Effects of microbial phytase supplementation of a barley-soybean meal diet on the performance and bone mineralization of growing-finishing pigs Eija Helander Helander, E. 1994. Effects of microbial phytase supplementation of a barley- soybean meal diet on the performance and bone mineralization of growing- finishing pigs. Agricultural Science in Finland 3: 439-448. (Department of Animal Science, P.O. Box 28, FIN-00014 University of Helsinki, Finland.) The study evaluated the effect of a phytase supplement, produced by Aspergillus niger , on the performance of 72 growing-finishingpigs (28-101 kg). Chemical and physical parameters of the tibia and fibula bones were measured. The control diet (diet 1) was formulated to be adequate for pigs with respect to all nutrients, and diets 2 and 3 with respect to all other nutrients except total phosphorus (P). The P source of the control diet was dicalciumphosphate (dihydrate), while no inorganic P was added to diets 2 and 3. Diet 3 was supplemented with phytase, 720 U/kg feed. The average daily gain (ADG) and the feed conversion ratio (FCR) of the control pigs were significantly better than those of the groups without inorganic P. Phytase improved the ADG (p <0.01) and the FCR during the first 45 days (28- 60 kg). The pigs on diet 2 were able to grow as well as the other pigs when they became heavier (between 60-100 kg), so that at the end of the trial there was no significant difference between diets 2 and 3 for these parameters. No signs of leg weakness were observed in the growing pigs on any of the diets. The density (p < 0.01) and the breaking strength (p < 0.01) of the tibia bone were reduced (p < 0.01) on low-P diets compared to control diet. Phytase supplementation im- proved the density of the tibia bone (p < 0.001) compared to diet 2 and tended to improve the breaking strength, but the difference was not significant. No differenc- es were found in the ash or P contents of the fibula bones. Phosphorus emissions were markedly reduced on diets 2 and 3. On the basis of the results it is not possible to recommend to replace inorganic P totally by phytase in growing-finish- ing pigs on barley-SBM diets. Key words: phosphorus, availability Introduction Approximately one percent of a mature pig’s body weight consists of phosphorus (Peo 1991). Four- fifths of it is concentrated in the skeleton while the rest can be found in soft tissues in different organic compounds participating in nearly all bio- chemical reactions occurring in the pig’s body (Cromwell 1989). In plants, phosphorus (P) is to a great extent in the form of organic calcium and magnesium salts of myo-inositol hexaphos- phate (phytic acid), the bioavailability of which varies depending on the species of plant and their phytic acid and intrinsic phytase content (Jong- bloed 1987). In diet formulation, the P availabil- ity is normally calculated to be only 0.30 in veg- etable feed ingredients. To ensure that the phos- phorus requirements of animals are met, feed mix- 439 Agricultural Science in Finland 3 (1994) https://www.c-info.fi/en/info/?token=KitKsWUGRyv7WiI9.0rDehYQkPjM-wx0UqEiRjg.l68H1pLIxyUsWQf819uVkNLYN1KkwqISSc6xsI9IQ4FpJOI6f5pha7-3h2u8DkTkhvwv6F5jxtZyWhmuC1z8AHV-3cn6iNcTWv7iWwZEGkXclfuReYB12KRKvJTqelkUQV8G4X6BiwGbmIT5MFZ6y1OJX-tE7MWG5gBWswTjxJI89Cz9FSAlXHiSbDKOnOaAgAvZGEP7ajyIC0SZUv_8jUGumpcl-VqqkJVMmbBHJ9fes21m6Ntam3-jhUir_wTTjSRGvw tures are usually supplemented with inorganic phosphorus. The low utilization of dietary P re- sults in a high amount of it excreted in manure. This can lead to an accumulation of P in the soil and a consequent disruption of the normal life cycle of various organisms in nature. Possibili- ties to decrease the use of P in pig feeds have therefore been studied for years. One of the key factors in decreasing the total phosphorus content of pig feeds is to make the organic phytic phosphorus of plants bioavailable for animals. Trials have included soaking the feed before feeding (Jongbloed 1987, Kemme and Jongbloed 1993, Näsi and Helander 1994) or adding microbiologically produced phytase to the feed. Most of the studies on the effects of micro- bial phytases have so far been conducted on young pigs on maize-soybean meal based diets (Simons et al. 1990, Jongbloed et al. 1991a, Beers and Jongbloed 1992a,b, Lei et al. 1992, Pallauf et al. 1992, Eeckhout and De Paepe 1992, Hoppe et al. 1993, Kemme and Jongbloed 1993). How- ever, 0.60-0.70 of the phosphorus excretion of pigs originates from the growing-finishing pigs. Attempts to reduce the excretion ofP should there- fore be especially directed at growing-finishing pigs (Jongbloed and Lenis 1993). In Finland and in many other areas the main cereal in pig diets is barley, the phytate and phytase contents of which