In the current poultry industry, phytases are commonly used as a feed supplement to increase the absorption of phosphorus, reduce the cost of feed and, consequently, to reduce phytate excretion into the environment [22]. In addition, there is evidence that the use of phytases as a feed additive has a positive effect on the productive characteristics of poultry [3]. This could be due to improved phytase-mediated utilization of phosphorus, Ca2+ and amino acids [23]. The AgpP Pantoea sp. 3.5.1 phytase used in our study belongs to the family of histidine acid 3-phytases [24], which includes enzymes successfully used as feed additives [18]. Our previous study showed that Pantoea sp. 3.5.1 phytase expressed in yeast Pichia pastoris (termed AgpP-P) has a high thermostability, a wide range of pH values and temperatures at which the activity and stability of the enzyme is maintained (Suleimanova, In Press). These properties make AgpP-P phytase a good candidate for the use as a feed additive. In this study we investigated the effect of AgpP-P phytase supplementation on productivity, nutrient availability, veterinary-sanitary assessment of meat and serum biochemical parameters of Hubboard broilers.
Effect of AgpP-P phytase supplementation on digestibility and absorption of nutrients and productivity of broilers
It is known that phytate can have an inhibitory effect on the activity of many endogenous digestive enzymes secreted in the gastrointestinal tract of animals by chelating the co-factors necessary for the activity of the enzymes [19]. Phytase addition can potentially improve utilization of nutrients in the feed. Our results showed that AgpP-P supplementation resulted in overall increase in digestibility coefficient of protein, dry matter, fiber and fat in phytase-fed broilers. It was particularly effective in the increase of digestibility of fiber (by 26.2%, P < 0.05, Table 2). Previously, Zhang et al. (2000) showed that phytase supplementation of broilers feed in concentration 250–2500 FTU/kg improves the digestibility of dry matter (DM) [25]. However, Lan et al. (2002) reported that phytase has to be added to the feed in a concentration range of at least 500–1000 U/kg to significantly increase DM digestibility. Improvements in digestibility of crude fiber and crude protein in broilers receiving phytase was showed by Attia et al. (2002) and Abd-Elsamee (2002) [27, 28]. In contrast, Ptak et al. (2013) reported that adding phytase did not affect fat digestibility [29].
Table 2
Effect of phytase supplementation on nutrient digestibility
Digestibility coefficient | Control group | Phytase-fed group |
Protein | 74 ± 0.6 | 74 ± 0.6 |
Dry matter | 72.6 ± 0.9 | 72.6 ± 0.9 |
Fiber | 10.3 ± 0.4 | 10.3 ± 0.4 |
Fat | 58.5 ± 0.9 | 58.5 ± 0.9 |
Phytate-bound phosphorus has the ability to bind and form insoluble complexes with minerals necessary for metabolism, and thus such complexes cannot be properly used by the organism [30]. We determined the effect of phytase on the balance and the absorption coefficients of Ca, P and N (Table 3). Supplementation of AgP-P increased P and Ca digestibility in broiler chickens by 38.2 and 4.4%, respectively (P < 0.05). Our results were similar to those obtained earlier by Selle et al. (2009) and Chung et al. (2013) [31, 32]. This increase in Ca and P availability is due to the fact that phytase liberates these minerals from the Ca-phytate complex, rendering them available for absorption [33]. In turn, phytase supplementation had no effect on absorption and digestibility of N, which was also shown by Ptak et al. (2013) and Hao et al. (2018) [29, 34].
Table 3
Effect of phytase supplementation on absorption of calcium, phosphorus and nitrogen
Indicator | Control group | Phytase-fed group |
| Ca | |
Ingested, g | 1.20 ± 0.01 | 1.20 ± 0.006 |
Excreted, g | 0.52 ± 0.007 | 0.49 ± 0.003 |
Digested, g | 0.68 ± 0.005 | 0.71 ± 0.008 |
Digestibility coefficient, % | 56.6 | 59.1 |
| P | |
Ingested, g | 0.75 ± 0.003 | 0.74 ± 0.003 |
Excreted, g | 0.53 ± 0.002 | 0.44 ± 0.002 |
Digested, g | 0.22 ± 0.006 | 0.30 ± 0.004 |
| N | |
Ingested, g | 3.17 ± 0.04 | 3.12 ± 0.03 |
Excreted, g | 1.48 ± 0.005 | 1.44 ± 0.005 |
Digested, g | 1.69 ± 0.008 | 1.68 ± 0.006 |
Digestibility coefficient, % | 53.3 | 53.8 |
Reduction of P excretion is particularly important in the reduction of P pollution by poultry manure. In the present study, the increase of P retention resulted in a significant decrease in P excretion. Compared to the control group, P excretion in broilers receiving phytase was reduced by 16.9% (P < 0.05). Earlier, Lan et al. (2002) reported that addition of phytase at a concentration of 500 U/ kg of feed reduced the excretion of phosphorus by 56.8% and 59.1% for days 11–13 and 18–20, respectively [26]. Therefore, we have shown that AgpP-P supplementation improves the digestibility and absorption of feed ingredients and reduces the excretion of phosphorus in broiler chickens.
