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Use of fixed calcium to phosphorus ratios in experimental diets may create bias in phytase efficacy responses in swine

Use of fixed calcium to phosphorus ratios in experimental diets may create bias in phytase... Translational Animal Science, 2022, 6, 1–9 https://doi.org/10.1093/tas/txac124 Advance access publication 29 August 2022 Non Ruminant Nutrition Use of fixed calcium to phosphorus ratios in experimental diets may create bias in phytase efficacy responses in swine †,1 ‡ † † † Hengxiao Zhai, Jon R. Bergstrom, JingchengZhang , Wei Dong, Zhenzhen Wang, ║ ║ Kostas Stamatopoulos, and Aaron J. Cowieson DSM (China) Animal Nutrition Research Center, Bazhou 065799, China DSM Nutritional Products, Parsippany, NJ 07054, USA DSM Nutritional Products, Kaiseraugst 4303, Switzerland Corresponding author: heng-xiao.zhai@dsm.com ABSTRACT The objective of this study was to investigate the effects of two dietary total Ca/P ratios on available P release by phytase, measured using growth performance and bone mineralization with 528 barrows and gilts according to a randomized complete block design. Three were 11 diets in a factorial of 2 by 4 plus 3, including 3 reference diets consisting of 0.25% (control), 0.70%, or 1.15% monocalcium phosphate (MCP) and 8 diets from combining 4 phytase doses (500, 1,000, 2,000, and 3,000 FYT/kg) with 0.25% MCP and 2 dietary Ca/P ratios (1.05 and 1.20). Each diet was fed to 6 pens of 8 pigs. All diets contained 3 g/kg TiO , and fecal samples were collected from each pen on d 13–15 of trial. At the end of trial, one pig per pen was sacrificed to collect a tibia and urine in the bladder. The results showed that MCP improved growth performance lin - early (P < 0.01), whereas both a linear and quadratic response was observed with the addition of phytase. The MCP increased the percent bone ash and weights of bone ash, Ca, and P linearly (P < 0.01). At both Ca/P ratios, increasing supplementation of phytase increased the percent bone ash and weights of bone ash, Ca, and P both linearly and quadratically (P < 0.05). Both MCP and phytase significantly increased digesti - bility of Ca and P as well as digestible Ca and P in diets and reduced the digestible Ca/P ratio. The dietary Ca/P ratio of 1.20 resulted in poorer feed utilization efficiency, more digestible Ca, greater percent bone ash, Ca, and P and heavier weights of bone Ca and P than the ratio of 1.05 (P < 0.05). The ratio of 1.20 elicited numerically higher available P release values from phytase, with percent bone ash and bone P weight as the response variables, but significantly lower values with gain:feed. The urinary concentration of Ca increased linearly ( P < 0.01) with increasing digestible Ca/P ratios whilst urinary concentration of P decreased quadratically (P < 0.01). In conclusion, fixing the same total Ca/total P ratio in diets supplemented with increasing phytase dosing created an imbalance of digestible Ca and P, which could have an adverse effect on bone mineralization and thus compromise the phytase efficacy relative to mineral P. Lay Summary Phytase is the most widely applied feed enzyme in livestock farming because of its main function of releasing phosphorus (P) bound by phytate in most plant feed ingredients which would otherwise not be utilized effectively by farm animals. This basic function enables phytase to become a proper supplement to an animal’s deficient phytate-processing system and to reduce the reliance of livestock farming on phosphate rocks. Phosphate rocks are a nonrenewable source of P for farm animals. The P contribution to a diet by phytase, therefore, needs to be measured for feed formulators to use phytase to its maximum potential and to meet the animal’s requirement for P precisely. Animal scientists have es- tablished different methods to determine either available or digestible P release from phytase, and each method has its own pros and cons. At the same time, this diversity of measurement methods creates confusion over selection of the appropriate P release values for phytase. This study explores the methodological differences with regard to measuring P release from phytase with the aim to shed light upon the underlying reasons and ultimately to facilitate more precise application of phytase in feed industry. Key words: available, bone, calcium, digestibility, phosphorus, phytase INTRODUCTION Increasing dietary Ca also increases bone mineralization until its maximum, as long as P and Ca are balanced (Létourneau- Phytase efficacy, in terms of available P release, was calcu - Montminy et al., 2012). Either excessive or inadequate supply lated from a standard response curve established by plotting of Ca in a diet has negative impacts on bone mineralization. a sensitive response variable such as bone ash against the Lagos et al (2021) summarized the standardized total tract level/intake of mineral P with an assumed P bioavailability digestible (STTD) Ca/STTD P ratios to be 1.70:1, 1.80:1, of 100% (Cromwell et al., 1993; Kornegay and Qian, 1996; 2:00:1, and 2.30:1 to maximize bone ash for 11–22, 20–50, Wensley et al., 2020a). An underlying assumption is that die- 50–85, and 100–130 kg pigs, respectively, when P was at ad- tary P supply, when it’s not exceeding the requirement and Ca equate. Theoretically, this digestible system can guarantee an is not limiting, is a good predictor for bone mineralization. Received July 16, 2022 Accepted August 29, 2022. © The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Zhai et al. optimal balance between Ca and P in diets for pigs. In the room temperature was 27 °C at the start and gradually field, the Ca and P homeostasis is worth monitoring to main - reduced to 23 °C at the end. The relative humidity was re- tain an optimal balance between Ca and P and to minimize corded to range from 40% to 60%. P footprint on the environment. The urinary concentrations of Ca and P should be explored as potential markers because Experimental Diets an oversupply of Ca or P was shown to trigger an increase The ingredient and nutrient composition of the basal diet in their excretion through urine (Rodehutscord et al., 1999; are presented in Table 1. There were 11 experimental diets. Stein et al., 2011). Three diets were established by including 0.25% (con- In pig studies to measure available P release by phytase, trol), 0.60%, or 0.95% MCP to establish the standard re- the dietary Ca levels (Cromwell et al., 1993) or ratios of total sponse curves. Four more diets were created by including the Ca/P were fixed. For example, a total Ca/P ratio of 2:1 was phytase at 500, 1,000, 2,000, or 3,000 FYT/kg feed in the used by Kornegay and Qian (1996), a ratio of 1.10:1 (0.97 to control diet. The test phytase (HiPhorius, DSM Nutritional 1.10 on analysis) by Wensley et al. (2020a), and a ratio be- Products, Switzerland) was encoded by a 6-phytase gene from tween 1.2 and 1.3 (1.31 to 1.39 on analysis) by Dersjant-Li Citrobacter braakii and expressed from a strain of Aspergillus et al. (2020). The total Ca and P do not reflect their different digestibility among feed ingredients, which implies that the same total Ca/P ratios in diets of different ingredient compo- Table 1. Ingredient and nutrient composition of the basal diet (g/kg of sition might correspond to disparate digestible Ca/P ratios. In feed, as-is basis) addition, the contribution of digestible Ca by phytase has not been described precisely, and the issue of dissimilar digestible Items Basal diet Ca/P ratios could be even worse between the reference diets and phytase diets in studies to determine the available P re- Ingredients lease for phytase. Corn 590.9 The aim of the current study, therefore, was to investigate Soybean meal 340.5 the effects of two total Ca/P ratios (1.05 and 1.20) on the Soybean oil 25.0 growth performance, digestibility of Ca and P, bone miner- NaCl 4.5 alization, and urinary concentrations of Ca and P in nursery NaHCO 1.5 pigs supplemented with different doses of a novel phytase. We hypothesized that the bone mineralization will be greater L-Lys·HCl 4.5 when there is slightly more Ca in the phytase-supplemented DL-Met 1.2 diets and, therefore, the measured available P release by L-Thr 1.5 phytase as benchmarked to monocalcium phosphate (MCP) L-Val 1.0 will be elevated. Limestone 6.8 Monocalcium phosphate 2.5 MATERIALS AND METHODS Vitamin-mineral premix 5.0 Rice hull 9.1 This study was conducted at DSM (China) Animal Nutrition Research Center Co. Ltd. (Bazhou, P. R. China), and its Benzoic acid 3.0 protocol was approved by the Animal Welfare Committee TiO 3.0 of DSM (China) Animal Nutrition Research Center Total 1,000.0 (AWCCAN). The guidelines in European Union council di- Calculated nutrients and energy rective 2010/63/EU for animal experiments were followed Net energy, kcal/kg 2,499 in this study. Metabolizable energy, kcal/kg 3,372 Crude protein 21.8 Animals and Facilities Total Ca 4.5 Five hundred and twenty-eight barrows and gilts (PIC L1050 × L337; initial body weight 7.4  ±  0.9  kg [mean ± standard Total P 4.2 deviation]) were used. The pigs were weaned at an average Phytate P 2.5 age of 21 d and transferred to a nursery facility for