Phosphorus (P) is an essential macro element that regulates the metabolic and physiological functions of a living organism. The P derived from plant components in the diet is only partly digestible by monogastric animals, because P is mainly stored in the form of phytate. Phytase enzymes are used in animal feeds to release P from phytate, and have been used commercially since the 1990s in both pig and poultry diets. Initially, phytase was added to reduce P excretion in litter and faecal material, although research has shown the enzyme could spare other nutrients in the diet, as phytate binds protein and various minerals, and has been shown to interfere with natural enzyme secretion activity (Selle et al., 2012). Moreover, as an essential nutrient, especially for growing animals, P is one of the most expensive feed components when provided as inorganic form of phosphates (30 - 50% of total P) and inclusion of phytase can reduce the inclusion of phosphates, leading to a financial benefit.
Positive effects with the application of exogenous phytase of different types and at various levels (FTU/kg) have been reported over the years. The first commercially available phytase was 3-phytase derived from Aspergillus niger, but in the last two decades, 6-phytase originating from Escherichia coli, Peniphora, Citrobacter, or Buttiauxella spp. have become commercially available. Phytase has been shown to improve performance, nutrient digestibility (ileal and faecal) and bone mineralisation in piglets and pigs in a dose responsive way (Dersjant-Li et al., 2017; Kühn et al., 2016; Kühn and Partanen, 2012; Torrallardona and Ader, 2016).
Commercial phytases can have different efficacies, but there are no standardised trial protocols to assess their potency, which makes it difficult to compare efficiency. Comparison of phytases at different inclusion levels has been mainly done in poultry (Leyva-Jiminez et al., 2019) while, in pigs, the smaller size of trial facilities limits such research. However, some publications exist on the topic. In a trial conducted by Guggenbuhl et al. (2016), starter and grower-finisher pigs were used to compare three phytases at varying inclusion levels. All three commercial phytases improved average daily gain (ADG), but effective levels differed in both piglets and grower-finisher pigs.
The effective inclusion rates for phytase has been the subject of much scrutiny, especially as the price of inorganic P sources has increased considerably in the last decade, and there has been a need for increased use of phytase to keep feed formulations economically viable. In a review (Dersjant-Li et al., 2015) dose response was examined in detail, and showed less than 60% phytate breakdown in the gut of pigs when 'standard' levels of 500 phytase units (FTU) were applied per kg of feed, and that higher doses were much more effective. Recently, it has been observed that the level and the source of calcium (Ca) has an impact on the efficiency of phytase in pigs, which means that this should be considered when evaluating the enzyme (Schlegel and Gutzwiller, 2016).
The aim of this study was to evaluate increasing levels of a novel 6-phytase derived from the E. coli appA2 gene (Huvepharma®, Belgium) at levels of 0, 125, 250, 500 amd 1,000 FTU/kg in the diet of piglets to determine the effects on performance, apparent total tract digestibility of Ca, P and crude protein (CP) and bone mineralisation.
Materials and methods
The experiment was approved by the Ethical Committee for the use of Animals in Feeding Studies at the Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Animal Sciences Unit, Belgium (ILVO; EC-2018/326).
In total, 150 piglets (male and female) obtained from crossing a Piétrain boar and a RA-SE genetics hybrid sow were used in this experiment. In one weaning round, six pens (three containing five barrows and three containing five gilts) were allocated to each of the five dietary treatments at weaning (four weeks of age; average body weight (BW) 8.2 kg), giving 30 pigs per treatment. All pigs were ear tagged for individual identification. The pens measured 1 m x 1.8 m to give 0.36m2/pig.
Feed and treatments
The trial compared five dietary treatments. The piglets received the weaner diets from four to six weeks of age and the pre-starter diets between six and 10 weeks of age. Feed and water were provided ad libitum throughout the study. Each diet was produced from the same batch of basal feed, and differed in the presence or absence and the concentration of the phytase (OptiPhos® Plus) developed from the appA2 gene from E. coli as described in Rodriguez et al., (1999) and expressed in Komatagaella phaffii (Huvepharma®, Belgium).
The five treatment diets were denoted:
- T1 - no added phytase (negative control diet)
- T2 - 125 FTU/kg
- T3 - 250 FTU/kg
- T4 - 500 FTU/kg
- T5 - 1,000 FTU/kg
The basal diet (T1) was reduced to 1.5 g digestible P in both weaner and pre-starter feed to create a deficiency, while Ca levels were maintained at 5.5 and 5.6 g/kg for weaner and pre-starter feed, respectively. Then, the basal feed batch was divided into five equal parts, and the four enzyme doses were premixed with 50 kg of feed before being added and mixed into the basal diet to obtain the respective concentrations. All diets were provided in pelleted form. A digestibility marker (0.5% titanium oxide; TiO2) was included in the pre-starter diet. The diets were formulated to meet commercial recommendations (CVB, 2016) except for digestible P and Ca, and were produced by ILVO Belgium. The basal composition is shown below in Table 1.
