Evaluation of Commercial 6-Phytases on Growth Performance, Bone Mineral Content, and Feed Digestibility of Broiler Chicks

Abstract

We evaluated the effects of 6-phytases on the growth performance of broilers (UK Chunky) under the recommended supplier-application dosage of each phytase. A nutritionally sufficient standard diet was administered as the base diets in the positive control feed. The diet in the negative control feed was designed by reducing total phosphorous, non-phytate phosphorus, and calcium by 0.1% to evaluate the effect of the nutrient restriction on broilers. Four 6-phytases were added to negative control feeds at the level of the recommended dosage of each product to compare the effect of phytases on broiler technical performance, tibia ash, and feed digestibility. Nine hundred one-day-old broiler chicks (males and females) were distributed in a completely randomized design composed of six treatments and three replicates of 50 chicks each. Chicks were fed ad libitum for 49 days. Body weight gain and feed intake were recorded on days 21 and 49, tibia ash was measured on day 21, and apparent ileal digestibility of dry matter, crude protein, and total phosphorus were analyzed on day 49. Birds reared with test feeds supplemented with phytase showed higher body weight gain and feed intake compared to those of the negative control birds. No significant differences in traits were observed among different phytase treatments. Similarly, the percentage of tibia ash increased when phytase was supplemented, resulting in higher bone levels compared to that of the positive control. The apparent ileal digestibility of crude protein and total phosphorus was enhanced by supplementing negative control diets with phytases.

Introduction

Plant materials in poultry feed store phosphorus (P) in the form of phytate-P (pP), which is largely unavailable to them and results in a reduction in nutrient digestibility and excretion of inorganic P into the environment (Kerr et al., 2010; Dersjant-Li et al., 2015). The enzyme phytase (myoinositol hexakisphosphate phosphohydrolase) catalyzes phytate hydrolysis and releases P, myo-inositol, and phytate-bound nutrients. It has been reported that 30%–40% of the excretion of inorganic P can be reduced without harming animal growth by the addition of phytase into feeds (NARO, 2012). Consequently, microbial phytase is commonly added to monogastric animal diets as a feed additive and accounts for 60% of the market share of enzymes for animal feeds (Adeola and Cowieson, 2011; Fuji Keizai Osaka market office, 2020). Since the first generation of commercial 3-phytase from Aspergillus niger was launched in 1996 in Japan, the number of registered products has increased (MAFF 2021). Recently, four 6-phytases have become commercially available in Japan as an alternative to 3-phytase (Fuji Keizai Osaka market office, 2020); however, the differences among the performance of 6-phytase products have not been evaluated. In this study, the effects of four 6-phytase products (administered at the supplier-recommended inclusion levels into feeds) on bird performance and ileal digestibility of nutrients were evaluated.

Materials and Methods

Experimental Chicks

One-day-old broiler chicks (UK chunky) were used in this study. Three hundred chicks (average body weight 42±0.19 g, 150 and 150 females, respectively) were classified into six groups. Each group contained 25 male and female chicks, and the total body weight of each group was similar between treatments. Fifty chicks were reared in a 3.3 m² pen with floor heating and 24 h lighting in a forced ventilation-type windowless poultry house. Feeds were provided ad libitum, and the floor was covered with sawdust. The experiments were repeated three times, and a total of 900 birds were used (6 treatments×3 repetitions×50 chicks).

The animals were reared according to the Guide for the Care and Use of Experimental Animals (Japan Scientific Feeds Association, ethical approval number: 819).

Feeds

Base diets were prepared by modifying the standard mixed feeds provided by the Japan Chunky Association (2014) (positive control; PC). The negative control (NC), quantity of total phosphorous (TP), non-phytate phosphorus (npP), and calcium (Ca) were reduced by 0.1% from the base diets by reducing dicalcium phosphate (Table 1).

Table 1. Composition and nutrient density of experimental diets.

Ingredients %	Starter feed 1–22 days of age		Grower feed 23–50 days of age
	PC	NC	PC	NC
Corn	54.84	55.88	61.04	62.16
Soybean meal	33.67	33.40	26.10	25.80
Rapeseed meal	4.00	4.00	4.00	4.00
Soybean oils	3.30	3.00	5.30	4.95
Dicalcium Phosphate	1.47	0.90	1.19	0.62
Calcium carbonate	1.02	1.10	0.88	0.95
Salt	0.36	0.36	0.37	0.37
Vitamin, mineral premix	0.25	0.25	0.25	0.25
Choline chloride	0.28	0.28	0.24	0.24
L-lysine hydrochloride	0.12	0.13	0.17	0.18
DL-methionine	0.33	0.33	0.23	0.23
L-threonine	0.07	0.07	0.02	0.03
L-arginine	0.18	0.19	0.17	0.18
L-isoleucine	0.06	0.06	0.03	0.03
L-valine	0.05	0.05	0.01	0.01
Total	100.00	100.00	100.00	100.00
Nutrient
CP (%)	21.550	21.537	18.520	18.506
ME (Mcal/kg)	3.006	3.007	3.202	3.201
Ca (%)	0.870	0.773	0.730	0.630
P (%)	0.659	0.559	0.577	0.477
NpP (%)	0.435	0.334	0.366	0.265

The composition is calculated from the Standard Tables of Feed Composition in Japan (2009).

