Traditionally, fishmeal has been the main protein source used in aquatic feeds (Luthada-Raswiswi et al., 2021) due to its high protein content, amino acid profile and palatability which make it suitable for piscivorous fish species (Rolland et al., 2015). However, the increased price of fishmeal and the inconsistency of global supply has meant that plant proteins have gained significant interest (Burel et al., 2000; Carter and Hauler, 2000; Chou and Cai, 2004; Hansen et al., 2007; Opstvedt et al., 2003; Øverland et al., 2009).
Plant-based proteins are an interesting alternative due to their high availability, competitive pricing, and nutritional profile (Gatlin et al., 2007; Hardy, 2010) and have shown promise in several species (Egerton et al., 2020; Hansen and Hemre, 2013; Hartviksen et al., 2014; Kaushik et al., 2004; Lassarotto et al., 2018; Taylor et al., 2019; Torrecillas et al., 2017; Vera et al., 2020). Despite noted success in aquafeeds, antinutritional factors present in plant materials present a constraint to their use (Kumar et al., 2012).
Phytate is a free form of inositol hexakisphosphate (IP6) and a polyanionic molecule with six phosphate groups. It is one of the major anti-nutrients in plant-based ingredients. Phytate has been shown to adversely affect the absorption and digestion of minerals in fish (Papatryphon et al., 1999) and its excretion has been shown to be environmentally damaging (Baruah et al., 2004). Despite negative effects, phytate acts as a potential source of phosphorus (P) within feeds, consisting of two thirds of the total P in plant-based ingredients (Singh, 2008). Current sources of inorganic P, such as monocalcium phosphate (MCP) are non-renewable and expensive (Mullaney et al., 2000; Lee et al., 2020) and so accessing phytate bound P has garnered a large amount of interest to maximise the nutritive values of plant-based ingredients as well as reducing the negative effects associated with their use.
The use of phytase supplementation is now established in the aquaculture industry (Adeola and Cowieson, 2011; Kumar et al., 2012) and though characterisation of phytase in vitro is widely available, a broad range of factors have been shown to influence the efficacy of phytases in vivo. Factors such as environmental temperature, pH, dose, fish species, complementary dietary ingredients and phytase source have all been shown to influence efficacy (Cao et al., 2007; Dersjant-Li et al., 2015; Greiner and Konietzny, 2006; Kumar et al., 2012; Lee et al., 2019).
The current trial assesses the efficacy of Escherichia coli phytase (OptiPhos; Huvepharma) on the growth performance, digestibility and retention in Atlantic salmon (Salmo salar).
Materials and methods
Diet
A basal diet was formulated with moderate levels of marine derived proteins and high levels of plant protein sources (Table 1). The trial comprised of three experimental diets:
- a positive control treatment (PC) consisting of a basal formulation supplemented with monocalcium phosphate (MCP)
- a negative control diet (NC) without MCP supplementation
- a phytate dosed diet containing 750 OTU/kg (1500 FTU/kg) of OptiPhos
Diets were manufactured by extrusion (pellet size 4.5 mm) at SPAROS - Portugal facilities, with a screw diameter of 55.5 mm and temperature range of 108 - 113oC. Upon extrusion, both batches of extruded feeds (PC and NC formulations) were dried in a vibrating fluid bed dryer and allowed to cool to room temperature. Subsequently OptiPhos and oil were applied by coating under vacuum conditions. For the post-extrusion coating procedure of enzyme and oils, the target amount of test enzyme (OptiPhos 8000L) was diluted in 2.5% demineralised water, emulsified with the oil on a high-shear mixer and sprayed onto the pellets under vacuum (760 mbar) for approximately 3 minutes. The PC and NC diets without enzyme supplementation were also coated with the oils, using the same procedure. Representative samples of each diet were taken for proximate composition analysis and quantification of supplemental enzyme. Throughout the duration of the trial, experimental feeds were stored in cold storage at room temperature.
Experimental setup and growth fish performance
The trial was conducted at Gildeskal Forskiningstasjon AS, Inndyr, Norway (GIFAS). A total of 984 Atlantic salmon (S. salar) were selected from GIFAS' production stock and were randomly allocated to twelve square 5x5x5 m experimental sea cages with a volume of 125 m2 (67.0319oN, 14.0281oE, Inndyr, Norway). Fish were subjected to a freshwater bath at transfer as a prophylactic measure against seawater and no mortalities were observed in association with the transfer to the experimental cages. Fish were placed in quarantine for 3 weeks for observation and acclimation to the new experimental conditions (temperature: 10 ± 1oC; dissolved oxygen: >8.2 mg/L). During this period fish were fed a 4.5 mm commercial salmon diet by hand in two daily meals at approximately 1.5% biomass daily.
Each triplicate group of 82 post-smolt Atlantic salmon, with a mean initial body weight (IBW) of 163 ± 14 g were fed one of three experimental diets (Table 1) over 61 days. During the trial period, cages were subjected to natural temperature, water quality and photoperiod conditions (temperature 12.8 ± 1.3oC; salinity: 32.4 ± 0.5 ppt; dissolved oxygen: >5.9 mg/l). Fish were hand fed to visual satiety over two meals per day (0800 and 1400h).
Digestibility and biological sampling
At the end of the growth performance trial and following all associated sampling, the remaining fish were used to determine the apparent digestibility coefficients (ADC) of P and phytate. Diets containing 0.5% yttrium oxide were fed to groups of the remaining fish (average weight 390 g). Fish were fed for one week to allow for adaptation to the feeds and then faeces were collected via stripping. Faeces from each fish were combined into a plastic container and stored frozen at -20oC prior to subsequent analysis.