differ from maize (Cromwell 1992). The aim of the present experiment was to eval- uate whether a Aspergillus niger phytase prepa- ration could improve the utilization of the intrin- sic phosphorus of commercial barley-soybean meal-based pelleted feed mixtures and thus re- place the added inorganic dietary P. The perform- ance of the growing-finishing pigs as well as dif- ferent bone parameters were used as response criteria of the enzyme effect. A digestibility and balance experiment was carried out simultaneously with the same feeds (NAsi and Helander 1994). Material and methods A feeding trial was conducted with 72 commer- cial crossbred pigs from 28 kg live weight to an average live weight of 101 kg at slaughter. The pigs were allocated into three blocks on the basis of weight. Eight pigs from each block were as- signed at random to one of three experimental diets. Four pigs were placed into each pen and each diet was tested on three pens of barrows and three pens of gilts. The animals were group-housed in concrete-floor pens and group-fed. No bed- ding was used. The pigs were fed pelleted (65°C, 4 mm diam- eter) barley-soybean meal based complete mix- tures. The same feed mixtures were used in a separate digestibility and balance experiment (NAsi and Helander 1994). The control diet (diet 1) was adequate for pigs with respect to all nutri- ents (Salo et al. 1990) and the experimental diets (diet 2 and diet 3) with respect to all other nutrients except total P. The inorganic P source on the control diet was dicalciumphosphate (di- hydrate). The experimental diets 2 and 3 were not supplemented with inorganic P. The control diet was calculated to contain 3.1 g and the ex- perimental diets 1.2 g digestible P per kg. The total P values of the feed raw materials needed for calculations were based on the results of sev- eral analyses obtained from the feed factory. The values were 3.1 g/kg for barley, 6.6 g/kg for soy- bean meal (440 g crude protein and 40 g crude fat per kg soybean meal, SBM) and 174 g/kg for dicalciumphosphate. Diet 3 was supplemented with a phytase prepared using Aspergillus niger (NatuphosR 5000, Gist-brocades, The Nether- lands), at a level of 0.24 g/kg (1200 U/kg). The calcium content was calculated to be 8.0 g/kg on all diets and the vitamin D content 800 HJ/kg feed. The composition of the diets is shown in Table 1. The pigs were restrictedly group-fed ac- cording to body weight using a scale from 1.5 to 2.85 feed units/pig/day (FU = 0.7 starch equiva- lents). The daily feed allowance was changed once a week. Feeds were offered twice a day, and wa- ter was available ad libitum. The pigs were weighed on days 0, 21, 45, 63, 84 from the be- ginning of the trial and again at the end of the trial. The feed consumption was registered pen- wise during the experiment. At slaughter, the carcass weight was recorded 440 Agricultural Science in Finland 3 (1994) Table I. Dietary ingredients (%), analyzed protein and calculated P, Ca and energy contents of the experimental feeds. Diet 1' Diet 2 Diet 3 Barley 76.0 76.8 76.8 Soybean meal 19.5 19.3 19.3 Dicalciumphosphate (dihydrate) 1.6 Limestone 0.7 1.7 1.7 Molasses 1.0 1.0 1.0 Serla Bondex (a pellet binder) 0.5 0.5 0.5 NaCl 0.4 0.4 0.4 Trace mineral mix 2 0.2 0.2 0.2 Vitamin mix' 0.1 0.1 0.1 Phytase 4 - 0.024 Crude protein, g/kg DM (anal.) 189 187 190 Phosphorus, g/kg (calc.) 6.5 3.7 3.7 Calcium, g/kg (calc.) 8.0 8.0 8.0 Feed unit (FU/kg) 5 1.0 1.0 1.0 ME, MJ/kg 12.5 12.5 12.5 1. Diet 1 = inorganic P supplementation, Diet 2 = no inorganic P supplementation, Diet 3 = like diet 2 + phytase addition. 2. Supplied per kg diet, 20 mg Fe, 21 mg Mn, 21 mg Cu, 73 mg Zn, 0.2 mg 1, 0.1 mg Se. 3. Supplied per kg diet, 5000 IU vitamin A, 800 IU vita- min D, 60 mg vitamin E, 2 mg vitamin K, 2 mg thia- min, 3 mg riboflavin, 20 pg vitamin 812, 50 pg biotin, 10 mg pantothenic acid, 20 mg niacin. 4. NatuphosR 5000 phytase (Gist-brocades, The Nether- lands). 5. FU = 0.7 kg starch equivalent. and the quality grade determined with an auto- matic Hennessy GP2 device. The lean content was estimated from measurements of back fat, eye muscle depth and carcass weight (English et al. 1988). A quality grading of 9 points was equiva- lent to a meat percentage over 61, 8 to 59-60, 7 to 52-58 and 6 below 51. The tibia and fibula bones from the left hind leg were removed from eight pigs per treatment. The bone samples were frozen (-18°C) to await chemical and physical analyses. Dry matter content, the concentration of ash, Ca, P and density of the diaphyses of the fibula bones, as well as density and breaking strength of the tibia bones, were determined. The densities of the bones were measured by weigh- ing the bones from which the soft tissues had been removed, placing