diet and was equal to that of adequate P diet. Walk et al. (2013) reported that super doses of phytase supplementation in low-P diet in 0- to 21-dayold broiler did not alter tibia ash percentage. Chung et al. (2013) observed that phytase supplementation
Journal of Animal Physiology and Animal Nutrition 100 (2016) 93–100 © 2015 Blackwell Verlag GmbH
DOI: 10.1111/jpn.12341
ORIGINAL ARTICLE
Effect of super dosing of phytase on growth performance, ileal digestibility and bone characteristics in broilers fed corn–soya-based diets M. Manobhavan, A. V. Elangovan, M. Sridhar, D. Shet, S. Ajith, D. T. Pal and N. K. S. Gowda National Institute of Animal Nutrition and Physiology, Bangalore, India
Summary A feeding trial was designed to assess the effect of super dosing of phytase in corn–soya-based diets of broiler chicken. One hundred and sixty-eight day-old broilers were selected and randomly allocated to four dietary treatment groups, with 6 replicates having 7 chicks per treatment group. Two-phased diets were used. The starter and finisher diet was fed from 0 to 3 weeks and 4 to 5 weeks of age respectively. The dietary treatments were consisted of normal phosphorus (NP) group without any phytase enzyme (4.5 g/kg available/non-phytin phosphorus (P) during starter and 4.0 g/kg during finisher phase), three low-phosphorus (LP) groups (3.2 g/kg available/non-phytin P during starter and 2.8 g/kg during finisher phase) supplemented with phytase at 500, 2500, 5000 FTU/kg diet, respectively, to full fill their phosphorus requirements. The results showed that super doses of phytase (at 2500 FTU and 5000 FTU/kg) on low-phosphorus diet improved feed intake, body weight gain, ileal digestibility (serine, aspartic acid, calcium, phosphorus), blood P levels and bone minerals such as calcium (Ca), P, magnesium (Mg) and zinc (Zn) content. It could be concluded that super doses of phytase in low-phosphorus diet were beneficial than the normal standard dose (at 500 FTU/kg) of phytase in diet of broiler chicken. Keywords bone, broiler, nutrient utilization, phosphorus, phytase Correspondence A. V. Elangovan, National Institute of Animal Nutrition and Physiology, Bangalore, India. Tel: +91-9341380858; Fax: +91-80-25711420; Emails: [email protected]; [email protected] Received: 11 December 2014; accepted: 31 March 2015
Introduction Phosphorus (P) is the most expensive essential mineral for poultry. In plant-based diets, two-third of P is bound to phytate and not available to poultry. The presence of phytate can result in reduced efficiency of nutrient utilization increasing the cost of feeding and lead to environmental pollution. The best way to improve phosphorus utilization is with judicious use of natural resources by phytase enzyme supplementation in the diet of monogastrics. Phytase is used extensively and the generally recommended dose of phytase in poultry is 500 FTU/ kg diet (Selle and Ravindran, 2007; Cowieson et al., 2009; Pirgozliev et al., 2012; Lalpanmawia et al., 2014). Shirley and Edwards (2003) observed a quadratic increase in phytate P disappearance of almost 95% with increasing phytase dose to 12 000 FTU/ kg in corn-based diets. Further, the use of higher doses of phytase has been gaining interest not only due to more P release but also leaving less residual phytate and generation of myo-inositol with
vitamin-like/lipotropic effects (Shirley and Edwards, 2003). The use of super doses of phytase (>1000 FTU/kg of diet) has been shown to improve nutrient availability in poultry diets (Cowieson et al., 2006). The high doses of phytase may result in more liberated phosphate or restoration of P/Ca proportionate release with less residual phytate in the gut (Cowieson et al., 2011). Phytate forms complexes with multivalent cations to produce insoluble salts, this Ca–phytate complex is the weakest, and it has been reported to be the most beneficial by dietary phytase supplementation (Angel and Applegate, 2001). Chung et al. (2013) reported that the impact of phytase on the availability of minerals other than P is not definite. It is well established that available P requirement is 0.45 and 0.40% during starter and finisher phase. Hence, low-P (available P, 0.30%) group without phytase supplementation was not taken as reference or negative control. Moreover, our study was to compare the advantage of conventionally fed 500 FTU over super dosing under practical farming condition.
