Dietary phosphorus deficiency impaired growth, intestinal digestion and absorption function of meat ducks.
It is known that phosphorus (P) plays an important role in nucleic acid synthesis, energy metabolism, muscle function, enzyme activity, lipid metabolism and bone mineralization . The effect of P on growth performance, bone development and bone quality in poultry has been well documented. Reduction of dietary non-phytate phosphorus (nPP) of broilers lead to poor bone mineralization and thus impaired animal welfare or increased processing losses . Valable et al  also found that a reduction of 23% of dietary nPP content reduced bone mineral content and gain, breaking strength, tibia and toe ash weight in broilers from 1 to 21 d of age. Recently, some studies reported that P deficiency decreased the intestinal digestion and absorption ability in Jian carp, and impaired the intestinal immune barrier and physical barrier function in grass carp, resulting in a decrease in production performance [4,5]. Our previous study  also found that dietary nPP levels affected the diversity and structure of cecal microbiota in meat ducks from 1 to 21 d of age, more specifically finding that increasing the dietary nPP levels influenced the cecal microbiota and positively affected the growth of meat ducks. Ducks are more sensitive to diet P deficiency. Rodehutscord et al  found that Pekin ducks require less available P than the NRC  recommended based on the growth rate and P retention data. And the NRC  recommended that the nPP requirement of Pekin ducks for 1 to 14 d and 15 to 35 d was 0.40% and 0.30%, respectively. However, to our knowledge, there are no reports which evaluated the requirement of nPP based on the intestinal digestion and absorption ability in meat ducks.
A major challenge for poultry nutritionists in recent years  has been to decrease feed costs while maintaining bird performance at a high level with minimal environmental pollution. To overcome this problem, a comprehensive and better understanding of the intestinal digestion, absorption and utilization of P in poultry is needed. Digestion and absorption of nutrients in the intestine can be evaluated by many factors, such as pH value, digestive enzyme activity, and transporter carrying gene expression. For example, absorption processes of nutrients in the intestine are mainly driven by [Na.sup.+]-[K.sup.+]-ATPase that generates a [Na.sup.+] and [K.sup.+] concentration gradient and electric potential difference, allowing the absorption of various molecules . The type IIb NaPi cotransporter (NaPi-IIb) is primarily expressed in the brush-border membranes of the duodenal epithelium, which is the major NaPi cotransporter and is responsible for active phosphate transport from the intestinal lumen into the blood . It was reported that in the small intestine of chickens, the duodenum is the main site of absorption of active P, followed by jejunum and ileum, which suggests that differences in gene expression are associated with sites in the small intestine . However, it remains unclear how dietary nPP levels regulate the pH value, digestive enzyme activity and gene expression of NaPi-IIb in the intestine of meat ducks. Therefore, the objective of this current study was to investigate the effect of dietary nPP levels on the intestinal digestion and absorption ability by determining the intestinal pH value, digestive enzyme activity, nutrient utilization, intestinal morphology and gene expression of NaPi-IIb in meat ducks from 1 to 21 d of age.
MATERIALS AND METHODS
Birds, diets and experimental design
The Institutional Animal Care and Use Committee of Sichuan Agricultural University approved all procedures used in this study. A total of 525 1-d-old male Cherry Valley ducks (Anas platyrhynchos) were obtained from a commercial hatchery (Mianying duck breeding farm, Sichuan Province, P. R. China), and randomly divided into one of five dietary treatments, each with seven replicate pens of 15 ducks, in a completely randomized design for 21d. Five experimental diets were formulated to contain 0.22%, 0.34%, 0.40%, 0.46%, and 0.58% nPP, supplemented with 0.09%, 0.21%, 0.27%, 0.33%, and 0.45% of inorganic P in the form of monocalcium phosphate (Ca[H.sub.2]P[O.sub.4]-2[H.sub.2]O), respectively. The analyzed total P (TP) content in the experimental diets was 0.54%, 0.72%, 0.79%, 0.84%, or 0.92%, respectively. Each diet contained a constant calcium content of approximately 0.90%. Therefore, the Ca:nPP in the five experimental diets was 4.09:1, 2.65:1, 2.25:1, 1.96:1, and 1.55:1, respectively.