The results of phytase supplementation on growth performance of poultry are presented in Tables 4 and 5. Results showed that addition of AgpP-P during the first 2 weeks of chicken life did not affect the weight gain. This could be linked to the low feed intake by chickens during the said period of growth. Starting from week 3, however, phytase supplement-fed chickens gained on average 18 grams more compared to the control group. By the end of week 5 the difference in weight gain between two groups reached 59.5 grams which correspond to 4% increase (P < 0.05). However, it is interesting that, along with such a small increase in broiler body weight gain, the amount of total feed consumed by broilers when AgpP-P was added to the diet of was reduced compared to the control groups (Table 5). We suppose that the phytase hydrolyses the phytate and reduces its anti-nutritional effects, therefore enhancing the efficient utilization of feed, which is very important from an economic point of view. The increase in poultry weight gain following the addition of phytase has been shown by several researchers. Shirley and Edward (2003) found that supplementing phytase from 0 to 12000 U/kg significantly increased body weight gain from 287 to 515 g/chick [35]. De Sousa et al. (2015) reported that broilers fed on the phytase supplemented diet showed 4.40, 11.04 and 7.14% (P < 0.05) improvement in feed intake, weight gain and feed conversion ratio, respectively [36]. Moreover, Lim et al. (2001) in a study demonstrated that inclusion of phytase in feed increased weight gain and reduced mortality [37].
Table 4
Change of broilers weight (g)
Indicator | Control group | Phytase-fed group |
0 w | 22.1 ± 0.21 | 22.5 ± 0.20 |
1 w | 297.6 ± 0.35 | 306.8 ± 0.36 |
2 w | 370.8 ± 1.14 | 373.6 ± 1.02 |
3 w | 572.3 ± 1.80 | 591.1 ± 2.20 |
4 w | 926.0 ± 4.55 | 978.5 ± 3.44 |
5 w | 1488.3 ± 8.67 | 1547.8 ± 7.98 |
Table 5
Growth performance of broilers
Indicator | Control group | Phytase-fed group |
Initial weight (g) | 22.1 ± 0.21 | 22.5 ± 0.20 |
Final weight (g) | 1488.3 ± 8.67 | 1547.8 ± 7.98 |
Absolute gain (g) | 1466.2 ± 3.46 | 1525.3 ± 3.98 |
Feed intake (g) | 2521 ± 7.48 | 2481 ± 8.01 |
Effect of AgpP-P phytase supplementation on biochemical parameters of chicken serum
Results of biochemical analysis of blood serum are shown in Fig. 1.The concentration of total serum calcium (Ca) in AgpP-P supplemented group was consistently higher compared to control group (Fig. 1A). In contrast, other authors have reported that phytase supplementation had no effect on Ca level in the serum [26]. AgpP-P supplementation in the experimental group also increased levels of inorganic phosphorus (P) in the serum. The level of P was significantly elevated in the serum of 20-old-days chickens feeding on AgpP-P diet and by day 35 post-hatch this indicator was higher by 12.2% (P < 0.05) in the serum compared to the age-matched chickens from the control group (Fig. 1B). In agreement with our data, multiple studies have also highlighted elevations of P concentrations in serum when chicken diet was supplemented with phytase [9, 26, 38].
Phytate supplementation of chicken feed increased the total serum protein content from days by the end of the experiment. In the experimental group, this indicator was higher by 5.6% (P < 0.05) in comparison with the control group (Fig. 1D). With the accumulation of protein in the blood, there was a decrease in the concentration of urea. From days 5 to 20, the urea content in the control and experimental groups decreased by 7.5% and 11.6% (P < 0.05), respectively. A similar observation was made when analyzing data for the 35th day (Fig. 1C). Urea is an indicator of the cost of the entire protein stock of the body. A decrease in the rate of urea synthesis thus reflects a higher accumulation of protein in the blood. In contrast with these findings, Wang et al. (2013) reported no effect of phytase on serum total protein and urea nitrogen [38].
Dietary inclusion of AgpP-P decreased serum aspartate aminotransferase (AST) activity by 2.9% at 35 d of age (P < 0.05) (Fig. 1E). However, in contrast to mammals, activity of AST is not liver-specific in birds [39]. Elevated activities usually indicate liver or muscle damage, but no particular significance is associated with low AST activity. The phytase supplementation caused an increase in serum alanine aminotransferase (ALT) activity by 3.7% (P < 0.05) (Fig. 1F). Plasma ALT activity has been reported to be low in all tissues of chickens [40], but ALT activities often increase due to damage in many tissues [41]. Therefore, specific diagnostic value of these enzymes in birds is poor. The phytase supplementation decreased serum alkaline phosphatase activity (Fig. 1G). Similar results have been reported by Atia et al. (2000) in turkeys and Viveros et al. (2002) in chickens [9, 42]. The decrease in serum alkaline phosphatase activity is associated with diets supplemented with phytase might reflect the downregulation of this enzyme resulting from the increased availability of phosphorus. However, Wang et al. (2013) showed that plasma alkaline phosphatase activity was not affected by phytase in broiler chicks [38].