an adap- Standardized total tract digestible P 2.0 tation period of 7 d using a common starter diet. The nursery Standardized ileal digestible facility was equipped with 80 pens (space/pen = 3.0 × 1.8 m ). Lys 12.9 Each pen had a plastic-coated wire floor and was equipped Met 3.9 with two water nipples and one stainless-steel feeder. After Thr 7.7 the adaptation period, the pigs were individually weighed and Trp 2.1 allotted into 66 pens based on their initial BW and gender (four barrows and four gilts per pen). Each pen of pigs was Val 8.3 assigned to 1 of 11 dietary treatments in a Randomized Premix supplied per kilogram of diet: vitamin A, 9,750 IU; vitamin D , Complete Block Design, resulting in 6 replicate pens per die- 3,000 IU; vitamin E, 63 mg; vitamin K , 3.0 mg; vitamin B , 3.0 mg; 3 1 tary treatments. The experimental diets were fed for 21 d, vitamin B , 9.6 mg; vitamin B , 4.5 mg; vitamin B , 36 μg; D-biotin, 240 2 6 12 with feed and water supplied ad libitum. At the end of trial, ug; D-calcium pantothenate, 30 mg; folic acid, 1.8 mg; niacin, 36 mg; Cu (tribasic copper chloride), 190 mg; I (potassium iodate), 0.6 mg; the pigs weighed 17.1 ± 2.1 kg. Fe (ferrous sulfate), 120 mg; Mn (manganese sulfate), 60 mg; Zn (zinc Room temperature and ventilation were controlled by a sulfate), 120 mg; Se (sodium selenite), 450 μg; choline (choline chloride), 300 mg; and Ca (calcium carbonate) 0.6 g. computer system to provide an optimal environment. The Bias in phytase efficacy responses 3 oryzae. In these seven diets, the formulated ratio of total Ca Digestibility of Ca and P was calculated using the following to total P was maintained at 1.05 by adjusting the inclusion equation: level of limestone. An additional four diets also included 500, 1,000, 2,000, or 3,000 FYT/kg phytase but with a formulated D =[1 − (T /T ) × (M /M )] × 100; i o o i total Ca/P ratio of 1.20 using adjusted inclusion levels of limestone. Titanium dioxide was included at 3 g/kg feed as an where D is the digestibility of Ca or P (%); T and T are the i o indigestible marker to enable the measurement of apparent titanium concentrations in diet and feces, respectively (% of total tract digestibility (ATTD) of Ca and P in all diets. The DM); M and M are the concentrations of Ca or P in diet i o net energy and nutrients, other than Ca and P, were above and feces (% of DM), respectively. The digestible Ca and P the recommendations in NRC (2012) for all the experimental were calculated by multiplying the concentrations of Ca diets. All diets were pelleted with a conditioning temperature and P in feed (g/kg feed) by their corresponding digestibility at 75 °C. coefficients. Available P release by phytase was calculated by referring to the method described by Wensley et al. (2020a). The Measurement and Sampling standard response curves were established by regressing ADG The pigs were individually weighed on day 0 and 21 of trial to against dietary P intake (g/d) and regressing gain:feed, bone obtain the total weight for each pen of pigs, and the feed con- ash percent, and bone P weight against dietary P concentra- sumption per pen was recorded during the trial to calculate tion (%). The feed intake was incorporated for the response average daily gain (ADG), average daily feed intake (ADFI), curve of ADG to achieve a greater fitting to the results. Using and gain:feed. the standard response equations, the dietary P concentration Fresh and clean fecal samples were grabbed from each pen equivalence was solved for each dose of phytase. These equiv- on day 13 to 15 of trial. All the pens were cleaned, and ex- alent values were corrected for the contribution by the con- isting feces removed before collection on each collection day. trol diet to generate the available P release values for phytase. A total of approximately 500  g of fresh feces was collected The data were analyzed using the GLM procedure of SAS per day per pen. All the fecal samples collected from each pen (SAS Inst. Inc., Cary, NC) with the model including the die- during the 3-d collection period were pooled and mixed to tary treatment as a fixed effect, replicate as a random effect, homogeneity with a hand-held blade mixer (TD-110, RuiBao and an error term. Polynomial orthogonal contrasts were Hardware Co. Ltd., Dongguan, P. R. China). A subsample of constructed to test the linear and quadratic effects of supple- around 400 g for each pen was collected and stored at −20 °C mentation of MCP and phytase, the effect of dietary Ca/P ratio until further processing. among phytase diets, and the interaction between phytase and The right tibia and urine from the bladder (if abundant at the Ca/P ratio. The least-square means were presented, and sampling) were collected from the pig in each pen with body the significance was defined at P < 0.05. weight closest to the average body weight per pen on d 21 of trial. The tibias were processed by referring to the non- defatting bone processing procedures described by Wensley RESULTS et al. (2020b). In short, the bones were autoclaved at 120 °C for 30 min to facilitate the removal of muscular tissues and Experimental Diets and the Analyses cartilaginous caps. The cleaned bones were left at room tem- The total P in diets with 0.25% MCP was analyzed to be perature for 1 d and then oven-dried at 105 °C for 7 d. In the 0.45%–0.47%, and incremental increases of 0.08%–0.09% end, the dried tibias were incinerated in a muffler oven for P agree with the increasing dietary inclusion of MCP from 72 h at 600 °C. All the samples were stored at −20 °C before 0.25% to 0.60% to 0.95% (Table 2). The analyzed total processing. Ca/P ratios ranged from 1.02 to 1.07 for the target of 1.05 and from 1.19 to 1.20 for 1.20. The analyzed marker Chemical Analyses concentrations ranged from 97% to 101% of the formulated value. The analyzed phytase activities were within ± 15% of The fecal samples were oven-dried to a constant weight and the intended doses. ground to pass through a 0.5-mm screen before analysis. The dietary and fecal samples were dried at 105 °C in an oven for 4 h for dry matter determination (method 934.01; AOAC Growth Performance and Bone Mineralization International, 2006). Titanium, Ca, and P were determined by The interaction between phytase and the Ca/P ratio was not Inductively Coupled Plasma-Optical Emission Spectrometry significant for growth performance. Increasing supplementa - (ICP-OES; Optima TM 8000, PerkinElmer, Shelton, USA; tion of MCP linearly improved (P < 0.01) final BW, ADG, method 985.01; AOAC International, 2006) after microwave ADFI and gain:feed, whereas both linear and quadratic digestion. Ten mL of each urine sample were dried at 60 °C responses, were observed with the addition of phytase (P < before the microwave digestion. One phytase unit (FYT) was 0.01; Table 3). Reducing the Ca/P ratio from 1.20 to 1.05 sig- defined as the amount of enzyme that releases 1 µmol of in - nificantly increased gain:feed from 766 to 778 g/kg. organic phosphate from 50 mM phytate per minute at 37 °C There was no significant interaction between phytase and pH 5.5. These analyses were performed in duplicate, ex- and the Ca/P ratio for bone mineralization. Incremental cept that phytase activity in the feed samples was determined increases in MCP linearly increased (P < 0.01) percent from three replicates. bone ash and weights of bone ash, Ca, and P (Table 4). Increasing the addition of phytase increased the percent Calculations and Statistical Analyses bone ash and weights of bone ash, Ca and P, both linearly The experiment was a randomized complete block design. and quadratically (P < 0.05). The bone Ca/P ratio decreased Each pen or pig was an experimental unit. linearly (P < 0.01) with increasing supplementation of MCP 4 Zhai et al. Table 2. Analyzed nutrients of the dietary treatments (as-is basis, %) Table 3. Growth performance of the pigs supplemented with monocalcium phosphate (MCP) or phytase Diet Ca/P ratio Ca, % P, % Ca/P ratio Phytase, U/kg 2 2 2 2 Diet IBW , FBW , ADG , ADFI , Gain: MCP, % kg kg g/d g/d feed, g/kg 0.25 1.05 0.50 0.47 1.07 0 MCP, % 0.60 1.05 0.57 0.55 1.05 0 0.25 7.4 14.9 359 521 691 0.95 1.05 0.67 0.64 1.05 0 0.60 7.4 16.0 410 563 729 Phytase, U/kg 0.95 7.4 16.8 447 591 757 500 1.05 0.47 0.46 1.02 511 Phytase, 1,000 1.05 0.50 0.47 1.06 1,009 U/kg 2,000 1.05 0.49 0.46 1.07 2,110 500 7.4 16.9 451 602 749 3,000 1.05 0.48 0.47 1.03 3,333 1,000 7.4 17.6 484 628 771 500 1.20 0.54 0.45 1.20 573 2,000 7.4 17.7 490 627 782 1,000 1.20 0.54 0.45 1.19 1,112 3,000 7.4 17.7 490 624 787 2,000 1.20 0.55 0.46 1.19 2,292 Ca/P 3,000 1.20 0.55 0.46 1.19 3,240 ratio 1.05 7.4 17.5 480 618 778 Phytase activity was analyzed in three replicates and the others in 1.20 7.4 17.4 477 623 766 duplicate. SD 0.09 0.43 20 26 19 P value and quadratically (P < 0.01) with supplemental phytase. MCP L 0.28 <0.01 <0.01 <0.01 <0.01 The dietary Ca/P ratio of 1.20 resulted in greater percent Q 0.60 0.46 0.52 0.61 0.63 bone ash Ca and P, a heavier weight of bone Ca, and a Phytase L 0.71 <0.01 <0.01 <0.01 <0.01 higher Ca/P ratio in bone than the dietary Ca/P ratio of 1.05 (P < 0.05). Q 0.40 <0.01 <0.01 <0.01 <0.01 Ca/P 0.68 0.64 0.59 0.55 <0.05 ratio Digestibility of