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phytate in cereals and cereal products. Journal of the Science of Food and Agriculture 34, 1423–1426. Ibrahim, S.; Jacob, J. P.; Blair, R., 1999: Phytase supplementation to reduce phosphorus excretion of broilers. The Journal of Applied Poultry Research 8, 414–425. ICAR, 2013: Nutrient Requirement of Animal – Poultry. Indian Council of Agricultural Research-NIANP Publication, India. Kocabagli, N., 2001: The effect of dietary phytase supplementation at different levels on tibial bone characteristics and strength in broilers. Turkish Journal of Veterinary and Animal Sciences 25, 797– 802. Kornegay, E. T.; Denbow, D. M.; Yi, Z.; Ravindran, V., 1996: Response of broilers to graded levels of microbial phytase added to maize-soybean-meal based diets containing three levels of nonphytate phosphorus. British Journal of Nutrition 75, 839–852. Lalpanmawia, H.; Elangovan, A. V.; Sridhar, M.; Shet, D.; Ajith, S.; Pal, D. T., 2014: Efficacy of phytase on growth performance, nutrient utilization and bone mineralization in broiler chicken. Animal Feed Science and Technology 192, 81– 89. Leeson, S.; Namkung, H.; Cottrill, M.; Forsberg, C. W., 2000: Efficacy of new bacterial phytase in poultry diets. Canadian Journal of Animal Science 80, 527– 528. Mansoori, B.; Acamovic, T., 1998: The influence of tannic acid on the amino acid digestibility in broilers. In: T. Garland, A. C. Barr (eds), Toxic Plants and Other Natural Toxicants. CABI Publishing, Wallingford, UK, pp. 106–110. Mohanna, C.; Nys, Y., 1999: Changes in zinc and manganese availability in broiler chicks induced by vegetal and microbial phytases. Animal Feed Science and Technology 77, 241–253. Musapuor, A.; Afsharmanesh, M.; Shahrbabak, H. M., 2006: Use of microbial phytase for decrease of pollutant due to environmental poultry excreta phosphorus. International Journal of Agriculture and Biology 8, 35–37. Onyango, E. M.; Bedford, M. R.; Adeola, O., 2005: Efficacy of an evolved Escherichia coli phytase in diets of broiler chicks. Poultry Science 84, 248–255. Pirgozliev, V.; Oduguwa, O.; Acamovic, T.; Bedford, M. R., 2008: Effects of dietary
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phytase on performance and nutrient metabolism in chickens. British Poultry Science 49, 144–154. Pirgozliev, V.; Karadas, F.; Pappas, A.; Acamovic, T.; Bedford, M. R., 2010: The effect on performance, energy metabolism and hepatic carotenoid content when phytase supplemented diets were fed to broiler chickens. Research in Veterinary Science 89, 203–205. Pirgozliev, V.; Bedford, M. R.; Acamovic, T.; Mares, P.; Allymehr, M., 2011: The effects of supplementary bacterial phytase on dietary energy and total tract amino acid digestibility when fed to young chickens. British Poultry Science 52, 245–254. Pirgozliev, V.; Bedford, M. R.; Oduguwa, O.; Acamovic, T.; Allymehr, M., 2012: The effect of supplementary bacterial phytase on dietary metabolisable energy, nutrient retention and endogenous losses in precision fed broiler chickens. Journal of Animal Physiology and Animal Nutrition 96, 52–57. Qian, H.; Veit, H. P.; Kornegay, E. T.; Ravindran, V.; Denbow, D. M., 1996: Effects of supplemental phytase and phosphorus on histological and other tibia bone characteristics and performance of broiler chickens fed semi purified diets. Poultry Science 75, 618–626. Ravindran, V.; Morel, P. C. H.; Partridge, G. G.; Hruby, M.; Sands, J. S., 2006: Influence of an Escherichia coli-derived phytase on nutrient utilization in broiler starters fed diets containing varying concentrations of phytic acid. Poultry Science 85, 82–89. Roberson, K. D.; Edwards, H. M., 1994: Effects of 1, 25-dihydroxicholecalciferol and phytase on zinc utilization in broiler chicks. Poultry Science 73, 1312–1326. Rousseau, X.; Letourneau-Montminy, M. P.; Meme, M.; Magnin, M.; Nys, Y.; Narcy, A., 2012: Phosphorus utilization in finishing broiler chickens: effects of dietary calcium and microbial phytase. Poultry Science 91, 2829–2837. Rutherfurd, S. M.; Chung, T. K.; Thomas, D. V.; Zou, M. L.; Moughan, P. J., 2012: Effect of a novel phytase on growth performance, AME and the availability of minerals and amino acids in a low-phosphorus corn-soybean meal diet for broilers. Poultry Science 91, 1118–1127. Sebastian, S.; Touchburn, S. P.; Chavez, E. R.; Lague, P. C., 1996: The effects of
supplemental microbial phytase on the performance and utilization of dietary calcium, phosphorus, copper and zinc in broiler chickens fed corn-soybean diets. Poultry Science 75, 729–736. Sebastian, S.; Touchburn, S. P.; Chavez, E. R.; Lague, P. C., 1997: Apparent digestibility of protein and amino acids in broiler chickens fed a cornsoybean diet supplemented with microbial phytase. Poultry Science 76, 1760–1769. Selle, P. H.; Ravindran, V., 2007: Microbial phytase in poultry nutrition. Animal Feed Science and Technology 35, 1–41. Selle, P. H.; Ravindran, V.; Caldwell, R. A.; Bryden, W. L., 2000: Phytate and phytase: consequences for protein utilization. Nutrition Research Reviews 13, 255– 278. Shirley, R. B.; Edwards, H. M., 2003: Graded levels of phytase past industry standards improves broiler performance. Poultry Science 82, 671–680. SPSS, 2010: SPSS Version 18. SPSS, Chicago, Illinois. Sukumar, D.; Jalaludeen, A.; Cyriac, S., 2013: Effect of mitigating environmental pollution by incorporation of phytase enzyme in feed of (Athulya) layer chicken. Wayamba Journal of Animal Science 5, 569–573. Sun, D.; Waters, J. K.; Mawhinney, T. P., 2000: Determination of thirteen common elements in food samples by inductively coupled plasma atomic emission spectrometry: comparison of five digestion methods. Journal of AOAC International 83, 1218–1224. Um, J. S.; Paik, I. K., 1999: Effects of microbial phytase supplementation on egg production, eggshell quality, and mineral retention of laying hens fed different levels of phosphorus. Poultry Science 78, 75–79. Walk, C. L.; Bedford, M. R.; Santos, T. S.; Paiva, D.; Bradley, J. R.; Wladecki, H.; Honaker, C.; McElroy, A. P., 2013: Extra-phosphoric effects of superdoses of a novel microbial phytase. Poultry Science 92, 719–725. Woyengo, T. A.; Emiola, A.; Asiedu, A. O.; Geunter, W.; Simmins, P. H.; Nyachoti, C. M., 2010: Performances and nutrient utilization responses in broilers fed phytase supplemented mash or pelleted corn-soybean meal based diets. The Journal of Poultry Science 47, 310–315.
