Combining NSP-Enzymes and Phytase: the formulation challenge in broiler nutrition!

In order to investigate the benefits of a multi-enzyme complex (RovabioTM Max) containing carbohydrolases and phytase activities on the performance and bone mineralization of broilers fed corn-soybean meal based diets, 2268 male Ross broiler chicks were allocated to 9 dietary treatments: a positive control diet formulated to be adequate in nutrients and four reduced nutrient diets (NC), with gradual decrease on AME, CP and digestible AA, available phosphorus (avP) and calcium contents, with or without supplementation of the enzymecomplex. Supplementation of the NC diets with the enzyme-complex improved feed intake, weight gain and feed conversion. The magnitude of the enzyme effect was stronger for the most reduced avP/Ca diets. The first limiting nutrient factor appeared to be the avP. With enzyme, there were no significant differences in growth of birds fed on NC or PC diets. Enzyme increased feed intake of birds fed on NC diets nearby the level of feed consumption of the positive control and resulted in better FCR. Enzyme restored bone mineralisation to that of the PC, with the low reduction on AME and CP and digestible AA. The results indicate that dietary supplementation with a multi-enzyme complex containing NSP-enzymes and phytase is efficient in reducing the phosphorus, energy, protein and amino acid specifications of a corn-soybean meal diet without performance losses, but not always fully restoring bone mineralisation.

In commercial broiler production, combinations of different enzymes or multi-enzyme complexes are used to increase nutrient and energy availability from feed ingredients, especially combinations of different carbohydrolases and phytase. The use of carbohydrolases, targeted to hydrolyse soluble NSP, is widely and successfully implemented when viscous-cereals (wheat, barley, rye, oats or triticale) are used. Although both corn and soybean meal are considered highly digestible ingredients, there is some room for the improvement of their nutritional value supporting the use of NSP-enzymes in such diets (Maisonnier et al., 2004). The use of phytase has become standard practice to reduce P levels in the environment and to compensate the drastic increase of the cost of inorganic phosphates. The effects of phytase in poultry have been extensively reviewed by Selle and Ravindran (2007). They reported that phytase increases P retention and tibia ash, but it has also positive effects on growth performance and digestibility parameters.
There are two strategies to benefit from enzyme supplementation: reducing the nutrient specifications of the complete feed appropriately or assigning specific nutritional values to the individual ingredients (Dalibard and Geraert, 2004). Both approaches require accurate knowledge of how much nutrients will be made available by adding the enzyme to the diet. Many factors can influence the response to an enzyme complex, since it depends on the enzyme specificity and concentration of the substrate, doses of enzymes and interactions between enzymes, ingredient quality and type, level of nutrients in the diet, age of animals.
The current study was designed to assess the reformulation matrix with the application of a multi-enzyme complex, combining carbohydrolase and phytase, in cornsoybean meal fed broilers, and to revise the different limiting factors supporting the performance benefits.

A multi-enzyme complex in liquid form was tested (RovabioTM Max, Adisseo, France), containing carbohydrolases produced from the fermentation of Penicillium funiculosum and bacterial 6-phytase (EC 3.1.3.2.6), derived from E. coli. The enzyme was applied after pelleting at a dose rate of 200 mL/tonne of feed to provide a minimum of 1100 visco-units of endo-β-1,4-xylanase, 100 AGL units of endo-1,3(4)-β-glucanase and 500 FTU (phytase units)/kg of feed, and pectinase, protease and mannanases side activities.
Five basal diets were tested: one positive control (PC) diet formulated to be adequate or to exceed all nutrients requirements, and four negative control (NC1 to NC4) diets with a combined reduction of AME, CP and digestible essential amino acids (CP-dAA), available phosphorus (avP) and calcium (Ca). The applied nutrients reduction was: NC1 (-0.27 MJ/kg, -1.5 % CP-dAA, -0.15 pcent point avP, -0.12 pcent point Ca); NC2 (-0.36 MJ/kg, -3.0 % CPdAA, -0.15 pcent point avP, -0.12 pcent point Ca); NC3 (-0.27 MJ /kg, -1.5 % CP-dAA, - 0.20 pcent point avP, -0.16 pcent point Ca); NC4 (-0.36 MJ/kg, -3.0 % CP-dAA, -0.20 pcent point avP, -0.16 pcent point Ca). PC diet provided 4.3 and 3.9 g/kg, NC1 and NC2 2.8 and 2.4 g/kg and NC3 and NC4 2.3 and 1.9 g/kg of avP from 0-21 d and 22-43 d, respectively. There were a total of nine dietary experimental treatments replicated six times each and allocated at random by blocks.
2268 one-day Ross 380 male broiler chickens were distributed into 54 floor pens, 42 chickens per pen. Feed, in pellets, and water were provided ad-libitum throughout the experiment. Average daily weight gain (WG), average daily feed intake (FI) and feed conversion ratio (FCR) were calculated for the periods 0-21, 22-42 and 0-42 d. Mortality was also recorded. At 43 d, twelve chickens per treatment were randomly selected and euthanized to determine the percentage of tibia ash as well as total phosphorus and calcium concentrations.
Data were analyzed as a randomized complete block design with a two-way analysis of variance (block and treatment) using the GLM procedure of SAS. Differences between PC and NC diets, with and without enzyme, were compared by a set of contrasts. Moreover, data without the PC group were analyzed by a 4 x 2 factorial analysis of the variance to determine the main effects of nutrient reduction and enzyme and their interaction.