Effect of AgpP-P phytase supplementation on veterinary-sanitary assessment of broiler meat
Veterinary and sanitary examination of broiler chicken meat was conducted according to Russian State Standards 7702.0–74, 7702.1–74 and 7702.2–74. (Tables 6, 7). Carcasses of chickens from the control and AgpP-P supplemented groups inspected 24 hours post sacrifice were found to have a similar appearance: dry whitish-yellow skin with a pink tint, the muscles were dense, elastic in texture, slightly moist on the site of cut. Chicken breast muscles and dark meat had a normal appearance. The odor from the surface and from the incision of muscles corresponded to that of a standard fresh meat. Chicken fat appeared to be pale yellow, elastic without any extrinsic odors. The broth, made from the meat of both groups, was transparent, fragrant, with a pleasant smell, drops of fat were observed on the surface of the broth. Analysis of fat tissue parameters (peroxide and acid number) from both groups met Russian State Standards for human consumption without any restrictions.
Interestingly, the pH value of the dark meet collected from the AgpP-fed chickens was slightly higher in comparison with pH of the dark meet collected from the control group. Previously it was notices that the meat of broilers fed on phytase supplements has decreased pH values Hao et al. (2018) (Table 6) [34]. It is known that the activity of muscle tissue peroxidase is manifested in weak acidic medium, which is seen in fresh meat. Therefore, the reaction to peroxidase is one of the important sanitary assessment indicators of meat quality. Reaction to peroxidase of the meat samples collected from both groups was positive. In a contrary, the reaction to the products of the primary breakdown of proteins, ammonia and ammonium salts was negative for both chicken groups (Table 6).
Table 6
Physico-chemical parameters of broiler chicken meat
Indicator | Control group | Phytase-fed group |
Leg muscle | Breast muscle | Leg muscle | Breast muscle |
рН value | 5.70 ± 0.04 | 5.71 ± 0.09 | 5.83 ± 0.03 | 5.65 ± 0.17 |
Peroxidase reaction | + | + | + | + |
Reaction to products of primary protein breakdown | - | - | - | - |
Reaction to ammonia and ammonium salts | - | - | - | - |
«+» positive reaction; «-» negative reaction |
The content of volatile fatty acids is another important indicator of the sanitary assessment of meat quality. According to Russian State Standard GOST 7702.1–74 the content of volatile fatty acids for fresh, high-quality poultry meat, should not exceed 4.5 mg KOH/g (GOST 7702.1–74 «Poultry meat. Methods for chemical and microscopic analysis of meat freshness»). The analysis showed that for the meat samples collected from both groups, this indicator was within 1.92–2.08 mg KOH/g. The acid content of fat collected from chilled and frozen carcasses of all types of poultry should not exceed 1 mg KOH/g according to the same Russian State Standard (GOST 7702.1–74 «Poultry meat. Methods for chemical and microscopic analysis of meat freshness»). In agreement, the analysis showed that for the fat of the chickens from the control and phytase-fed groups, the acid numbers were 0.55 ± 0.02 KOH/g, and 0.59 ± 0.02 KOH/g, respectively. Microscopic analysis of the muscle surface in the samples collected from both groups showed the presence of isolated cocci and Gram-positive rod-shaped bacteria in the microscope field, which is an indicator of the freshness of the meat (SanPin 2.3.2.1078-01, Hygienic requirements for safety and nutrition value of food products, Health and hygiene rules and standards, 2002).
As shown in Table 7, indicators of the relative biological value of the meat of the experimental and control groups did not have any significant differences. The fluctuation of indicators occurred within a 99.0-99.2% range. The analysis showed that meat from any of the groups was not toxic for ciliate Tetrahymena pyriformis (Table 7). Normally, the number of altered cell forms ranges between 0.1 and 1.0% (Methodological Guidelines for the Toxic-Biological Evaluation of Meat, Meat Products and Milk using Tetrahymena pyriformis (1997)).
Table 7
The biological value of broiler chicken meat
Indicator | Control group | Phytase-fed group |
Relative biological value,% | 99.0 ± 0.2 | 99.2 ± 0.3 |
Toxicity to Tetrahymena pyriformis,% | 0.2 ± 0.02 | 0.1 ± 0.02 |
The analysis for heavy metal contamination (lead, cadmium, mercury, and arsenic) came back negative for samples collected from both groups of birds.
Thus, the meat of broiler chickens, which additionally received AgpP-P preparation to the ration meets the standards for human consumption and corresponds to Russia State Standards (GOST) for fresh, good-quality meat according to organoleptic, physicochemical and bacterioscopic characteristics.