To assess whole body P and bone ash content, whole fish from the initial stock and whole fish at the end of the trial were sampled and stored at -20oC for subsequent analysis. Fish vertebrae (feed from soft tissue) were collected at the start and at the end of the trial and frozen at -20oC for analysis of bone ash.
Measurements
Analysis of feed, whole body and faeces were carried out with analytical duplicated methods following the methodology described by AOAC (2006).
Analysis of dry matter, total ash, crude protein, crude lipid, gross energy, total P, calcium, phytate-P and yttrium were performed at the University of Porto, Portugal.
Phytase activity in the feeds was measured by Biovet, Bulgaria. Activity was measured with a unit of phytase activity (OTU) being defined as the amount of enzyme that catalyses the release of 1.0 micromole of inorganic phosphate per minute from a 5.1 mM sodium phytate in pH 5.5 citrate buffer at 37oC., measured as the blue molybdate complex colour at 820 nm.
Performance parameters were calculated as follows, where FBW represents final mean body weight (g) and IBW represents initial mean body weight (g).
Specific growth rate (SGR, %/day): LnFBW-LnIBW) x 100 / days
Feed intake (% BW/day): ((crude feed intake/(IBW + FBW)/2))/days) x 100
Feed conversion ratio (FCR): crude feed intake / weight gain
Protein efficiency ratio (PER): wet weight gain / crude protein intake
Protein retention: 100 x (FBW x final body P content - IBW x initial body P content) / P intake
Digestibility parameters were calculated as follows, where FBW represents final mean body weight (g) and IBW represents initial mean body weight (g).
Apparent digestibility coefficient (ADC, %):
Daily P gain: ((final body P content - initial body P content) / (IBW + FBW) / 2) / days
Daily P intake: (P intake / (IBW + FBW) / 2) / days
Daily faecal P losses: daily P intake x (100 - ADC P) / 100
Daily metabolic P losses: daily P intake - (daily P gain + daily faecal P losses)
Results
Performance
No external lesions or apparent abnormalities in the internal organs were observed.
Fish fed the PC and PHY-750 diets showed significant increases compared to the NC diet in specific growth rate (SGR), final body weight (FBW), feed conversion ratio (FCR) and protein efficiency ratio (PER; Table 2). No significant differences were seen in feed intake (FI; Table 2).
SGR = specific growth rate, FCR = feed conversion ratio, FI = feed intake, PER = protein efficiency ratio. Different superscript letters denote statistical differences (p < 0.05).
Digestibility and biological sampling
Different superscript letters denote statistical differences (p < 0.05)
Digestibility of dry matter was not affected by the dietary treatments. Significant differences were seen between all treatments in P digestibility. The PHY-750 diet showed significantly higher digestibility of phytate than both control diets (Table 3).
No significant differences in body P were seen between the diets. The PC and PHY-750 diets showed significantly higher bone ash than the NC diet (Table 3).
The PHY-750 diet showed significantly higher phytase retention than the NC and PC treatments (Table 3).
Discussion
Performance
Across all studies available in Atlantic salmon, additions of phytase to diets showed reduced performance compared to those seen in the present trial. Previous studies investigating the effect of microbial phytases on performance required post-production inclusion levels in diets of at least 2000 FTU.
The additions of P. pastori phytase at 1900 FTU from a competitor showed no significant performance improvement over a fishmeal control or basal diet (Denstadli et al., 2007).
Though lower inclusion levels of phytase were effective in the present study, the capacity of phytase to increase the availability of micro- and macronutrients in different plant ingredients has been noted in salmonids and the differences in the ingredients used in studies may have some impact on the availability of phytate bound P (Cheng and Hardy, 2002; Denstadli et al., 2006, 2007; Sajjadi and Carter, 2004a).
The improved action of OptiPhos to function in a high plant-based protein diet at lower temperatures may be a result of the characteristics of different phytases. The optimal conditions for OptiPhos activity are 58oC and a pH of 3.4 compared to 65oC and a pH of 2.0 in A. niger phytase (Dersjant-Li et al., 2015). This increased activity at higher pH, closer to the active pH of the stomach of salmon (Krogdahl et al., 2015) and lower optimal temperature may account for the increased performance seen.
Digestibility
The application of OptiPhos resulted in significant improvement in P digestibility over both the NC and the MCP dosing, suggesting active increases in P digestibility were a direct action of phytate breakdown as evidenced by the significantly higher phytate digestibility seen in the test diet.
Bone ash in the current study was significantly higher at 750 OTU dosing by phytase and MCP dosed diets, suggesting sufficient P availability as seen in other studies conducted with rainbow trout (Vielma et al., 2002) and Atlantic salmon (Sajjadi and Carter, 2004a). Reductions in bone P in the NC diet were not reflected in whole body P content and a reduction in bone ash content was seen in the NC diet, evidencing supplementation of P deficiency from bone bound P.
Conclusion
The inclusion of phytase OptiPhos at 750 OTU proved to be an effective strategy to enhance P utilisation in high plant content feeds.
Supplementation resulted in an enhanced digestibility and retention compared to inorganic P dosing.
The benefits associated with phytase use make it a valuable nutritional tool to reduce waste effluents as well as reduce the use of expensive inorganic P sources.
Though OptiPhos was found to enhance P utilisation in high plant content feeds, further investigation into appropriate dose responses of specific phytases in similar feed formulations would be advantageous.