Piglets were monitored daily to record health, mortality and performance. Piglets were weighed individually at the start of the trial (at four weeks of age) and at the end of the experimental feeding period (10 weeks of age). The feed intake per pen was recorded at the same time points and average daily feed intake (DFI) was calculated. Feed conversion ratio (FCR) was calculated per pen.
During the fifth week of the trial (when piglets were nine weeks old), faecal samples from the rectum were collected daily from two randomly selected piglets per pen for four consecutive days and frozen at -20 oC. The samples were pooled per pen, freeze-dried, ground and stored ready for analysis.
Determination of bone parameters
Four days after the final weighing at 10 weeks of age (pre-starter feeding was continued), one piglet (closest to the average pen BW) per pen was euthanised to assess the effect of the dietary treatments on bone quality. The right front foot from the animal was excised and stored at -20oC. Frozen feet were placed in a beaker (500 ml) filled with warm water with the claws upwards to prevent charring of the metacarpal bones. The beaker was then placed in a warm water bath (75oC) to soak for 24 hours. After 24 hours, the metacarpal bone IV was collected and surrounding tissue removed. The bone was weighed, oven dried for 16.45 hours at 65oC, and reweighed. The length of metacarpus IV (interior side) was measured using digital callipers. The metacarpal bone was gently cracked and stored at -20oC. The cracked metacarpal bone was defatted by extraction with petroleum ether (boiling point 40 - 60oC, ISO 6492A), dried at 103oC and incinerated at 650oC to constant weight according to the methodology described by Bikker et al. (2013). The ash content of the fat free dry matter was calculated based on the weighed bone before and after incineration.
Feed and faecal samples were ground through a 1 mm screen and analysed using accredited methods at the Animalab of IVLO-Animal Sciences Unit, Belgium. Crude protein (N x 6.25) was determined according to Kjeldahl (ISO, 2009a), TiO2 by colorimetry (Myers et al., 2004), phytate P by the method of Haug and Lantzsch (1983) and total Ca and P by inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 511 VDV, Agilent Technologies, Inc., Santa Clara, CA, USA). Moisture was determined by drying at 103oC (EC, 1971), crude ash was obtained by incineration at 550oC (ISO, 2002), crude fat was extracted with petroleum ether after hydrolysis with HCl (ISO, 1999), crude fibre was obtained using an Ankom Fiber Analyser (Ankom Technology, Macedon NY, USA) after boiling subsequently with sulphuric acid and sodium hydroxide (EC, 1992). The results of the feed analysis are shown in Table 2 and were in line with the calculated nutrient composition.
The final trial diets were analysed at Biovet JSC (Sofia, Bulgaria) for phytase activity (ISO, 2009b). A small amount was found in the negative control diets due to endogenous phytase from feed ingredients (Table 3). The actual included levels were then corrected for this endogenous activity, and were higher than expected. However, control of the good manufacturing procedure for feed production showed that the right amount of phytase was added to each treatment. It is well known that phytase analysis can provide quite variable results particularly at lower levels. For instance, two collaborative studies between 14 (study 1) and 13 (study 2) laboratories to validate a colorimetric assay for determination of microbial phytase activity in feed at levels of 200 to 400 FTU/kg (in different types of feed) showed that reproducibility relative standard deviation values ranged from 14.1 to 27.6% in study 1 and 14.0 to 20.5% in study 2 (Engelen et al., 2001).
Apparent total tract digestibility
Faeces and feeds were analysed for Ca, P, CP and TiO2 as external marker for deriving digestibility. The faecal digestibility coefficients (%) were calculated using the following formula:
Statistical analysis was performed using R (R Core Team, 2017). Each pen was considered as an experimental unit. A linear and quadratic dose-response model was fitted with the supplemented phytase as independent continuous variable (0, 125, 250, 500 and 1,000 FTU/kg), and used to calculate the responses per increment of phytase added to feed. Bodyweight at the start (for analysis of performance) or bodyweight at the end (for analysis of bone mineralisation) were considered as covariables. Results were considered as significant where P < 0.05.
Results and discussion
During the trial, one piglet in the T3 diet group died in the weaner phase, and three piglets (one each from treatments T1, T2 and T4) died in the pre-starter phase. This was not related to the dietary treatment. At the end of the trial, some joint stiffness was observed at levels of 38, 3, 0, 3 and 0% in piglets in T1, T2, T3, T4 and T5 fed groups, respectively. This problem was highest in piglets receiving the negative control diet (T1) with reduced P levels, and improved with the addition of phytase in all the other groups.
Across the whole trial period, the statistical model predicted a linear increase for DFI, ADG and BW with rising levels of phytase (from 0 to 1,000 FTU/kg; Table 4, Figure 1). BW at the end of the trial increased by 824 g per increase of 250 FTU/kg feed of phytase (p < 0.001). ADG and DFI followed the same pattern, increasing by 19 g/day (p < 0.001) and 18 g/day (p < 0.05) per increase of 250 FTU/kg feed of phytase, respectively. In addition, as the effect on ADG and final BW was slightly higher at the lowest doses, with diminishing effect on higher doses, a significant quadratic effect for these parameters was observed, within the measured range of phytase levels in this study (p = 0.029 and p = 0.028, respectively). A reduction of 0.03 in FCR was observed per increase of phytase level with 250 FTU/kg feed (p < 0.01; Table 4, Figure 1).