Journal of Animal Physiology and Animal Nutrition 100 (2016) 93–100 © 2015 Blackwell Verlag GmbH
ORIGINAL ARTICLE
Effect of super dosing of phytase on growth performance, ileal digestibility and bone characteristics in broilers fed corn–soya-based diets M. Manobhavan, A. V. Elangovan, M. Sridhar, D. Shet, S. Ajith, D. T. Pal and N. K. S. Gowda National Institute of Animal Nutrition and Physiology, Bangalore, India
Summary A feeding trial was designed to assess the effect of super dosing of phytase in corn–soya-based diets of broiler chicken. One hundred and sixty-eight day-old broilers were selected and randomly allocated to four dietary treatment groups, with 6 replicates having 7 chicks per treatment group. Two-phased diets were used. The starter and finisher diet was fed from 0 to 3 weeks and 4 to 5 weeks of age respectively. The dietary treatments were consisted of normal phosphorus (NP) group without any phytase enzyme (4.5 g/kg available/non-phytin phosphorus (P) during starter and 4.0 g/kg during finisher phase), three low-phosphorus (LP) groups (3.2 g/kg available/non-phytin P during starter and 2.8 g/kg during finisher phase) supplemented with phytase at 500, 2500, 5000 FTU/kg diet, respectively, to full fill their phosphorus requirements. The results showed that super doses of phytase (at 2500 FTU and 5000 FTU/kg) on low-phosphorus diet improved feed intake, body weight gain, ileal digestibility (serine, aspartic acid, calcium, phosphorus), blood P levels and bone minerals such as calcium (Ca), P, magnesium (Mg) and zinc (Zn) content. It could be concluded that super doses of phytase in low-phosphorus diet were beneficial than the normal standard dose (at 500 FTU/kg) of phytase in diet of broiler chicken. Keywords bone, broiler, nutrient utilization, phosphorus, phytase Correspondence A. V. Elangovan, National Institute of Animal Nutrition and Physiology, Bangalore, India. Tel: +91-9341380858; Fax: +91-80-25711420; Emails: [email protected]; [email protected] Received: 11 December 2014; accepted: 31 March 2015
Introduction Phosphorus (P) is the most expensive essential mineral for poultry. In plant-based diets, two-third of P is bound to phytate and not available to poultry. The presence of phytate can result in reduced efficiency of nutrient utilization increasing the cost of feeding and lead to environmental pollution. The best way to improve phosphorus utilization is with judicious use of natural resources by phytase enzyme supplementation in the diet of monogastrics. Phytase is used extensively and the generally recommended dose of phytase in poultry is 500 FTU/ kg diet (Selle and Ravindran, 2007; Cowieson et al., 2009; Pirgozliev et al., 2012; Lalpanmawia et al., 2014). Shirley and Edwards (2003) observed a quadratic increase in phytate P disappearance of almost 95% with increasing phytase dose to 12 000 FTU/ kg in corn-based diets. Further, the use of higher doses of phytase has been gaining interest not only due to more P release but also leaving less residual phytate and generation of myo-inositol with
vitamin-like/lipotropic effects (Shirley and Edwards, 2003). The use of super doses of phytase (>1000 FTU/kg of diet) has been shown to improve nutrient availability in poultry diets (Cowieson et al., 2006). The high doses of phytase may result in more liberated phosphate or restoration of P/Ca proportionate release with less residual phytate in the gut (Cowieson et al., 2011). Phytate forms complexes with multivalent cations to produce insoluble salts, this Ca–phytate complex is the weakest, and it has been reported to be the most beneficial by dietary phytase supplementation (Angel and Applegate, 2001). Chung et al. (2013) reported that the impact of phytase on the availability of minerals other than P is not definite. It is well established that available P requirement is 0.45 and 0.40% during starter and finisher phase. Hence, low-P (available P, 0.30%) group without phytase supplementation was not taken as reference or negative control. Moreover, our study was to compare the advantage of conventionally fed 500 FTU over super dosing under practical farming condition.