Ca and P, Digestible Ca and P in Experimental Diets, and Urinary Concentrations of There were six replicates of eight pigs. Ca and P 2 IBW, initial body weight; FBW, final body weight; ADG, average daily gain; ADFI, average daily feed intake. The interaction between phytase and the Ca/P ratio was SD, standard deviation. significant for digestibility of Ca and digestible Ca. This 4 Linear and quadratic effects of monocalcium phosphate. Linear and quadratic effects of phytase. significant interaction was due to continued increase in The effect of Ca/P ratio compares phytase diets between the ratios of 1.05 digestibility of Ca and digestible Ca beyond the phytase and 1.20, and no significant interaction between phytase and the Ca/P dose of 1,000 FYT/kg at the Ca/P ratio of 1.20 in con- ratio was observed. trast to the plateaued response at the Ca/P ratio of 1.05. Increasing MCP or phytase in the diets, irrespective of the whereas a linear (P < 0.01) increase was observed based on dietary Ca/P ratio, improved digestibility of P and digestible gain: feed and percent bone ash. The Ca/P ratio of 1.05 tended P, but reduced the digestible Ca/P ratio, both linearly and to increase available P release based on gain: feed (0.221 vs quadratically (P < 0.05; Table 5). Digestible Ca was line- 0.192) when compared to the ratio of 1.20, but the opposite arly increased (P < 0.01) with increasing dietary inclusion tendency (0.167 vs 0.186) was shown by bone P weight (P < of MCP. The high dietary Ca/P ratio significantly increased 0.10). the digestible Ca/P ratio compared to the low dietary Ca/P ratio (1.28 vs 1.10). The urinary concentration of Ca increased linearly (P < DISCUSSION 0.01) with increasing digestible Ca/P ratio whilst urinary con- The Diets and Animals centration of P increased quadratically (P < 0.01; Figure 1). In general, the analyzed dietary concentrations of Ca and P were slightly higher than the formulated values, but the agree- Standard Response Curves ment between the calculated and formulated ratios lent cre- Y = 76.6 × X + 168.8 The standard regression equations were dence to the experimental diets to test the hypothesis of this 2 2 (r = 0.94), Y = 396.3 × X + 507.8 (r = 0.62), study. Moreover, the analyzed phytase activities were con- 2 2 Y = 2.08 × X + 0.45 Y = 39.2 × X + 25.1 (r = 0.61), and (r sistent with their intended values. The stepwise increases in = 0.78), respectively, for ADG, gain:feed, percent bone ash both analyzed Ca and P agreed with the incremental additions and bone P weight, respectively. of MCP, which are prerequisites for establishing the standard response curves. The animals were in good health throughout The Comparison of Available P Release by Phytase the trial. The culling rate was only 1.0%. The interaction between phytase and the Ca/P ratio was not Phytase Efficacy significant ( Table 6). Using ADG and bone P weight as re- sponse variables, available P release increased both linearly The measured growth performance, digestibility of Ca and and quadratically (P < 0.05) with increasing phytase dose, P, and bone mineralization in this study followed a classical, Bias in phytase efficacy responses 5 Table 4. Bone mineralization of the pigs supplemented with monocalcium phosphate (MCP) or phytase Diet Bone ash, % Bone ash Ca, % BoneashP, % Bone ash, g Bone Ca, g Bone P, g Bone Ca/P ratio MCP, % 0.25 43.7 33.8 16.9 3.18 1.07 0.53 2.00 0.60 46.0 33.6 17.0 4.01 1.34 0.68 1.98 0.95 50.1 33.4 17.2 5.09 1.70 0.88 1.94 Phytase, U/kg 500 48.0 33.9 17.3 4.51 1.51 0.78 1.96 1,000 48.2 34.0 17.6 4.87 1.64 0.85 1.92 2,000 50.6 34.4 17.6 5.28 1.80 0.93 1.94 3,000 50.2 34.6 17.7 5.36 1.84 0.94 1.95 Ca/P ratio 1.05 49.0 33.6 17.4 4.95 1.65 0.86 1.93 1.20 49.5 34.8 17.8 5.06 1.75 0.90 1.96 SD 2.20 1.44 0.59 0.41 0.15 0.07 0.04 P value MCP L <0.01 0.67 0.32 <0.01 <0.01 <0.01 <0.01 Q 0.41 0.98 0.70 0.53 0.61 0.45 0.46 Phytase L <0.01 0.14 <0.01 <0.01 <0.01 <0.01 0.09 Q <0.01 0.92 0.08 <0.01 <0.01 <0.01 <0.01 Ca/P ratio 0.41 <0.01 0.03 0.36 0.03 0.06 0.04 There were six replicates of one pig. SD, standard deviation. Linear and quadratic effects of monocalcium phosphate. Linear and quadratic effects of phytase. The effect of Ca/P ratio compares phytase diets between the ratios of 1.05 and 1.20, and no significant interaction between phytase and the Ca/P ratio was observed. curvilinear trend in response to the increasing levels of phytase, considered that the ratio of Ca/P in bone is about 2.1:1 which which attests to the basic function of phytase to liberate Ca is tightly regulated due to the finite chemical structure of hy - and P. The improvement in body weight gain with added droxyapatite of bone (Cromwell, 2005). Thirdly, our Ca/P phytase was attributed to an increase in both feed intake and ratios in bone are much lower than the ratios reported by feed utilization efficiency. A deficiency of P can cause a poor González-Vega et al. (2016) and Lagos et al. (2019), which appetite in pigs (Jongbloed, 1987), so the addition of phytase indicates that there is still space for even higher dietary Ca/P to a P-deficient diet should restore appetite by providing bi - ratios to elicit greater bone development. oavailable P. The improvement in feed utilization efficiency The Effects of Dietary Total Ca to Total P Ratio is supported by the close relationship between whole-body P mass and whole-body N mass (NRC, 2012). Soft tissue devel- In the current study, increasing dietary Ca/P ratio from 1.05 opment is dependent mainly on P even though bone mineral- to 1.20 impaired feed utilization efficiency, but increased di - ization requires both Ca and P (Létourneau-Montminy et al., gestibility of Ca as well as the amount of digestible Ca, and 2012). Moreover, the preference of most exogenous phytases consequently increased the amounts of Ca and P deposited in for breaking down myo-inositol hexakisphosphate (IP ) and bone. These results indicate that slightly more Ca in phytase- IP (Wyss et al., 1999; Pontoppidan et al., 2012) means there supplemented diets could result in more Ca digested and ab- will be less residual phytate exerting antinutritional effects sorbed without diminishing the efficacy of phytase in terms of because IP and IP are more capable of binding protein and P release and greater bone mineralization could be realized. 6 5 minerals than other IP esters (Yu et al., 2012). In addition, This agrees with the finding by Létourneau-Montminy et al. myo-inositol released from complete destruction of phytate (2010) that reducing the dietary Ca/P ratio from 1.9 to 1.3 at high phytase doses might provide some extra-phosphoric in a practical diet containing 0.56% P did not improve the effects (Schmeisser et al., 2017; Lu et al., 2019). efficiency of phytase in releasing P, but impaired bone miner - There were some interesting observations about bone min- alization in weanling pigs. Increasing dietary Ca will increase eralization. First, phytase increased percentage and weight of the amount of retained P as long as Ca and P are balanced bone ash irrespective of the dietary Ca/P ratio, but a further or until bone mineralization reaches a plateau (Létourneau- improvement in the percentage of Ca and P in bone ash was Montminy et al., 2012). The impairment of gain:feed by observed only at the dietary Ca/P ratio of 1.20. This implies the high Ca/P ratio agrees with the finding by Johnston et that weights of Ca and P in bone ash are more sensitive than al. (2004) that the reduction in dietary Ca and P was just bone ash weight. Second, more available Ca at the dietary as effective as dietary phytase addition in increasing the di- Ca/P ratio of 1.20 than at 1.05 led to a higher Ca/P ratio in gestibility of nutrients. The antinutritional effects of phytate the bone. It appears that bone has a certain degree of plasticity could be aggravated by more Ca present in diet. It is well in terms of the ratio of Ca/P in it. However, it is generally known that phytate negatively affects amino acids availability 6 Zhai et al. Table 5. Apparent total tract digestibility (ATTD) of Ca and P in the experimental diets supplemented with monocalcium phosphate (MCP) or phytase Diet Ca/P ratio ATTD ofCa, % ATTD ofP, % Digestible Ca, g Digestible P, g Digestible Ca/P MCP, % 0.25 1.05 56.2 38.9 2.82 1.83 1.55 0.60 1.05 62.8 50.3 3.58 2.74 1.31 0.95 1.05 64.1 53.5 4.28 3.39 1.26 Phytase, U/kg 500 1.05 71.3 62.6 3.33 2.88 1.16 1,000 1.05 77.9 72.0 3.86 3.37 1.15 2,000 1.05 75.7 75.9 3.70 3.48 1.06 3,000 1.05 80.1 81.9 3.86 3.82 1.01 500 1.20 73.7 65.4 3.96 2.94 1.35 1,000 1.20 76.4 71.2 4.15 3.23 1.28 2,000 1.20 80.3 78.0 4.41 3.58 1.23 3,000 1.20 84.8 80.7 4.68 3.73 1.25 SEM 0.94 0.65 0.05 0.03 0.02 P value MCP L <0.01 < 0.01 < 0.01 < 0.01 < 0.01 Q 0.03 < 0.01 0.59 < 0.01 < 0.01 Phytase (1.05) L < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Q < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Phytase (1.20) L < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Q < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Ca/P ratio < 0.01 0.12 < 0.01 0.50 < 0.01 There were six replicate pens. SEM, standard error of the mean. Linear and quadratic effects of monocalcium phosphate. Linear and quadratic effects of phytase at the Ca/P