These results were in agreement with the findings reported by Dersjant-Li et al. (2017), who found that supplementation with 500, 1,000 and 2,000 FTU/kg Buttiauxella phytase in a P and Ca-deficient diet (1.5 g/kg) increased ADG and gain to feed (G:F) ratio in piglets 14 days post weaning. This result had been previously observed by Zeng et al. (2015), who found that supplementation with 500, 1,000 and 20,000 FTU/kg Buttiauxella phytase to a feed reduced in Ca and total P (-1.6 g/kg) linearly improved ADG and reduced feed:gain ratio in a four-week feeding trial. Over a six-week trial using weaned piglets, Torrallardona and Ader (2016) statistically confirmed significant linear responses for the addition of phytase (125, 250, 500 and 1,000 FTU/kg) on ADG (p < 0.001), DFI (p < 0.01) and G:F (p < 0.05) in a feed reduced to 1.7 to 1.9 g P per kg.
The performance improvements seen in the current trial and from published data can be attributed to the release of phytate-bound P replacing the deficient levels in the negative control diet. This trial did not have a positive control diet (e.g. where P levels were present at adequate quantities), however other studies (Dersjant-Li et al., 2017; Torrallardona and Ader, 2016; Zeng et al., 2015) showed that phytase supplementation regained the performance lost in the negative control diet back to match the performance seen in the positive control diets.
The digestibility results for each of the five treatment diets are shown below in Table 5. Apparent total tract digestibility (ATTD) of P, Ca and CP linearly improved with increasing levels of phytase (0 to 1,000 FTU/kg feed). From the regression analysis, each increase of 250 FTU/kg phytase resulted in an increase of 6.5, 2.8 and 0.4% in ATTD of P (p < 0.001), Ca (p = 0.005) and CP (p < 0.05), respectively. For P, a quadratic effect could be observed (p = 0.005), indicating a diminishing response with increasing doses.
These findings were in agreement with other trials (Dersjant-Li et al., 2017; Guggenbuhl et al., 2016; Kühn and Partanen, 2012; Rutherfurd et al., 2014). In a trial using 6-phytase supplementation at levels of 250, 500, 1,000 and 2,000 FTU/kg feed, Velayudhan et al., (2015) showed a significant increase in ATTD of Ca and P of 18.2, 30.4, 24.5 amd 33.8% and 46.8, 98.4, 99.7 and 125.3%, respectively with increasing phytase inclusion (p < 0.05). In their trial, a significant increase in CP digestibility was noted at all inclusion levels, which was not the case in the current trial. A higher, but not significant, ATTD for CP was observed by Zeng et al., (2015) in weaned piglets, using the same phytase at 500 and 1,000 FTU/kg as Velayudhan et al. (2015). However, in the study by Zeng et al. ( 2015), phytase significantly increased ATTD of Ca from 46.0 to 60.7% and ATTD of P from 37.7 to 59.3% at an inclusion level of 1,000 FTU/kg. The higher Ca digestibility due to phytase could be linked to the liberation of Ca complexed on the phytase molecule (Rutherford et al., 2014).
A treatment effect was observed in the dry weight and crude ash of the metacarpus IV (right front leg), whereby bone weights and ash concentrations linearly increased as phytase level increased from 0 to 1,000 FTU/kg feed (Table 6). From the regression analysis, each increase of 250 FTU/kg feed of phytase increased bone dry weight by 131 g (p < 0.05) and ash concentration by 1.9% fat free dry matter (p < 0.001). For dry weight, a quadratic effect was observed (p = 0.013), indicating a diminishing response with increasing doses. A quadratic effect of treatment on bone length was observed (p = 0.014).
The effect on bone development was logical, considering the increased digestibility of P and Ca (Table 5), and was in line with other research, whether assessed in metacarpal bones (Gourley et al., 2018; Torrallardana and Ader, 2016; Zeng et al., 2015) or any other part of the skeleton as e.g. ribs (Kühn et al., 2016). As supplementation with phytase had no effect on fresh weight of the bone, it appeared that Ca and P deficiency resulted in a lower dry matter content in the bone, while phytase supplementation led to better mineralisation of the bone and, thereby, improved bone strength.
Increasing the dietary level of phytase (OptiPhos® Plus) in graded levels up to 1,000 FTU/kg linearly improved growth performance (BW, ADG, DFI and FCR), which was attributed to a linearly increased digestibility of P, Ca and CP. This resulted in better bone mineralisation (dry weight and crude ash concentration) in comparison to a down-specified Ca and P negative control diet.
References are available on request.
This article originally appeared in the Journal of Applied Animal Nutrition, 2020 online.