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Further, it may be difficult to completely replace inorganic P source from diet as the phytase supplementation also depends on the phytate content of the diet. This study was conducted to test the effect of super dosing of phytase on growth performance, ileal digestibility of nutrients and bone characteristics in broiler chicken. Materials and methods Experimental design
The animal care and use protocol (No. 5/2012) was reviewed and approved by the Institutional Animal Ethics committee of National Institute of Animal Nutrition and Physiology, Bangalore, India. The experiment was conducted in a completely randomized design. A total of one hundred sixty-eight, unsexed, day-old Cobb broiler chicks, vaccinated against Newcastle disease were obtained from commercial hatchery (Venkateswara hatchery). The chicks with a mean body weight of 45.8 g 1.46 (SD) were distributed into four treatments; each treatment had six replicates with seven chicks in each replicate. The chicks were housed in battery cages (72 cm wide 9 48 cm long 9 38 cm high) as groups in random manner, fitted with heating arrangements, feeders, waterer and dropping trays, with 24 h light and proper air ventilation, and reared under standard managemental conditions. The temperature inside the cage was maintained at 33 °C on day 1 and gradually reduced 24–25 °C by the end of the third week and maintained. The feed and fresh drinking water were provided ad libitum during the entire experimental period. Experimental diets
The dietary treatments consisted of one normal phosphorus (NP) diet, without any phytase enzyme (0.45% available P during starter and 0.40% during finisher phase), and three low-phosphorus diets (0.32% available P during starter and 0.28% during finisher phase) supplemented with phytase (commercial phytase, Finase EC from AB Vista, Pune, India, 5000 FTU/g) at 500, 2500 and 5000 FTU/kg diet designated as LP 500, LP 2500 and LP 5000 respectively. Ingredient and nutrient composition of experimental diets are given in Table 1. The diets were formulated based on Nutrient Requirement of Poultry published by ICAR (2013) using Excel-based feed formulation. The phytate phosphorus content of maize and soya bean meal was estimated by the method described by Haug and Lantzsch (1983). The Ca and P in feed 94
were analysed by inductively coupled plasma-optical emission spectroscopy (ICP-OES) using a Perkin Elmer instrument. The amino acid content of feed and digesta was analysed at Amino Lab, Evonik Industries (Nordic European Centre, Singapore). Measurements and laboratory analysis
Body weight gain and feed intake (FI) were recorded at weekly intervals. The feed conversion ratio (FCR) was calculated. Prior to sacrificing, blood samples of birds (two birds/replicate) were collected from jugular vein into a sterile centrifuge tube containing heparin (20 IU/ml). The blood was centrifuged at 1550 g for 10 min, and plasma was separated and stored at 20◦C for estimation of Ca and phosphorous by commercial diagnostic kits (Span diagnostics, Gujarat, India). At the end of 35 days, four birds were selected randomly per pen and sacrificed by cervical Table 1 Ingredient and nutrient composition of experimental diets during starter (0–3 week) and finisher (4–5 week) phase Starter (0–3 week)
Finisher (4–5 week)
NP
LP
NP
LP
584.2 360 20 16 10 0 3.5 2.5 1.3 2.5
612 325 25 10 15 5 3.5 1 1 2.5
612.5 325 25 17 7.5 5 3.5 1 1 2.5
12.6 223 13.7 4.9 10.7 6.30 3.12 3.18
12.8 193 11.3 4.3 10.1 6.96 2.98 3.97
12.8 199 11.3 4.3 10.3 5.68 2.98 2.69
Ingredient composition (g/kg) Corn 583.4 Soya bean meal (CP, 46.1%) 359.3 Sunflower oil 20 Limestone 10 Di calcium phosphate 17.5 Celite 0 Salt 3.5 Lysine 2.5 Methionine 1.3 Vitamin mineral premix* 2.5 Determined nutrient composition (g/kg) ME (MJ/kg)† 12.6 Crude protein 222 Lysine 13.7 Methionine 4.9 Calcium 10.9 Total phosphorus 7.57 Phytate phosphorus 3.12 Available phosphorus† 4.45
NP, normal phosphorus; LP, low phosphorus. *Trace mineral premix, 1 g/kg; Vit. Premix, 1 g/kg and choline 0.5 g/kg. Trace mineral premix supplied mg/kg diet: Mg, 300; Mn, 55; I, 0.4; Fe, 56; Zn, 30; Cu, 4. The vitamin premix supplied per kg diet: Vit. A, 8250 IU; Vit. D3, 1200 ICU; Vit. K, 1 mg; Vit. E, 40 IU; Vit. B1, 2 mg; Vit. B2 4 mg; Vit. B12, 10 mcg; niacin, 60 mg; pantothenic acid, 10 mg; choline, 500 mg. †Calculated.
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dislocation for evaluation of ileal digestibility and bone minerals. After sacrificing the birds, the contents of the ileum from Meckel’s diverticulum to ileo-caecal junction were pooled within a pen, collected and frozen immediately (20 °C). The digesta samples were freeze-dried, ground and stored in an air tight container for estimation of crude protein (AOAC, 1990), amino acids (Amino Lab, Evonik Industries), acid insoluble ash (AIA) (AOAC, 1990) and minerals (ICP). The P measured in the ileal digesta was used for calculating the total P in the excreta/faeces. The apparent ileal digestibility coefficient (AIDC) of the nutrients was calculated from the equation: AIDC ¼1 ½ðileal nutrient=ileal AIAÞ = ðdiet nutrient=diet AIAÞ: The left tibia bones (two birds per replicate) were collected after removing the muscles and adhering structures. The length and width of the tibia bone were measured with vernier callipers. Each tibia was defatted for 16 h in petroleum spirit (boiling point 60– 80 °C) using Soxhlet apparatus, dried and weighed before ashing. The ash samples were digested with dilute hydrochloric acid (1:2), and the mineral extract was used for estimation of minerals by inductively coupled plasma-optical emission spectroscopy (ICPOES) using a Perkin Elmer instrument (Sun et al., 2000). Statistical analysis
The data were subjected to one-way analysis of variance (ANOVA) for completely randomized design and tested for significance between the dietary treatments means employing Tukey’s HSD post hoc test (SPSS, 2010 Version 18.0).