ratio of 1.05. Linear and quadratic effects of phytase at the Ca/P ratio of 1.20. The effect of Ca/P ratio compares phytase diets between the ratios of 1.05 and 1.20, and a significant interaction effect between phytase and the Ca/P ratio was observed for digestibility of Ca and digestible Ca. Figure 1. Urinary concentrations of Ca (linear effect: P < 0.01) and P (quadratic effect: P < 0.01) with increasing ratios of apparent total tract digestible (ATTD) Ca over ATTD P. (Pontoppidan et al., 2007), starch digestion (Thompson, indicated by the significant interaction between phytase and 1987), and fat utilization (Govers and Van der Meer, 1993), the Ca/P ratio on digestible Ca. and is associated with greater endogenous losses of amino These results with different dietary Ca/P ratios in the acids and minerals (Cowieson et al., 2009). The adverse effects current study should be interpreted with prudence because of excess Ca could be mitigated by high doses of phytase as we investigated only a narrow range of dietary Ca/P ratios Bias in phytase efficacy responses 7 Table 6. Comparison of available P release values based on different response variables, g/kg feed Response variable 1.05 Ca/P 1.20 Ca/P SD P value Phytase, FYT/kg feed Phytase, FYT/kg feed Phytase Ca/P ratio 3 3 500 1,000 2,000 3,000 500 1,000 2,000 3,000 L Q ADG , g/d 0.158 0.210 0.199 0.221 0.139 0.180 0.216 0.204 0.03 < 0.01 < 0.01 0.12 Gain:feed, g/kg 0.177 0.234 0.212 0.262 0.119 0.176 0.249 0.226 0.05 < 0.01 0.06 0.05 Bone ash, % 0.117 0.110 0.192 0.182 0.132 0.149 0.193 0.180 0.05 < 0.01 0.18 0.39 Bone P, g 0.125 0.148 0.198 0.197 0.131 0.184 0.207 0.221 0.04 < 0.01 0.04 0.09 There were six replicates. SD, standard deviation. Linear and quadratic effects of phytase. The effect of Ca/P ratio compares between the ratios of 1.05 and 1.20, and no significant interaction between phytase and the Ca/P ratio was observed. ADG, average daily gain. corresponding to a difference in Ca level of only 0.05%. noted when using percent bone ash and bone P weight. This When the Ca/P ratio was examined in a wider range, different generality reflects the different effects of Ca/P ratios on growth conclusions could be made. Qian et al. (1996) investigated performance and bone mineralization as discussed above. It is three total Ca/P ratios of 1.2:1, 1.6:1, and 2.0:1 in diets apparent that Ca plays a very important role in defining the supplemented with 700 or 1,050 U phytase/kg and reported P release values of phytase. The P release values of phytase adverse effects of wide Ca/P ratios on growth performance, should relate to the dietary supply of Ca, which depends on bone characteristics, and P digestibility in weanling pigs. the production goals: growth performance vs bone develop- In growing-finishing pigs fed diets supplemented with 500 ment. More research is warranted to understand and address phytase U/kg, lowering the dietary Ca/P ratio from 1.5:1 to the plausible dilemma for nutritionists that different dietary 1.3:1 to 1.0:1 improved growth performance and bone miner- levels of Ca are required for optimal growth performance and alization (Liu et al., 1998). In nursery pigs, a linear decrease in bone development. Using greater levels of phytase seems to growth performance and bone mineral content was observed be one possible solution. Separating feeding of Ca did not when the Ca/available P ratio increased from 1.25 to 2.75 in appear to be a valid solution (Pointillart and Guéguen, 1993). diets with 250 U/kg phytase added (Becker et al., 2020). These Different Ca sources should be investigated and compared in adverse effects associated with the wide Ca/P ratios could be terms of their dissolution in gut and absorbability. due to a reduction in phytase efficacy ( Qian et al., 1996), the formation of insoluble Ca-P complexes in the gastrointestinal The Homeostasis of Ca and P as Indicated by tract (Stein et al., 2011), and the antinutritional effects of re- Urinary Concentrations of Ca and P sidual phytate due to low phytase dosing coupled with excess The urinary concentration of Ca increased with increasing Ca. The extra supply of Ca can be expelled through urine, but ATTD Ca/P ratio, which agrees with the results by Stein et has a negative impact on P digestibility (Stein et al., 2011). al. (2011) that increasing Ca supply from 55 to 173% of the In the current study, a slight increase in Ca supply didn’t de- requirement increased urinary excretion of Ca. Calcium ho- press P digestibility and the improved digestible Ca/P ratio meostasis is mainly regulated in kidney rather than in gut in happened to be conducive to bone mineralization. pigs (Stein et al., 2011). On the contrary, P homeostasis can be regulated at both renal and intestinal levels (Rodehutscord Measuring Available P Release of Phytase et al., 1999). The urinary concentration of P increased strik- A fixed dietary Ca/P ratio was usually used to determine ingly at the ratios of 1.01 and 1.06 when compared to the available P release from phytase. A ratio of 2:1 was used by ratio of 1.15 or above, indicating the excretion of excess P Kornegay and Qian (1996), a ratio of 1.10:1 (0.97 to 1.10 on through urine when there was a deficiency of Ca in relation to analysis) by Wensley et al. (2020a), and a ratio between 1.2 P. Pointillart and Fontaine (1983) also found more digestible and 1.3 (1.31 to 1.39 on analysis) by Dersjant-Li et al. (2020). P lost in urine because of a lack of Ca in the diet. The combi- The total Ca and P do not reflect the different digestibility nation of concentrations of Ca and P in urine can be used as among feed ingredients, and the same total Ca/P ratios might a powerful indicator for the balance of Ca and P supply for result in different digestible Ca/P ratios. In the current study, pigs. More research should be conducted to link the urinary the digestible Ca/P ratio decreased with increasing supple- concentrations of Ca and P with different degrees of bone mentation of either MCP or phytase, and the digestible Ca/P mineralization. This relationship, if coupled with a real-time ratios were more closely correlated with the systemic home- portable device to measure urinary Ca and P concentrations, ostasis of Ca and P as indicated by urinary concentrations of can be a useful tool to gauge the balance of Ca and P in diet Ca and P. Of note, only at the total Ca/P ratio of 1.20 did the for pigs. digestible Ca/P ratios in the diets with phytase remained sim- ilar to the ratios in the reference diets with a total Ca/P ratio of 1.05. It appears that fixing the total Ca/P ratios in diets to CONCLUSION compare MCP and phytase is not “fair” for phytase. In general, the available P release values were higher at the In this study, increasing the dietary Ca/P ratio from 1.05 total Ca/P ratio of 1.05 than at 1.20 when ADG and gain:feed to 1.20 in diets supplemented with 500 to 3,000 FYT/kg were used as response variables, whereas the opposite was phytase impaired feed utilization efficiency, but increased 8 Zhai et al. dietary digestible calcium on growth performance, bone mineral- the digestible Ca in diet and the deposition of Ca and P in ization, plasma calcium, and abundance of genes involved in in- bone. This led to a tendency to elicit higher available P re- testinal absorption of calcium in pigs from 11 to 22  kg fed diets lease values from phytase at the Ca/P ratio of 1.20 than at with different concentrations of digestible phosphorus. J. Anim. Sci. 1.05 when using bone P weight as the response variable. Biotechnol. 10:47. doi:10.1186/s40104-019-0349-2. Therefore, fixing the same total Ca/total P ratio in diets Lagos, L. V., A. L. Su, M. R. Bedford, and H. H. Stein. 2021. Formulating supplemented with increasing phytase dosing created an im- diets based on digestible calcium instead of total calcium does not balance of digestible Ca and P, which could have an adverse affect growth performance or carcass characteristics, but microbial effect on bone mineralization and should be corrected with phytase ameliorates bone resorption caused by low calcium in diets the appropriate addition of Ca. More research is warranted fed to pigs from 11 to 130 kg. J. Anim. Sci. 99:1–11. doi:10.1093/ to precisely quantify the contribution of digestible Ca and jas/skab057. Létourneau-Montminy, M. P., C. Jondreville, D. Sauvant, and A. Narcy. P by phytase under different dietary and animal conditions, 2012. Meta-analysis of phosphorus utilization by growing pigs: ef- and this will enable a more precise supply of adequate Ca fect of dietary phosphorus, calcium and exogenous phytase. Ani- and P for nutrition. mal. 6:1590–1600. doi:10.1017/S1751731112000560. Létourneau-Montminy, M. P., A. Narcy, M. Magnin, D. Sauvant, J. F. Bernier, C. Pomar, and C. Jondreville. 2010. Effect of reduced die- Conflict of Interest Statement tary calcium concentration and phytase supplementation on cal- The authors are employees of DSM Nutritional Products. cium and phosphorus utilization in weanling pigs with modified mineral status. J. Anim. Sci. 88:1706–1717. doi:10.2527/jas.2008- LITERATURE CITED Liu, J., D. W. Bollinger, D. R. Ledoux, and T. L. Veum. 1998. Lowering AOAC International. 2006. Official methods of analysis . 18th ed. the dietary calcium to total phosphorus ratio increases phos- Arlington (VA): Association of Official Analytical Chemists. phorus utilization in low-phosphorus corn-soybean meal diets Becker, S. L., S. A. Gould, A. L. Petry, L. M. Kellesvig, and J. F. Patience. supplemented with microbial phytase for growing-finishing pigs. J. 2020. 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Sci. 98:1–8. doi:10.1093/jas/skaa251. 3866. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Use of fixed calcium to phosphorus ratios in experimental diets may create bias in phytase efficacy responses in swine