The effect of diets and enzyme-complex on performance from 0 to 42 d is presented in Table 1. Growth of birds fed NC1 and NC2 diets was not significantly different from growth of birds fed PC diet. Further avP and Ca reduction resulted in a lower (P < 0.001) WG compared with the PC, by 13% for NC3 and 16% for NC4 diet. With enzyme, there were no significant differences in growth of birds fed on NC with the PC diets. Feed intake of birds fed on NC diets were lower (P < 0.05) compared with PC, and in all cases, enzyme supplementation increased it nearby the level of feed consumption of the PC. Enzyme supplementation to NC diets resulted in all cases in better (P < 0.05) FCR than the PC. Mortality of birds fed NC3 and NC4 diets was higher (P < 0.05) compared with the PC and it was reduced by the enzyme. The magnitude of the enzyme effect in increasing WG and FI was stronger for the most reduced avP and Ca diets (NC3 and NC4). No significant differences were detected between the two levels of energy and CP/dAA reduction, only significant differences between the two levels of avP/Ca could be detected. This might indicate that without enzyme supplementation, avP/Ca reduction affected more greatly the growth performance than the energy and CP/dAA reduction.
Tibia ash of birds fed NC diets was lower (P < 0.05) compared to birds fed PC (Table 1). Enzyme supplementation to NC1 and NC3 diets returned bone mineralisation to that of the PC, whereas tibia ash in birds fed NC2 and NC4, the most energy and CP-dAA reduced diets, remained lower (P < 0.05).
Overall and with the multi-enzyme-complex, performance of birds fed reduced nutrient diets was not different than that of broilers fed PC diet, even better with NC1 and NC2 diets. Enzyme-complex increased feed intake and weight gain and improved FCR, and the strongest effects were observed with the lowest avP and Ca levels. This might indicate that the first limiting nutrient was P and one time the P deficiency was overcome by phytase and feed intake was restored, the carbohydrase enzymes could increase the nutritive value of diet compensating the reduction in AME (by 2.8%) and CP/dAA (by 1.5-3.0%). Cowieson et al. (2006) suggested the feasibility of reducing -0.61 MJ/kg AME, 0.13% P, 0.12% Ca and 1 to 2% amino acids of a corn-soybean meal diet without significant impairment of performance at 42 d, by the use of a combination of xylanase, amylase, protease and phytase. The mode of action of the enzymes in corn-soybean meal diets has been linked to the disruption of the cell wall matrix, facilitating the release of encapsulated nutrients and the digestive enzymes access, and also to the modification of the intestinal microbiota communities (Bedford, 1996; Cowieson, 2005, Classen, 2006). Some works also suggested that carbohydrolases, that are able to breakdown the cell wall NSP-matrix, can facilitate the access of phytase to phytate molecule (Olukosi et al., 2007), supporting the thought that the use of a combination of enzyme can fully strengthen their effects.

In summary, results of the present experiment indicate that the use of a multi-enzyme complex containing xylanase, β-glucanase and phytase as main activities, and pectinase, protease and mannanases side activities, allows the reduction of the AME, CP-dAA, avP and Ca contents of a corn-soybean meal diet without penalising performance, but not always restoring fully the bone mineralisation.

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Classen HL (2006) in Direct-fed microbial, enzymes & forage additive compendium. Michael Howie (ed.) 8th edition. Miller Publishing Company, Minnetonka, MN, pp. 20-24.
Cowieson AJ, Singh DN, Adeola O (2006) British Poultry Science 47, 477-489.
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