Results Growth performance
Chicks fed the LP 2500 and LP 5000 ate more and gained more (p < 0.001) than chicks fed the LP 500 or NP during starter, finisher and overall phase (Table 2). There was no difference in body weight gains of chicks fed NP and LP 500 during whole experimental period. The feed intake followed the similar pattern as that of body weight gain. The feed conversion ratio was nonsignificant (p > 0.05) during the experimental period. Apparent ileal digestibility
There was no (p > 0.05) effect of diets on AIDC of crude protein and amino acids except serine and aspartic acid (Table 3). Broilers fed LP 2500 had higher AIDC for serine (p < 0.05) and aspartic acid (p < 0.05). Ca digestibility was higher (p < 0.01) in broilers fed LP 500 or LP 5000 compared to broilers fed NP, but this was not different than broilers fed LP 2500 (Table 4). P digestibility was higher (p < 0.001) in broilers fed LP 2500 in comparison with broilers fed NP. The AIDC of Mg, Mn and Zn were non-significant (p > 0.05) among the different treatment groups. Plasma minerals
Plasma Ca content was higher (p < 0.001) in broilers fed the NP or LP 500 compared with broilers fed LP 2500 or LP 5000 (Table 5). Plasma P content was higher (p < 0.001) in broilers fed LP 5000 compared with broilers fed all other diets (Table 5). Bone morphometry and mineralization
The bone weight was higher (p < 0.01) in broilers fed LP 2500 compared with broilers fed all other diets
Table 2 Growth performance of broiler chicken
Body weight gain (g/bird)
Feed conversion ratio, Feed intake (g/bird)/Body weight gain (g/bird)
Feed intake (g/bird)
Groups
0–3 week
4–5 week
0–5 week
0–3 week
4–5 week
0–5 week
0–3 week
4–5 week
0–5 week
NP LP 500 LP 2500 LP 5000 SEM Significance (p value)
717b 725b 798a 825a 11.0 0.001
952b 917b 1007a 1013a 11.8 0.003
1669b 1642b 1805a 1838a 20.6 0.001
984b 1024b 1104a 1177a 17.8 0.001
1713b 1610b 1803a 1850a 24.1 0.001
2697b 2634b 2907a 3027a 37.7 0.001
1.37 1.41 1.38 1.43 0.010 0.178
1.80 1.76 1.79 1.83 0.011 0.326
1.62 1.61 1.61 1.65 0.008 0.327
SEM: standard error of means; NP: normal phosphorus; LP 500: low phosphorus + phytase at 500 FTU/kg; LP 2500: low phosphorus + phytase at 2500 FTU/kg; LP 5000: low phosphorus + phytase at 5000 FTU/kg. Means in the same column bearing different superscript differ significantly.
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Table 3 Apparent ileal digestibility coefficient (AIDC) of crude protein and amino acids at 5 week of age Groups Amino acids
NP
LP 500
LP 2500
LP 5000
SEM
Significance (p value)
Crude protein Cystine Serine Proline Alanine Aspartic acid Glutamic acid Methionine Lysine Threonine Arginine Isoleucine Leucine Valine Histidine Phenylalanine Glycine
0.824 0.804 0.867ab 0.911 0.853 0.88ab 0.913 0.914 0.913 0.814 0.902 0.876 0.893 0.857 0.886 0.845 0.819
0.813 0.783 0.852b 0.899 0.837 0.865b 0.896 0.911 0.907 0.808 0.886 0.865 0.875 0.847 0.861 0.837 0.813
0.823 0.823 0.89a 0.915 0.861 0.901a 0.928 0.932 0.931 0.831 0.906 0.887 0.903 0.866 0.887 0.845 0.825
0.815 0.818 0.877ab 0.914 0.831 0.888ab 0.916 0.916 0.91 0.817 0.905 0.879 0.893 0.856 0.879 0.844 0.814
0.0049 0.0061 0.0053 0.0032 0.0055 0.0048 0.0048 0.0035 0.0036 0.0041 0.0035 0.0038 0.0044 0.0035 0.0044 0.0033 0.0033
0.8747 0.0670 0.0324 0.2589 0.2084 0.0193 0.1046 0.1524 0.0619 0.2623 0.1636 0.2576 0.1483 0.3905 0.1247 0.8417 0.6617
SEM: standard error of means; NP: normal phosphorus; LP 500: low phosphorus + phytase at 500 FTU/kg; LP 2500: low phosphorus + phytase at 2500 FTU/kg; LP 5000: low phosphorus + phytase at 5000 FTU/kg. Means in the same row bearing different superscript differ significantly.