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Translational Animal Science, 2022, 6, 1–9 https://doi.org/10.1093/tas/txac124 Advance access publication 29 August 2022 Non Ruminant Nutrition Use of fixed calcium to phosphorus ratios in experimental diets may create bias in phytase efficacy responses in swine †,1 ‡ † † † Hengxiao Zhai, Jon R. Bergstrom, JingchengZhang , Wei Dong, Zhenzhen Wang, ║ ║ Kostas Stamatopoulos, and Aaron J. Cowieson DSM (China) Animal Nutrition Research Center, Bazhou 065799, China DSM Nutritional Products, Parsippany, NJ 07054, USA DSM Nutritional Products, Kaiseraugst 4303, Switzerland Corresponding author: heng-xiao.zhai@dsm.com ABSTRACT The objective of this study was to investigate the effects of two dietary total Ca/P ratios on available P release by phytase, measured using growth performance and bone mineralization with 528 barrows and gilts according to a randomized complete block design. Three were 11 diets in a factorial of 2 by 4 plus 3, including 3 reference diets consisting of 0.25% (control), 0.70%, or 1.15% monocalcium phosphate (MCP) and 8 diets from combining 4 phytase doses (500, 1,000, 2,000, and 3,000 FYT/kg) with 0.25% MCP and 2 dietary Ca/P ratios (1.05 and 1.20). Each diet was fed to 6 pens of 8 pigs. All diets contained 3 g/kg TiO , and fecal samples were collected from each pen on d 13–15 of trial. At the end of trial, one pig per pen was sacrificed to collect a tibia and urine in the bladder. The results showed that MCP improved growth performance lin - early (P < 0.01), whereas both a linear and quadratic response was observed with the addition of phytase. The MCP increased the percent bone ash and weights of bone ash, Ca, and P linearly (P < 0.01). At both Ca/P ratios, increasing supplementation of phytase increased the percent bone ash and weights of bone ash, Ca, and P both linearly and quadratically (P < 0.05). Both MCP and phytase significantly increased digesti - bility of Ca and P as well as digestible Ca and P in diets and reduced the digestible Ca/P ratio. The dietary Ca/P ratio of 1.20 resulted in poorer feed utilization efficiency, more digestible Ca, greater percent bone ash, Ca, and P and heavier weights of bone Ca and P than the ratio of 1.05 (P < 0.05). The ratio of 1.20 elicited numerically higher available P release values from phytase, with percent bone ash and bone P weight as the response variables, but significantly lower values with gain:feed. The urinary concentration of Ca increased linearly ( P < 0.01) with increasing digestible Ca/P ratios whilst urinary concentration of P decreased quadratically (P < 0.01). In conclusion, fixing the same total Ca/total P ratio in diets supplemented with increasing phytase dosing created an imbalance of digestible Ca and P, which could have an adverse effect on bone mineralization and thus compromise the phytase efficacy relative to mineral P. Lay Summary Phytase is the most widely applied feed enzyme in livestock farming because of its main function of releasing phosphorus (P) bound by phytate in most plant feed ingredients which would otherwise not be utilized effectively by farm animals. This basic function enables phytase to become a proper supplement to an animal’s deficient phytate-processing system and to reduce the reliance of livestock farming on phosphate rocks. Phosphate rocks are a nonrenewable source of P for farm animals. The P contribution to a diet by phytase, therefore, needs to be measured for feed formulators to use phytase to its maximum potential and to meet the animal’s requirement for P precisely. Animal scientists have es- tablished different methods to determine either available or digestible P release from phytase, and each method has its own pros and cons. At the same time, this diversity of measurement methods creates confusion over selection of the appropriate P release values for phytase. This study explores the methodological differences with regard to measuring P release from phytase with the aim to shed light upon the underlying reasons and ultimately to facilitate more precise application of phytase in feed industry. Key words: available, bone, calcium, digestibility, phosphorus, phytase INTRODUCTION Increasing dietary Ca also increases bone mineralization until its maximum, as long as P and Ca are balanced (Létourneau- Phytase efficacy, in terms of available P release, was calcu - Montminy et al., 2012). Either excessive or inadequate supply lated from a standard response curve established by plotting of Ca in a diet has negative impacts on bone mineralization. a sensitive response variable such as bone ash against the Lagos et al (2021) summarized the standardized total tract level/intake of mineral P with an assumed P bioavailability digestible (STTD) Ca/STTD P ratios to be 1.70:1, 1.80:1, of 100% (Cromwell et al., 1993; Kornegay and Qian, 1996; 2:00:1, and 2.30:1 to maximize bone ash for 11–22, 20–50, Wensley et al., 2020a). An underlying assumption is that die- 50–85, and 100–130 kg pigs, respectively, when P was at ad- tary P supply, when it’s not exceeding the requirement and Ca equate. Theoretically, this digestible system can guarantee an is not limiting, is a good predictor for bone mineralization. Received July 16, 2022 Accepted August 29, 2022. © The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Zhai et al. optimal balance between Ca and P in diets for pigs. In the room temperature was 27 °C at the start and gradually field, the Ca and P homeostasis is worth monitoring to main - reduced to 23 °C at the end. The relative humidity was re- tain an optimal balance between Ca and P and to minimize corded to range from 40% to 60%. P footprint on the environment. The urinary concentrations of Ca and P should be explored as potential markers because Experimental Diets an oversupply of Ca or P was shown to trigger an increase The ingredient and nutrient composition of the basal diet in their excretion through urine (Rodehutscord et al., 1999; are presented in Table 1. There were 11 experimental diets. Stein et al., 2011). Three diets were established by including 0.25% (con- In pig studies to measure available P release by phytase, trol), 0.60%, or 0.95% MCP to establish the standard re- the dietary Ca levels (Cromwell et al., 1993) or ratios of total sponse curves. Four more diets were created by including the Ca/P were fixed. For example, a total Ca/P ratio of 2:1 was phytase at 500, 1,000, 2,000, or 3,000 FYT/kg feed in the used by Kornegay and Qian (1996), a ratio of 1.10:1 (0.97 to control diet. The test phytase (HiPhorius, DSM Nutritional 1.10 on analysis) by Wensley et al. (2020a), and a ratio be- Products, Switzerland) was encoded by a 6-phytase gene from tween 1.2 and 1.3 (1.31 to 1.39 on analysis) by Dersjant-Li Citrobacter braakii and expressed from a strain of Aspergillus et al. (2020). The total Ca and P do not reflect their different digestibility among feed ingredients, which implies that the same total Ca/P ratios in diets of different ingredient compo- Table 1. Ingredient and nutrient composition of the basal diet (g/kg of sition might correspond to disparate digestible Ca/P ratios. In feed, as-is basis) addition, the contribution of digestible Ca by phytase has not been described precisely, and the issue of dissimilar digestible Items Basal diet Ca/P ratios could be even worse between the reference diets and phytase diets in studies to determine the available P re- Ingredients lease for phytase. Corn 590.9 The aim of the current study, therefore, was to investigate Soybean meal 340.5 the effects of two total Ca/P ratios (1.05 and 1.20) on the Soybean oil 25.0 growth performance, digestibility of Ca and P, bone miner- NaCl 4.5 alization, and urinary concentrations of Ca and P in nursery NaHCO 1.5 pigs supplemented with different doses of a novel phytase. We hypothesized that the bone mineralization will be greater L-Lys·HCl 4.5 when there is slightly more Ca in the phytase-supplemented DL-Met 1.2 diets and, therefore, the measured available P release by L-Thr 1.5 phytase as benchmarked to monocalcium phosphate (MCP) L-Val 1.0 will be elevated. Limestone 6.8 Monocalcium phosphate 2.5 MATERIALS AND METHODS Vitamin-mineral premix 5.0 Rice hull 9.1 This study was conducted at DSM (China) Animal Nutrition Research Center Co. Ltd. (Bazhou, P. R. China), and its Benzoic acid 3.0 protocol was approved by the Animal Welfare Committee TiO 3.0 of DSM (China) Animal Nutrition Research Center Total 1,000.0 (AWCCAN). The guidelines in European Union council di- Calculated nutrients and energy rective 2010/63/EU for animal experiments