the whole bones into wa- ter and recording the quantity of water replaced by the bones, after which the weight-to-volume ratio was calculated. The breaking strength was determined with an Instron Testing Instrument (Model 1112) by recording the amount of force applied at a constant speed of 5 cm/min required to break a 3.7 cm piece of the mid-diaphysis placed in a horizontal position on a support. Two samples from each bone were measured, and the mean value was used for the statistical analysis. For dry matter determination, small pieces of the fibula bones’ diaphyses from which the visible fat had been removed, were dried in a 105°C oven overnight. The ash percentage was deter- mined by placing the driedbones in a 525°C muf- fle furnace for 16 hours. The chemical analyses of the feeds were con- ducted by standard methods (AOAC 1984). In the determination of phytic acid, ICP-AES meth- od was used to determine P (Plaami and Kumpu- lainen 1991). In this method the P content of the sample is indirectly measured by atomic emis- sion spectrophotometry after burning the sample by inductively coupled plasma. P from the feeds and bones was analyzed after dry ashing colori- metrically by the vanadomolybdate procedure of Tayssky and Shorr (1953). The calcium con- tents of the feeds and bones were measured with a Perkin-Elmer 5100 PC atomic-absorption spec- trophotometer. The phytase activity of the main raw materials, of diets and of Natuphos was meas- ured spectrophotometrically as free phosphate from phytate after incubating the sample in a 0.1 M sodium acetate buffer, pH 5.0, at 35°C for 30 minutes (Puhakka, Cultor Research Center). A phytase unit is defined as the amount of enzyme that liberates 1 pmol of inorganic P from sodi- um-phytate in one minute. The data were subjected to an analysis of vari- ance using the GLM procedure of SAS (1985). A split-plot design was used for the statistical ana- lysis of periodical average daily gain (ADG) and feed conversion ratio (FCR). In the performance data the mean of each pen was used as an obser- vation for the statistical analysis. The following 441 Agricultural Science in Finland 3 (1994) model was used in analyzing the performance data (Snedecor and Cochran 1989); Y.. = u + B + T. + S, + (T*S).. + e..., whereijk • i j k v 'jk ijk (i = overall mean B = effect of block ii T. = effect of treatment j j J Sk = effect of sex k (T*S) k = interaction between sex and treatment e .., = residual term ijk The mean initial weight of a pen was used as a covariate in analyzing days in the experiment, weight gain, ADG, feed consumption and FCR. In analyzing the bone data the final live weight was used as a covariate. Differences were com- pared using orthogonal contrasts as follows: Cl = inorganic P supplement vs. without P supple- ment (Diet 1 vs. Diets 2 and 3), C 2 = no phytase supplement vs. phytase supplement (Diet 2 vs. Diet 3). Results and discussion Analyses The detailed chemical composition of the diets has been published earlier by Näsi and Helan- der (1994). The total P content was 0.7 g/kg higher and the Ca content 0.3 g/kg lower in diet 1 than calculated (6.5 g/kg P and 8.0 g/kg Ca). In addition, the P content in diets 2 and 3 was 0.5 g/kg higher and Ca content 0.7 g/kg low- er than calculated. Thus, the Ca to P ratios of the diets were lower than expected (Table 2). The analyzed total P content of the barley batch used in this trial was 3.6 g/kg and of soybean meal 4.8 g/kg on average. The P content of barley was on the same level as reported by Oksbjerg (1988) and Veevoedertabel (1991) but was lower than the values measured by Pointillart (1988) and Jongbloed and Kemme (1990). The P content of SBM was lower than the values reported by Poin- tillart (1988), Jongbloed and Kemme (1990), Veevoedertabel (1991) Jmd Ketaren et al. (1993). In Finnish feedtables (Salo et al. 1990) the val- ues of P are 3.5 and 7.3 g/kg DM for barley and SBM, respectively. Using the measured digestibility coefficients 0.55, 0.45 and 0.65 for P (Näsi and Helander 1994) in diets 1, 2 and 3, respectively, and the determined total P contents, it can be calculated that diet 1 contained 3.9 g, diet 2 1.8 g, and diet 3 2.6 g digestible P/kg. The interpretation of the enzyme activity meas- urements is somewhat questionable, because the assays were performed according to methods which have not been validated (Puhakka, Cultor Research Centre). The method is not sensitive Table 2. Analyzed P and Ca contents of the experimental diets, barley and soybean meal (SBM). Treatment Diet I 1 Diet 2 Diet 3 Barley SBM Composition, g/kg Phosphorus 7.2 4.1 4.1 3.6 4.8 Phytic acid 9.3 9.3 9.9 8.1 10.1 P from phytic P(*0.282) 2.6 2.6 2.8 2.3 2.8 Phytic P % of total P 36.1 63.4 68.3 63.9 58.3 Phytase, U/kg