CP: crude protein, ME: metabolizable energy, Ca: calcium, P: phosphate, NpP: non-phytate phosphorus.

Feeds for 1–22-day-old chicks contained 0.66% TP, 0.44% npP, 0.87% Ca, 21.6% CP, 1.15% av. Lys, and 3010 kcal/kg metabolizable energy (PC). The NC feed was similar, although it contained 0.56% TP, 0.33% npP, and 0.77% Ca. Feeds for 23–50-day-old chicks contained 0.58% TP, 0.37% npP, 0.73% Ca, 18.5% CP, 1.01% av. Lys, and 3200 kcal/kg metabolizable energy. The NC feed was similar although it contained 0.48% TP, 0.27% npP, and 0.63% Ca.

6-phytase

Four commercially available 6-phytase products were used in this study: Ronozyme HiPhos granule (DSM, Netherlands) as phytase A, Natuphos E granule (BASF, Germany), phytase B; Axtra PHY TPT2 (Dupont, USA) as phytase C, and OptiPhos G (Huvepharma, Belgium) as phytase D (Table 2). The recommended dose of each phytase was added to the NC feed.

Table 2. Overview of the four different 6-phytase used to supplement feeds.

	Product name	Distributer	Recommended dose
Phytase A	Ronozyme HiPhos	DSM Japan K.K (Tokyo, Japan)	1000 (FYT/kg)*
Phytase B	Natuphos E granule	BASF Japan Ltd. (Tokyo, Japan)	500 (FTU/kg)**
Phytase C	Axtra Phy 20000 TPT2	Dupont Japan Ltd. (Tokyo, Japan)	500 (FTU/kg)**
Phytase D	OptiPhos 4000 G	Huvepharma Japan Inc. (Kyoto, Japan)	250 (FTU Phytex/kg)***

^*One FYT is the amount of enzyme that liberates 1 µmol inorganic phosphate from phytate per minute under reaction conditions with a phytate concentration of 5.0 mM at pH 5.5, and 37°C.

^**One FTU is the amount of enzyme which liberates 1 µmol inorganic phosphate per minute from sodium phytate at pH 5.5 and 37°C.

^***One FTU-phytex is the amount of enzyme that catalyzes the release of 1 µmol inorganic phosphate per minute from 5,1 mM sodium phytate in pH 5.5 citrate buffer at 37°C, measured as the blue P-molybdate complex color at 820 nm.

Performance Test

Chicks were grown for 49 days from May 13, 2020, with feeds as described above. In the test feeds, each 6-phytase was added to the NCfeed at the supplier-recommended dose. Chromium oxide (Cr₂O₃) was added as an indigestible marker for ileal digestibility analysis in feeds of 23–50-day-old chicks at 0.1%. The body weight gain (BWG) and feed intake (FI) of the chicks in each pen were recorded every seven days during the trial. The data were then summed as the initial body weight and body weight on days 21 and 49. The feed conversion ratio (FCR) was calculated as FI/BWG.

Tibia Ash and Apparent Ileal Digestibility of TP

The left tibia was collected from four chicks with average weights (two male and two female each) from each pen on the day 21^st of rearing. Then, all bones were defatted by soaking in ethanol for 20 h, further petroleum ether for 20 h, drying for 4 h, and weight and ash contents measured following AOAC(2000). On day 49, the ileum from the vitelline diverticulum to a point 40 mm proximal to the ileocecal junction was dissected. The apparent ileal digestibility (AID) was estimated for 18 chicks (8 males and 10 females). DM, CP, and TP were measured following the AOAC(2000). Cr₂O₃ was measured according to the method of Takemasa (1992). AID was calculated using the following formula:

Statistical Analysis

Data were analyzed by ANOVA and significant (p<0.05) results were analyzes further by using the Tukey-Kramer HSD test (Yoshida, 1998).