were followed Net energy, kcal/kg 2,499 in this study. Metabolizable energy, kcal/kg 3,372 Crude protein 21.8 Animals and Facilities Total Ca 4.5 Five hundred and twenty-eight barrows and gilts (PIC L1050 × L337; initial body weight 7.4  ±  0.9  kg [mean ± standard Total P 4.2 deviation]) were used. The pigs were weaned at an average Phytate P 2.5 age of 21 d and transferred to a nursery facility for an adap- Standardized total tract digestible P 2.0 tation period of 7 d using a common starter diet. The nursery Standardized ileal digestible facility was equipped with 80 pens (space/pen = 3.0 × 1.8 m ). Lys 12.9 Each pen had a plastic-coated wire floor and was equipped Met 3.9 with two water nipples and one stainless-steel feeder. After Thr 7.7 the adaptation period, the pigs were individually weighed and Trp 2.1 allotted into 66 pens based on their initial BW and gender (four barrows and four gilts per pen). Each pen of pigs was Val 8.3 assigned to 1 of 11 dietary treatments in a Randomized Premix supplied per kilogram of diet: vitamin A, 9,750 IU; vitamin D , Complete Block Design, resulting in 6 replicate pens per die- 3,000 IU; vitamin E, 63 mg; vitamin K , 3.0 mg; vitamin B , 3.0 mg; 3 1 tary treatments. The experimental diets were fed for 21 d, vitamin B , 9.6 mg; vitamin B , 4.5 mg; vitamin B , 36 μg; D-biotin, 240 2 6 12 with feed and water supplied ad libitum. At the end of trial, ug; D-calcium pantothenate, 30 mg; folic acid, 1.8 mg; niacin, 36 mg; Cu (tribasic copper chloride), 190 mg; I (potassium iodate), 0.6 mg; the pigs weighed 17.1 ± 2.1 kg. Fe (ferrous sulfate), 120 mg; Mn (manganese sulfate), 60 mg; Zn (zinc Room temperature and ventilation were controlled by a sulfate), 120 mg; Se (sodium selenite), 450 μg; choline (choline chloride), 300 mg; and Ca (calcium carbonate) 0.6 g. computer system to provide an optimal environment. The Bias in phytase efficacy responses 3 oryzae. In these seven diets, the formulated ratio of total Ca Digestibility of Ca and P was calculated using the following to total P was maintained at 1.05 by adjusting the inclusion equation: level of limestone. An additional four diets also included 500, 1,000, 2,000, or 3,000 FYT/kg phytase but with a formulated D =[1 − (T /T ) × (M /M )] × 100; i o o i total Ca/P ratio of 1.20 using adjusted inclusion levels of limestone. Titanium dioxide was included at 3 g/kg feed as an where D is the digestibility of Ca or P (%); T and T are the i o indigestible marker to enable the measurement of apparent titanium concentrations in diet and feces, respectively (% of total tract digestibility (ATTD) of Ca and P in all diets. The DM); M and M are the concentrations of Ca or P in diet i o net energy and nutrients, other than Ca and P, were above and feces (% of DM), respectively. The digestible Ca and P the recommendations in NRC (2012) for all the experimental were calculated by multiplying the concentrations of Ca diets. All diets were pelleted with a conditioning temperature and P in feed (g/kg feed) by their corresponding digestibility at 75 °C. coefficients. Available P release by phytase was calculated by referring to the method described by Wensley et al. (2020a). The Measurement and Sampling standard response curves were established by regressing ADG The pigs were individually weighed on day 0 and 21 of trial to against dietary P intake (g/d) and regressing gain:feed, bone obtain the total weight for each pen of pigs, and the feed con- ash percent, and bone P weight against dietary P concentra- sumption per pen was recorded during the trial to calculate tion (%). The feed intake was incorporated for the response average daily gain (ADG), average daily feed intake (ADFI), curve of ADG to achieve a greater fitting to the results. Using and gain:feed. the standard response equations, the dietary P concentration Fresh and clean fecal samples were grabbed from each pen equivalence was solved for each dose of phytase. These equiv- on day 13 to 15 of trial. All the pens were cleaned, and ex- alent values were corrected for the contribution by the con- isting feces removed before collection on each collection day. trol diet to generate the available P release values for phytase. A total of approximately 500  g of fresh feces was collected The data were analyzed using the GLM procedure of SAS per day per pen. All the fecal samples collected from each pen (SAS Inst. Inc., Cary, NC) with the model including the die- during the 3-d collection period were pooled and mixed to tary treatment as a fixed effect, replicate as a random effect, homogeneity with a hand-held blade mixer (TD-110, RuiBao and an error term. Polynomial orthogonal contrasts were Hardware Co. Ltd., Dongguan, P. R. China). A subsample of constructed to test the linear and quadratic effects of supple- around 400 g for each pen was collected and stored at −20 °C mentation of MCP and phytase, the effect of dietary Ca/P ratio until further processing. among phytase diets, and the interaction between phytase and The right tibia and urine from the bladder (if abundant at the Ca/P ratio. The least-square means were presented, and sampling) were collected from the pig in each pen with body the significance was defined at P < 0.05. weight closest to the average body weight per pen on d 21 of trial. The tibias were processed by referring to the non- defatting bone processing procedures described by Wensley RESULTS et al. (2020b). In short, the bones were autoclaved at 120 °C for 30 min to facilitate the removal of muscular tissues and Experimental Diets and the Analyses cartilaginous caps. The cleaned bones were left at room tem- The total P in diets with 0.25% MCP was analyzed to be perature for 1 d and then oven-dried at 105 °C for 7 d. In the 0.45%–0.47%, and incremental increases of 0.08%–0.09% end, the dried tibias were incinerated in a muffler oven for P agree with the increasing dietary inclusion of MCP from 72 h at 600 °C. All the samples were stored at −20 °C before 0.25% to 0.60% to 0.95% (Table 2). The analyzed total processing. Ca/P ratios ranged from 1.02 to 1.07 for the target of 1.05 and from 1.19 to 1.20 for 1.20. The analyzed marker Chemical Analyses concentrations ranged from 97% to 101% of the formulated value. The analyzed phytase activities were within ± 15% of The fecal samples were oven-dried to a constant weight and the intended doses. ground to pass through a 0.5-mm screen before analysis. The dietary and fecal samples were dried at 105 °C in an oven for 4 h for dry matter determination (method 934.01; AOAC Growth Performance and Bone Mineralization International, 2006). Titanium, Ca, and P were determined by The interaction between phytase and the Ca/P ratio was not Inductively Coupled Plasma-Optical Emission Spectrometry significant for growth performance. Increasing supplementa - (ICP-OES; Optima TM 8000, PerkinElmer, Shelton, USA; tion of MCP linearly improved (P < 0.01) final BW, ADG, method 985.01; AOAC International, 2006) after microwave ADFI and gain:feed, whereas both linear and quadratic digestion. Ten mL of each urine sample were dried at 60 °C responses, were observed with the addition of phytase (P < before the microwave digestion. One phytase unit (FYT) was 0.01; Table 3). Reducing the Ca/P ratio from 1.20 to 1.05 sig- defined as the amount of enzyme that releases 1 µmol of in - nificantly increased gain:feed from 766 to 778 g/kg. organic phosphate from 50 mM phytate per minute at 37 °C There was no significant interaction between phytase and pH 5.5. These analyses were performed in duplicate, ex- and the Ca/P ratio for bone mineralization. Incremental cept that phytase activity in the feed samples was determined increases in MCP linearly increased (P < 0.01) percent from three replicates. bone ash and weights of bone ash, Ca, and P (Table 4). Increasing the addition of phytase increased the percent Calculations and Statistical Analyses bone ash and weights of bone ash, Ca and P, both linearly The experiment was a randomized complete block design. and quadratically (P < 0.05). The bone Ca/P ratio decreased Each pen or pig was an experimental unit. linearly (P < 0.01) with increasing supplementation of MCP 4 Zhai et al. Table 2. Analyzed nutrients of the dietary treatments (as-is basis, %) Table 3. Growth performance of the pigs supplemented with monocalcium phosphate (MCP) or phytase Diet Ca/P ratio Ca, % P, % Ca/P ratio Phytase, U/kg 2 2 2 2 Diet IBW , FBW , ADG , ADFI , Gain: MCP, % kg kg g/d g/d feed, g/kg 0.25 1.05 0.50 0.47 1.07 0 MCP, % 0.60 1.05 0.57 0.55 1.05 0 0.25 7.4 14.9 359 521 691 0.95 1.05 0.67 0.64 1.05 0 0.60 7.4 16.0 410 563 729 Phytase, U/kg 0.95 7.4 16.8 447 591 757 500 1.05 0.47 0.46 1.02 511 Phytase, 1,000 1.05 0.50 0.47 1.06 1,009 U/kg 2,000 1.05 0.49 0.46 1.07 2,110 500 7.4 16.9 451 602 749 3,000 1.05 0.48 0.47 1.03 3,333 1,000 7.4 17.6 484 628 771 500 1.20 0.54 0.45 1.20 573 2,000 7.4 17.7 490 627 782 1,000 1.20 0.54 0.45 1.19 1,112 3,000 7.4 17.7 490 624 787 2,000 1.20 0.55 0.46 1.19 2,292 Ca/P 3,000 1.20 0.55 0.46 1.19 3,240 ratio 1.05 7.4 17.5 480 618 778 Phytase activity was analyzed in three replicates and the others in 1.20 7.4 17.4 477 623 766 duplicate. SD 0.09 0.43 20 26 19 P value and quadratically (P < 