Table 4 Apparent ileal digestibility coefficient (AIDC) of minerals at 5 week of age Groups Minerals
NP
LP 500
LP 2500
LP 5000
SEM
Significance (p value)
Ca P Mg Mn Zn
0.574b 0.464c 0.272 0.471 0.455
0.635a 0.518b 0.275 0.511 0.467
0.602ab 0.557a 0.281 0.542 0.516
0.626a 0.533ab 0.245 0.511 0.457
0.0073 0.0079 0.0242 0.0177 0.0195
0.006 0.001 0.961 0.599 0.082
Means in the same row bearing different superscript differ significantly.
(Table 6). The bone length, proximal epiphysis width, diaphysis and distal epiphysis width were higher (p < 0.001) in broilers fed LP 2500 and LP 5000 compared to broilers fed NP and LP 500. The ash, Ca, P, Mg and Zn content of bone were higher (p < 0.001) in broilers fed LP 2500 and LP 5000 compared to broilers fed NP and LP 500 (Table 7). The bone Fe content was higher (p < 0.05) in LP 500. The Mn content of bone showed no significant (p > 0.05) difference among the treatment groups. Phosphorus intake and excretion
The total P intake was higher (p < 0.001) in broilers fed NP followed by LP 5000, LP 2500 and lower in LP 96
Table 5 Plasma Calcium and Phosphorus content of broiler chicken at 5 week of age
Groups
Calcium (mg/dl)
Phosphorus (mg/dl)
NP LP 500 LP 2500 LP 5000 SEM Significance (p value)
10.3a 11.2a 9.09b 8.94b 0.268 0.001
4.39b 4.06b 4.88b 5.35a 0.236 0.001
Means in the same column bearing different superscript differ significantly.
500. The P excretion g/kg feed intake differed significantly (p < 0.001). It was higher in broilers fed NP followed by LP 500, LP 5000, and least excretion was noticed in broilers fed LP 2500 (Table 8). Discussion It is well documented that the phytase supplementation in low-P diet increases growth performance in broiler chicken (Selle and Ravindran, 2007). Shirley and Edwards (2003) observed that broilers consuming a P-deficient corn–soya bean meal diet can achieve maximum performance with phytase supplementation of 12 000 U/kg in diet and also suggested that
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Table 6 Bone morphometry (left tibia) of broiler chicken at 5 week of age
Groups NP LP 500 LP 2500 LP 5000 SEM Significance (p value)
Bone weight (g/kg live weight 3.19ab 3.08b 3.36a 3.33ab 0.029 0.01
Bone length (cm)
Bone width (cm) proximal epiphysis
Bone width (cm) diaphysis
Bone width (cm) distal epiphysis
8.53b 8.35b 8.89a 9.00a 0.053 0.001
2.06b 2.06b 2.26a 2.27a 0.019 0.001
0.830b 0.833b 0.876a 0.892a 0.009 0.021
1.65b 1.67b 1.80a 1.78a 0.014 0.001
Means in the same column bearing different superscript differ significantly.
phytase supplementation levels within the poultry industry need to be re-evaluated. Onyango et al. (2005) reported increase in body weight gain of 555 and 569 g/bird by supplementing phytase at 500 and 1000 FTU/kg, respectively, in low-P diet, which was comparable with normal P diet (568 g/bird) and significantly higher than low-P group without phytase (476 g/bird). Cowieson et al. (2006) reported improvements in body weight gain (15.47–17.59 g/b/ day) as phytase supplementation was increased in logarithmic doses from 150 to 24 000 FTU/kg in low-P diet and was higher than normal P diet (16.07 g/b/ day). Pirgozliev et al. (2008) reported increase in feed intake as the level of phytase increased from 250, 500 and 2500 FTU/kg which in turn increased body weight gain 39.6, 41.1 and 44.0 g/b/day accordingly. The same authors (Pirgozliev et al., 2011), on phytase supplementation of 12 500 in low-P diet, observed higher weight gain (33.1 g/b/day) than normal P (30.8 g/b/day) and low-P diet with phytase 500 FTU/ kg (28.1 g/b/day). Chung et al. (2013) reported that there was no effect (p > 0.05) on feed intake and weight gain of broiler chicken fed low-P diet with
Table 8 Total P intake and excretion of broiler chicken
Groups
NP
LP 500 LP 2500 LP 5000
SEM
Significance (P value)
Total P intake (g/bird) Total P in excreta/ faeces (g/bird) P excretion (g/kg feed intake)
19.4a
15.6c
0.304
0.001
10.4a
3.85a
17.2b
17.9b
7.52c
7.62c
8.37b
0.245
0.001
2.86b
2.62d
2.77c
0.101
0.001
Means in the same row bearing different superscript differ significantly.