Results

Performance

BWG and FI were significantly higher in the PC and all phytase-administered groups than those in the NC group. There was no significant difference between the PC and phytase-supplemented groups. All phytases improved growth performance, although there was no significant difference in BWG, yet there were considerable numerical differences among phytases (Table 3). FCR did not differ between the treatments.

Table 3. Results of growth performance and apparent ileal digestibility.

	P value (ANOVA)	Positive control	Negative control	Phytase A	Phytase B	Phytase C	Phytase D
BWG (g/chick)* 1–22 days of age	<0.001	856±21a	764±10b	876±25a	855±24a	883±13a	871±16a
BWG (g/chick)* 1–50 days of age	<0.001	3498±41a	3184±79b	3430±54a	3476±81a	3556±90a	3610±106a
FI (g/chick)* 1–50 days of age	0.015	5637±48a	5109±148b	5555±95a	5600±72a	5696±172a	5782±168a
FCR*	0.808	1.61±0.01	1.61±0.01	1.62±0.02	1.61±0.03	1.60±0.01	1.60±0.01
Tibia (g/chick)**	<0.001	2.31±0.21b	2.10±0.15c	2.41±0.30ab	2.40±0.20ab	2.57±0.20a	2.47±0.18ab
Tibia (g/100 g BW)**	0.127	0.266±0.015	0.255±0.013	0.268±0.025	0.265±0.015	0.274±0.015	0.272±0.017
Tibia Ash (Fat-free basis, %)**	0.003	37.8±1.8b	33.8±2.8c	40.1±2.9ab	39.6±1.3ab	41.0±1.0a	40.9±1.4a
AID DM (%)***	<0.001	71.0±1.9b	74.9±1.5a	74.7±2.6a	74.5±1.6ab	75.4±1.5a	75.9±2.0a
AID CP (%)***	<0.001	75.5±2.2b	78.6±2.6ab	80.2±2.7a	79.5±1.7ab	80.9±0.9a	80.7±2.2a
AID TP (%)***	<0.001	33.7±4.7c	44.8±4.4b	58.3±4.1a	58.3±4.1a	57.0±6.4a	58.4±5.7a

Except for body weight gain (BWG), the data reflect the final value after 49 days of treatment.

^*average value±SD (trial repetition, n=3)

^**average value±SD (number of chicks, n=12)

^***average value±SD (number of chicks, n=6)

Different alphabets indicate significance (p<0.05, Tukey-Kramer HSD test)

FI, feed intake; FCR, feed conversion ratio; AID, apparent ileal digestible; DM, dry matter; CP, crude protein; TP, total phosphorus

Tibia Bone Ash

Tibia ash content was significantly higher in the PC than in the NC. Similarly, tibia bone ash was significantly higher in phytase C and D groups than that in the PC group (Table 3, p<0.05). All phytase groups showed a higher tibia ash percentage than that of the NC group.

Apparent Ileal Digestibility of DM, CP and TP

Adding 6-phytase to the diet significantly increased the AID of DM, CP, and TP (p<0.05) compared to the PC, except for the phytase B group (Table 3). No significant differences were observed among the phytase groups or within the phytase groups and NC for AID of DM and CP. All phytase treatments improved the AID of TP by 13% compared to that of the NC.

Discussion

The effects of 6-phytases on broiler growth performance and DM, CP, and P digestibility were evaluated using four commercially available products in Japan at the recommended dose. All 6-phytase in feeds improved BWG and FI, but not FCR, of broiler chickens when phytase was added to the NC diet. No statistical difference in relative tibia weights indicated the well-balanced growth of the chicks in this study, and the dry weight of the tibia ash response supported the increase in AID of P observed (Table 3). This study demonstrated that the supply of 6-phytase accelerated the utility of phytic acid in feeds and effectively improved growth performance and P digestibility. In this study, we evaluated the effects of four 6-phytase products on growth performance. The recommended dose of each 6-phytase was as follows: phytase A, 1000 FYT/kg; phytase B and C, 500 FTU/kg; and phytase D, 250 FTU-phytex/kg. Although a different value with a unique unit was officially registered as a recommended dose, this was regulated to release 1 µmol of inorganic phosphate from phytate per minute under reaction conditions (Table 1) (MAFF 2021). Thus, it is natural that the results showed supplementation with phytases was efficient in restoring growth performance and tibia ash to the same level as those observed in the PC; however, no significant difference in any criterion among the 6-phytase addition groups was observed. Alternatively, some variations were noted in the AID of DM, CP, and tibia ash among the 6-phytase groups, which reflect the features of the products. The negative effect of reducing dietary TP, npP, and Ca on growth was alleviated via FI, AID of TP, and tibia ash.

Conflicts of Interest

The authors declare no conflicts of interest.