0.01) with supplemental phytase. MCP L 0.28 <0.01 <0.01 <0.01 <0.01 The dietary Ca/P ratio of 1.20 resulted in greater percent Q 0.60 0.46 0.52 0.61 0.63 bone ash Ca and P, a heavier weight of bone Ca, and a Phytase L 0.71 <0.01 <0.01 <0.01 <0.01 higher Ca/P ratio in bone than the dietary Ca/P ratio of 1.05 (P < 0.05). Q 0.40 <0.01 <0.01 <0.01 <0.01 Ca/P 0.68 0.64 0.59 0.55 <0.05 ratio Digestibility of Ca and P, Digestible Ca and P in Experimental Diets, and Urinary Concentrations of There were six replicates of eight pigs. Ca and P 2 IBW, initial body weight; FBW, final body weight; ADG, average daily gain; ADFI, average daily feed intake. The interaction between phytase and the Ca/P ratio was SD, standard deviation. significant for digestibility of Ca and digestible Ca. This 4 Linear and quadratic effects of monocalcium phosphate. Linear and quadratic effects of phytase. significant interaction was due to continued increase in The effect of Ca/P ratio compares phytase diets between the ratios of 1.05 digestibility of Ca and digestible Ca beyond the phytase and 1.20, and no significant interaction between phytase and the Ca/P dose of 1,000 FYT/kg at the Ca/P ratio of 1.20 in con- ratio was observed. trast to the plateaued response at the Ca/P ratio of 1.05. Increasing MCP or phytase in the diets, irrespective of the whereas a linear (P < 0.01) increase was observed based on dietary Ca/P ratio, improved digestibility of P and digestible gain: feed and percent bone ash. The Ca/P ratio of 1.05 tended P, but reduced the digestible Ca/P ratio, both linearly and to increase available P release based on gain: feed (0.221 vs quadratically (P < 0.05; Table 5). Digestible Ca was line- 0.192) when compared to the ratio of 1.20, but the opposite arly increased (P < 0.01) with increasing dietary inclusion tendency (0.167 vs 0.186) was shown by bone P weight (P < of MCP. The high dietary Ca/P ratio significantly increased 0.10). the digestible Ca/P ratio compared to the low dietary Ca/P ratio (1.28 vs 1.10). The urinary concentration of Ca increased linearly (P < DISCUSSION 0.01) with increasing digestible Ca/P ratio whilst urinary con- The Diets and Animals centration of P increased quadratically (P < 0.01; Figure 1). In general, the analyzed dietary concentrations of Ca and P were slightly higher than the formulated values, but the agree- Standard Response Curves ment between the calculated and formulated ratios lent cre- Y = 76.6 × X + 168.8 The standard regression equations were dence to the experimental diets to test the hypothesis of this 2 2 (r = 0.94), Y = 396.3 × X + 507.8 (r = 0.62), study. Moreover, the analyzed phytase activities were con- 2 2 Y = 2.08 × X + 0.45 Y = 39.2 × X + 25.1 (r = 0.61), and (r sistent with their intended values. The stepwise increases in = 0.78), respectively, for ADG, gain:feed, percent bone ash both analyzed Ca and P agreed with the incremental additions and bone P weight, respectively. of MCP, which are prerequisites for establishing the standard response curves. The animals were in good health throughout The Comparison of Available P Release by Phytase the trial. The culling rate was only 1.0%. The interaction between phytase and the Ca/P ratio was not Phytase Efficacy significant ( Table 6). Using ADG and bone P weight as re- sponse variables, available P release increased both linearly The measured growth performance, digestibility of Ca and and quadratically (P < 0.05) with increasing phytase dose, P, and bone mineralization in this study followed a classical, Bias in phytase efficacy responses 5 Table 4. Bone mineralization of the pigs supplemented with monocalcium phosphate (MCP) or phytase Diet Bone ash, % Bone ash Ca, % BoneashP, % Bone ash, g Bone Ca, g Bone P, g Bone Ca/P ratio MCP, % 0.25 43.7 33.8 16.9 3.18 1.07 0.53 2.00 0.60 46.0 33.6 17.0 4.01 1.34 0.68 1.98 0.95 50.1 33.4 17.2 5.09 1.70 0.88 1.94 Phytase, U/kg 500 48.0 33.9 17.3 4.51 1.51 0.78 1.96 1,000 48.2 34.0 17.6 4.87 1.64 0.85 1.92 2,000 50.6 34.4 17.6 5.28 1.80 0.93 1.94 3,000 50.2 34.6 17.7 5.36 1.84 0.94 1.95 Ca/P ratio 1.05 49.0 33.6 17.4 4.95 1.65 0.86 1.93 1.20 49.5 34.8 17.8 5.06 1.75 0.90 1.96 SD 2.20 1.44 0.59 0.41 0.15 0.07 0.04 P value MCP L <0.01 0.67 0.32 <0.01 <0.01 <0.01 <0.01 Q 0.41 0.98 0.70 0.53 0.61 0.45 0.46 Phytase L <0.01 0.14 <0.01 <0.01 <0.01 <0.01 0.09 Q <0.01 0.92 0.08 <0.01 <0.01 <0.01 <0.01 Ca/P ratio 0.41 <0.01 0.03 0.36 0.03 0.06 0.04 There were six replicates of one pig. SD, standard deviation. Linear and quadratic effects of monocalcium phosphate. Linear and quadratic effects of phytase. The effect of Ca/P ratio compares phytase diets between the ratios of 1.05 and 1.20, and no significant interaction between phytase and the Ca/P ratio was observed. curvilinear trend in response to the increasing levels of phytase, considered that the ratio of Ca/P in bone is about 2.1:1 which which attests to the basic function of phytase to liberate Ca is tightly regulated due to the finite chemical structure of hy - and P. The improvement in body weight gain with added droxyapatite of bone (Cromwell, 2005). Thirdly, our Ca/P phytase was attributed to an increase in both feed intake and ratios in bone are much lower than the ratios reported by feed utilization efficiency. A deficiency of P can cause a poor González-Vega et al. (2016) and Lagos et al. (2019), which appetite in pigs (Jongbloed, 1987), so the addition of phytase indicates that there is still space for even higher dietary Ca/P to a P-deficient diet should restore appetite by providing bi - ratios to elicit greater bone development. oavailable P. The improvement in feed utilization efficiency The Effects of Dietary Total Ca to Total P Ratio is supported by the close relationship between whole-body P mass and whole-body N mass (NRC, 2012). Soft tissue devel- In the current study, increasing dietary Ca/P ratio from 1.05 opment is dependent mainly on P even though bone mineral- to 1.20 impaired feed utilization efficiency, but increased di - ization requires both Ca and P (Létourneau-Montminy et al., gestibility of Ca as well as the amount of digestible Ca, and 2012). Moreover, the preference of most exogenous phytases consequently increased the amounts of Ca and P deposited in for breaking down myo-inositol hexakisphosphate (IP ) and bone. These results indicate that slightly more Ca in phytase- IP (Wyss et al., 1999; Pontoppidan et al., 2012) means there supplemented diets could result in more Ca digested and ab- will be less residual phytate exerting antinutritional effects sorbed without diminishing the efficacy of phytase in terms of because IP and IP are more capable of binding protein and P release and greater bone mineralization could be realized. 6 5 minerals than other IP esters (Yu et al., 2012). In addition, This agrees with the finding by Létourneau-Montminy et al. myo-inositol released from complete destruction of phytate (2010) that reducing the dietary Ca/P ratio from 1.9 to 1.3 at high phytase doses might provide some extra-phosphoric in a practical diet containing 0.56% P did not improve the effects (Schmeisser et al., 2017; Lu et al., 2019). efficiency of phytase in releasing P, but impaired bone miner - There were some interesting observations about bone min- alization in weanling pigs. Increasing dietary Ca will increase eralization. First, phytase increased percentage and weight of the amount of retained P as long as Ca and P are balanced bone ash irrespective of the dietary Ca/P ratio, but a further or until bone mineralization reaches a plateau (Létourneau- improvement in the percentage of Ca and P in bone ash was Montminy et al., 2012). The impairment of gain:feed by observed only at the dietary Ca/P ratio of 1.20. This implies the high Ca/P ratio agrees with the finding by Johnston et that weights of Ca and P in bone ash are more sensitive than al. (2004) that the reduction in dietary Ca and P was just bone ash weight. Second, more available Ca at the dietary as effective as dietary phytase addition in increasing the di- Ca/P ratio of 1.20 than at 1.05 led to a higher Ca/P ratio in gestibility of nutrients. The antinutritional effects of phytate the bone. It appears that bone has a certain degree of plasticity could be aggravated by more Ca present in diet. It is well in terms of the ratio of Ca/P in it. However, it is generally known that phytate negatively affects amino acids availability 6 Zhai et al. Table 5. Apparent total tract digestibility (ATTD) of Ca and P in the experimental diets supplemented with monocalcium phosphate (MCP) or phytase Diet Ca/P ratio ATTD ofCa, % ATTD ofP, % Digestible Ca, g Digestible P, g Digestible Ca/P MCP, % 0.25 1.05 56.2 38.9 2.82 1.83 1.55 0.60 1.05 62.8 50.3 3.58 2.74 1.31 0.95 1.05 64.1 53.5 4.28 3.39 1.26 Phytase, U/kg 500 1.05 71.3 62.6 3.33 2.88 1.16 1,000 1.05 77.9 72.0 3.86 3.37 1.15 2,000 1.05 75.7 75.9 3.70 3.48 1.06 3,000 1.05 80.1 81.9 3.86 3.82 1.01 500 1.20 73.7 65.4 3.96 2.94 1.35 1,000 1.20 76.4 71.2 4.15 3.23 1.28 2,000 1.20 80.3 78.0 4.41 3.58 1.23 3,000 1.20 84.8 80.7 4.68 3.73 1.25 SEM 0.94 0.65 0.05 0.03 0.02 P value MCP L <0.01 < 0.01 < 0.01 < 0.01 < 0.01 Q 0.03 < 0.01 0.59 < 0.01 < 0.01 Phytase (1.05) L < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Q < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Phytase (1.20) L < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Q < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Ca/P ratio < 0.01 0.12 < 0.01 0.50 < 0.01 There were six replicate pens. SEM, standard error of the mean. Linear and quadratic effects of monocalcium phosphate. Linear and quadratic effects of phytase at the Ca/P ratio of 1.05. Linear and quadratic effects of phytase at the Ca/P ratio of 1.20. The effect of Ca/P ratio compares phytase diets between the ratios of 1.05 and 1.20, and a significant interaction effect between phytase and the Ca/P ratio was observed for digestibility of Ca and digestible Ca. Figure 1. Urinary concentrations of Ca (linear effect: P < 0.01) and P (quadratic effect: P < 0.01) with increasing ratios of apparent total tract digestible (ATTD) Ca over ATTD P. (Pontoppidan et al., 2007), starch digestion (Thompson, indicated by the significant interaction between phytase and 1987), and fat utilization (Govers and Van der Meer, 1993), the Ca/P ratio on digestible Ca. and is associated with greater endogenous losses of amino These results with different dietary Ca/P ratios in the acids and minerals (Cowieson et al., 2009). The adverse effects current study should be interpreted with prudence because of excess Ca could be mitigated by high doses of phytase as we investigated only a narrow range of dietary Ca/P ratios Bias in phytase efficacy responses 7 Table 6. Comparison of available P release values based on different response variables, g/kg feed Response variable 1.05 Ca/P 1.20 Ca/P SD P value Phytase, FYT/kg feed Phytase, FYT/kg feed Phytase Ca/P ratio 3 3 500 1,000 2,000 3,000 500 1,000 2,000 3,000 L Q ADG , g/d 0.158 0.210 0.199 0.221 0.139 0.180 0.216 0.204 0.03 < 0.01 < 0.01 0.12 Gain:feed, g/kg 0.177 0.234 0.212 0.262 0.119 0.176 0.249 0.226 0.05 < 0.01 0.06 0.05 Bone ash, % 0.117 0.110 0.192 0.182 0.132 0.149 0.193 0.180 0.05 < 0.01 0.18 0.39 Bone P, g 0.125 0.148 0.198 0.197 0.131 0.184 0.207 0.221 0.04 < 0.01 0.04 0.09 There were six replicates. SD, standard deviation. Linear and quadratic effects of phytase. The effect of Ca/P ratio compares between the ratios of 1.05 and 1.20, and no significant interaction between phytase and the Ca/P ratio was observed. ADG, average daily gain. corresponding to a difference in Ca level of only 0.05%. noted when using percent bone ash and bone P weight. This When the Ca/P ratio was examined in a wider range, different generality reflects the different effects of Ca/P ratios on growth conclusions could be made. Qian et al. (1996) investigated performance and bone mineralization as discussed above. It is three total Ca/P ratios of 1.2:1, 1.6:1, and 2.0:1 in diets apparent that Ca plays a very important role in defining the supplemented with 700 or 1,050 U phytase/kg and reported P release values of phytase. The P release values of phytase adverse effects of wide Ca/P ratios on growth performance, should relate to the dietary supply of Ca, which depends on bone characteristics, and P digestibility in weanling pigs. the production goals: growth performance vs bone develop- In growing-finishing pigs fed diets supplemented with 500 ment. More research is warranted to understand and address phytase U/kg, lowering the dietary Ca/P ratio from 1.5:1 to the plausible dilemma for nutritionists that different dietary 1.3:1 to 1.0:1 improved growth performance and bone miner- levels of Ca are required for optimal growth performance and alization (Liu et al., 1998). In nursery pigs, a linear decrease in bone development. Using greater levels of phytase seems to growth performance and bone mineral content was observed be one possible solution. Separating feeding of Ca did not when the Ca/available P ratio increased from 1.25 to 2.75 in appear to be a valid solution (Pointillart and Guéguen, 1993). diets with 250 U/kg phytase added (Becker et al., 2020). These Different Ca sources should be investigated and compared in adverse effects associated with the wide Ca/P ratios could be terms of their dissolution in gut and absorbability. due to a reduction in phytase efficacy ( Qian et al., 1996), the formation of insoluble Ca-P complexes in the gastrointestinal The Homeostasis of Ca and P as Indicated by tract (Stein et al., 2011), and the antinutritional effects of re- Urinary Concentrations of Ca and P sidual phytate due to low phytase dosing coupled with excess The urinary concentration of Ca increased with increasing Ca. The extra supply of Ca can be expelled through urine, but ATTD Ca/P ratio, which agrees with the results by Stein et has a negative impact on P digestibility (Stein et al., 2011). al. (2011) that increasing Ca supply from 55 to 173% of the In the current study, a slight increase in Ca supply didn’t de- requirement increased urinary excretion of Ca. Calcium ho- press P digestibility and the improved digestible Ca/P ratio meostasis is mainly regulated in kidney rather than in gut in happened to be conducive to bone mineralization. pigs (Stein et al., 2011). On the contrary, P homeostasis can be regulated at both renal and intestinal levels (Rodehutscord Measuring Available P Release of Phytase et al., 1999). The urinary concentration of P increased strik- A fixed dietary Ca/P ratio was usually used to determine ingly at the ratios of 1.01 and 1.06 when compared to the available P release from phytase. A ratio of 2:1 was used by ratio of 1.15 or above, indicating the excretion of excess P Kornegay and Qian (1996), a ratio of 1.10:1 (0.97 to 1.10 on through urine when there was a deficiency of Ca in relation to analysis) by Wensley et al. (2020a), and a ratio between 1.2 P. Pointillart and Fontaine (1983) also found more digestible and 1.3 (1.31 to 1.39 on analysis) by Dersjant-Li et al. (2020). P lost in urine because of a lack of Ca in the diet. The combi- The total Ca and P do not reflect the different digestibility nation of concentrations of Ca and P in urine can be used as among feed ingredients, and the same total Ca/P ratios might a powerful indicator for the balance of Ca and P supply for result in different digestible Ca/P ratios. In the current study, pigs. More research should be conducted to link the urinary the digestible Ca/P ratio decreased with increasing supple- concentrations of Ca and P with different degrees of bone mentation of either MCP or phytase, and the digestible Ca/P mineralization. This relationship, if coupled with a real-time ratios were more closely correlated with the systemic home- portable device to measure urinary Ca and P concentrations, ostasis of Ca and P as indicated by urinary concentrations of can be a useful tool to gauge the balance of Ca and P in diet Ca and P. Of note, only at the total Ca/P ratio of 1.20 did the for pigs. digestible Ca/P ratios in the diets with phytase remained sim- ilar to the ratios in the reference diets with a total Ca/P ratio of 1.05. It appears that fixing the total Ca/P ratios in diets to CONCLUSION compare MCP and phytase is not “fair” for phytase. In general, the available P release values were higher at the In this study, increasing the dietary Ca/P ratio from 1.05 total Ca/P ratio of 1.05 than at 1.20 when ADG and gain:feed to 1.20 in diets supplemented with 500 to 3,000 FYT/kg were used as response variables, whereas the opposite was phytase impaired feed utilization efficiency, but increased 8 Zhai et al. dietary digestible calcium on growth performance, bone mineral- the digestible Ca in diet and the deposition of Ca and P in ization, plasma calcium, and abundance of genes involved in in- bone. This led to a tendency to elicit higher available P re- testinal absorption of calcium in pigs from 11 to 22  kg fed diets lease values from phytase at the Ca/P ratio of 1.20 than at with different concentrations of digestible phosphorus. J. Anim. Sci. 1.05 when using bone P weight as the response variable. Biotechnol. 10:47. doi:10.1186/s40104-019-0349-2. Therefore, fixing the same total Ca/total P ratio in diets Lagos, L. V., A. L. Su, M. R. Bedford, and H. H. Stein. 2021. Formulating supplemented with increasing phytase dosing created an im- diets based on digestible calcium instead of total calcium does not balance of digestible Ca and P, which could have an adverse affect growth performance or carcass characteristics, but microbial effect on bone mineralization and should be corrected with phytase ameliorates bone resorption caused by low calcium in diets the appropriate addition of Ca. More research is warranted fed to pigs from 11 to 130 kg. J. Anim. Sci. 99:1–11. doi:10.1093/ to precisely quantify the contribution of digestible Ca and jas/skab057. Létourneau-Montminy, M. P., C. Jondreville, D. Sauvant, and A. Narcy. P by phytase under different dietary and animal conditions, 2012. Meta-analysis of phosphorus utilization by growing pigs: ef- and this will enable a more precise supply of adequate Ca fect of dietary phosphorus, calcium and exogenous phytase. 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Journal

Translational Animal ScienceOxford University Press

Published: Aug 29, 2022

Keywords: available; bone; calcium; digestibility; phosphorus; phytase

References