phytase supplementation (at 1500 and 3000 FTU/kg). The growth-promoting effect in the present study can be partially attributed to the increase in feed intake and better utilization of phosphorus. Shirley and Edwards (2003) reported that graded dose of phytase (0–12 000 FTU) improved feed intake from 381 to 595 g/chick. Pirgozliev et al. (2008) stated that 14.5% increase in feed intake was observed with 500 FTU/kg phytase supplementation compared to low-P diet and the response was 22% more, when the phytase level was increased to 2500 FTU/kg. Pirgozliev et al. (2010) observed that the feed intake of control diet, 49.7 g/b/day, was comparable with low-P diet supplemented with graded level of phytase at 250, 500, 12 500 FTU/kg with 47.4, 46.6 and 48.8 g/b/day feed intake respectively. In the present study, the increased phytase dose improved feed intake as observed in many of the earlier studies. Cowieson et al. (2006) reported that chickens fed on low-P diets supplemented with either 2400 or 24 000 FTU had 14% better feed conversion ratio compared to birds fed a diet with adequate supplies of P. Pirgozliev et al. (2010) reported 9.4% better FCR
Table 7 Bone ash and mineral content of tibia bone at 5 week of age Groups Ash and minerals
NP
LP 500
LP 2500
LP 5000
SEM
Significance (p value)
Ash (%) Ca (%) Mg (%) P (%) Zn (ppm) Fe (ppm) Mn (ppm)
43.4c 19.2bc 0.403b 9.15c 147bc 422ab 40.9
43.37c 18.23c 0.394b 8.67d 133c 447a 40.5
46.45b 19.93ab 0.453a 9.67b 165ab 390b 31.3
48.96a 20.83a 0.473a 10.22a 178a 405ab 31.3
0.4247 0.1992 0.0069 0.1008 3.41 7.08 1.89
0.001 0.001 0.001 0.001 0.001 0.022 0.147
Means in the same row bearing different superscript differ significantly.
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compared to low-P diet with phytase supplementation at 500 FTU/kg and even 10.1% better FCR compared to normal P diet when phytase was supplemented at 12 500 FTU/kg in low-P diet. Rutherfurd et al. (2012) observed that there was no effect (p > 0.05) on the feed intake to weight gain ratio for the birds fed phytase (at 1000 and 2000 FTU/kg)-supplemented low-P diet compared with unsupplemented low-P diet such that performance was equal to that of the adequate P diet. The apparent ileal digestibility of CP was not altered by phytase supplementation (Woyengo et al., 2010; Rutherfurd et al., 2012; Lalpanmawia et al., 2014). However, Ravindran et al. (2006) reported improved ileal CP digestibility in broilers fed phytase of 500 FTU/kg from medium to high concentrations of phytate. Cowieson et al. (2006) reported that broilers fed with low-P diet with phytase showed improved (p < 0.001) apparent ileal digestibility of amino acids when compared to normal P diet. Pirgozliev et al. (2011) observed that higher dose (12 500 FTU) of phytase supplementation in low-P diet improved the total tract amino acid digestibility when compared to phytase supplemented at 500 FTU/kg. Rutherfurd et al. (2012) and Chung et al. (2013) reported that the apparent ileal digestibility of amino acids was improved with phytase supplementation in low-P diets. Sebastian et al. (1997) reported that microbial phytase supplementation had no effect (p < 0.065) on the apparent ileal digestibility (AID) of CP or any AA except methionine and phenylalanine in male broiler chicken, whereas, in females, adding phytase increased the AID of all AA except lysine, methionine, phenylalanine and proline. Effect of phytase on amino acids digestibility is varied (Selle et al., 2000; Adeola and Sands, 2003), and it is clear that when phytase influences amino acids digestibility coefficients, it does not do so to the same extent for all amino acids. This may be linked to differential interactions between amino groups and phytate or it may be associated with the ability of phytate to increase the loss of endogenous compounds, such as mucins, that are rich in certain amino acids (Mansoori and Acamovic, 1998; Cowieson et al., 2004). Improved P digestibility and Ca digestibility in phytase-supplemented diet were reported by several researchers (Ravindran et al., 2006; Lalpanmawia et al., 2014). This is probably related to the release of minerals from the phytate bond as well as prevention of bounding with phytate. Further, Chung et al. (2013) also observed that the impact of phytase on the availability of minerals other than P is not definite. 98
Ayasan and Okan (1999) reported a value of 15.47– 20.68 mg of Ca/100 ml and 5.33–7.71 mg of P/100 ml of blood on feeding dietary levels of different calcium and phosphorus in laying Japanese quails. Shirley and Edwards (2003) reported that supplementation of phytase of 0–12 000 FTU/kg in low-P diet increased blood P level and reduced blood Ca level. It may be observed higher doses of phytase in low-P diet increased plasma p values probably more P released from phytate. However, reduced blood Ca levels indicated more utilization of Ca. Qian et al. (1996) observed higher tibial length with phytase supplementation at 600 FTU/kg in comparison with low-P diet. The bone length was higher probably due to the higher live weight of the birds pertaining to super dosing groups. Kocabagli (2001) found that the tibio-tarsal weight/length index was found to be significantly higher in phytase-supplemented groups than in control group. Rutherfurd et al. (2012) and Guo et al. (2009) reported that tibial weight was significantly higher in phytase-supplemented group. Rousseau et al. (2012) reported that tibial diameter/mid-shaft width was improved by phytase supplementation of 500 FTU/kg in low-P diets but no improvement in tibial bone length. Shirley and Edwards (2003) observed that phytase supplementation (0–12 000 FTU) in low-P diets improved tibia ash from 26 to 41%, tibia ash weight from 0.200 to 0.601 g/tibia, total phosphorus retention from 51 to 80%. The increase in tibial ash is considered to be a good indicator of bone mineralization (Sebastian et al., 1996; Leeson et al., 2000). This improvement in ash percentage in tibia can be related to the increase in Ca and P retention. Cowieson et al. (2006) observed that phytase supplementation (0–24 000 FTU) in low-P diet improved (p < 0.001) apparent retention of P, K, Na, Mg, Ca, S, Cu, Fe and Mn, which was higher than normal P-fed group. Guo et al. (2009) reported that phytase supplementation of 500 and 1000 FTU/kg in diets containing 2.0 g non-phytate phosphorus/kg linearly or quadratically increased performance and tibia ash of broilers during the whole growing period. Rutherfurd et al. (2012) reported that phytase supplementation of 1000 and 2000 FTU/kg in low-P diet increased both tibial mineral concentration and density by 35% and 24%, respectively, compared to low-P diet and was equal to that of adequate P diet. Walk et al. (2013) reported that super doses of phytase supplementation in low-P diet in 0- to 21-dayold broiler did not alter tibia ash percentage. Chung et al. (2013) observed that phytase supplementation
Journal of Animal Physiology and Animal Nutrition 100 (2016) 93–100 © 2015 Blackwell Verlag GmbH
DOI: 10.1111/jpn.12341
ORIGINAL ARTICLE
Effect of super dosing of phytase on growth performance, ileal digestibility and bone characteristics in broilers fed corn–soya-based diets M. Manobhavan, A. V. Elangovan, M. Sridhar, D. Shet, S. Ajith, D. T. Pal and N. K. S. Gowda National Institute of Animal Nutrition and Physiology, Bangalore, India
Summary A feeding trial was designed to assess the effect of super dosing of phytase in corn–soya-based diets of broiler chicken. One hundred and sixty-eight day-old broilers were selected and randomly allocated to four dietary treatment groups, with 6 replicates having 7 chicks per treatment group. Two-phased diets were used. The starter and finisher diet was fed from 0 to 3 weeks and 4 to 5 weeks of age respectively. The dietary treatments were consisted of normal phosphorus (NP) group without any phytase enzyme (4.5 g/kg available/non-phytin phosphorus (P) during starter and 4.0 g/kg during finisher phase), three low-phosphorus (LP) groups (3.2 g/kg available/non-phytin P during starter and 2.8 g/kg during finisher phase) supplemented with phytase at 500, 2500, 5000 FTU/kg diet, respectively, to full fill their phosphorus requirements. The results showed that super doses of phytase (at 2500 FTU and 5000 FTU/kg) on low-phosphorus diet improved feed intake, body weight gain, ileal digestibility (serine, aspartic acid, calcium, phosphorus), blood P levels and bone minerals such as calcium (Ca), P, magnesium (Mg) and zinc (Zn) content. It could be concluded that super doses of phytase in low-phosphorus diet were beneficial than the normal standard dose (at 500 FTU/kg) of phytase in diet of broiler chicken. Keywords bone, broiler, nutrient utilization, phosphorus, phytase Correspondence A. V. Elangovan, National Institute of Animal Nutrition and Physiology, Bangalore, India. Tel: +91-9341380858; Fax: +91-80-25711420; Emails: [email protected]; [email protected] Received: 11 December 2014; accepted: 31 March 2015
Introduction Phosphorus (P) is the most expensive essential mineral for poultry. In plant-based diets, two-third of P is bound to phytate and not available to poultry. The presence of phytate can result in reduced efficiency of nutrient utilization increasing the cost of feeding and lead to environmental pollution. The best way to improve phosphorus utilization is with judicious use of natural resources by phytase enzyme supplementation in the diet of monogastrics. Phytase is used extensively and the generally recommended dose of phytase in poultry is 500 FTU/ kg diet (Selle and Ravindran, 2007; Cowieson et al., 2009; Pirgozliev et al., 2012; Lalpanmawia et al., 2014). Shirley and Edwards (2003) observed a quadratic increase in phytate P disappearance of almost 95% with increasing phytase dose to 12 000 FTU/ kg in corn-based diets. Further, the use of higher doses of phytase has been gaining interest not only due to more P release but also leaving less residual phytate and generation of myo-inositol with
vitamin-like/lipotropic effects (Shirley and Edwards, 2003). The use of super doses of phytase (>1000 FTU/kg of diet) has been shown to improve nutrient availability in poultry diets (Cowieson et al., 2006). The high doses of phytase may result in more liberated phosphate or restoration of P/Ca proportionate release with less residual phytate in the gut (Cowieson et al., 2011). Phytate forms complexes with multivalent cations to produce insoluble salts, this Ca–phytate complex is the weakest, and it has been reported to be the most beneficial by dietary phytase supplementation (Angel and Applegate, 2001). Chung et al. (2013) reported that the impact of phytase on the availability of minerals other than P is not definite. It is well established that available P requirement is 0.45 and 0.40% during starter and finisher phase. Hence, low-P (available P, 0.30%) group without phytase supplementation was not taken as reference or negative control. Moreover, our study was to compare the advantage of conventionally fed 500 FTU over super dosing under practical farming condition.
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