The basal diet (1 to 21 d) (Table 1, 2-mm-diameter-pellet) was formulated under a digestible amino acid basis to meet the nutrient requirements of Pekin ducks suggested in the NRC  and Han et al  except for P. Ducks were reared in pens (2.2 mx1.2 mx0.9 m) in a temperature- and humidity-controlled room with a 24-h constant light schedule and free access to water and feed.
Data collection and sampling
At the end of the experiment, and after 12 h of feed withdrawal, ducks were weighed, and feed consumption was obtained for each pen. Body weight (BW) and body weight gain (BWG), feed intake (FI), and feed to gain ratio (F:G) were calculated accordingly at the following intervals: 1 to 14 d, 15 to 21 d, and 1 to 21 d. Feed waste was recorded daily, and the data were used in the calculations of feed consumption. Birds that died during the experiment were weighed, and the data were used in the calculations of F:G.
Then, on d 14 and 21, one duck per pen with a weight closest to the pen average was selected and bled from the jugular vein, respectively. The blood samples (n = 7) were centrifuged at 3,000 g/15 min at 4[degrees]C, and serum was collected and stored at -20[degrees]C for Ca, P content and alkaline phosphatase (ALP) activity determination. After that, all ducks were euthanized by cervical dislocation. The duodenal and jejunal segments were quickly isolated and flushed with ice cold saline solution. Then, the duodena land jejuna mucosa (n = 7) were scraped off with an ice-cold microscope slide, immediately frozen and stored in liquid nitrogen for assay of NaPi-IIb gene expression. Then, the digestive contents of the jejunum (n = 7) were collected by gently squeezing and stored at -20[degrees]C for digestible enzyme activities. The acidity of the digesta in gizzard, duodenum, jejunum, ileum and cecum was immediately measured by direct insertion of a pH electrodes indicated by Liao et al . After collecting digesta, 1.5-cm sections of mid duodenum and jejunum were separated for morphological assessment. The procedures and equipment used were the same as described by Han et al .
Serum Ca, P concentration and alkaline phosphatase activity
Serum Ca and P concentrations were analyzed using a Biochemistry Analyzer (Yellow Springs Instrument Co., Inc., Yellow Springs, OH, USA). The serums ALP activity was determined by conversion of p-nitrophenyl phosphate to p-nitrophenol and monitored at 405 nm using a Gemini XPS Microplate Reader (Molecular Devices, LLC., Sunnyvale, CA, USA) as indicated by Zhang et al . The kits in this trial were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).
Jejunal enzyme activity
Enzyme activities of ALP and [Na.sup.+]-[K.sup.+]-ATPase in digesta from the jejunum were determined according to the analytical kits (Nanjing Jiancheng Bioengineering Institute, China) after digesta was homogenized with a cold medium (weight:volume = 1:9), provided by the kit, in an ice-water bath according to Zhu et al  and Liu et al .
RNA isolations and real-time polymerase chain reaction
The RNA isolation and real-time polymerase chain reaction (PCR) were performed as previously described in our laboratory . Briefly, total RNA was isolated from the duodenal and jejunal mucosa by using a TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA concentration was measured by using the Nano Drop ND-1000 spectrofluorometer (Nano-Drop Technologies, Wilmington, DE, USA), and the quality of total RNA was determined in agarose gels stained with ethidium bromide. One microgram of total RNA was subjected to reverse transcription with the Super Script First-Strand Synthesis System (Invitrogen, USA). Real-time PCR reactions were performed on an ABI 7500 real-time PCR system using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The primer sequences for NaPi-IIb mRNA were GG TAAAGCAGCAGGGGACAT (Forward) and CTGAGGT GCCAATGTTTGCC (Reverse). The primer sequences for [beta]-actin (housekeeping gene) mRNA were AAGTACCCCATT GAACACGGT (Forward) and TCTGTTGGCTTTGGGG TTCA (Reverse). Gene-specific amplification was determined by melting curve analysis and agarose gel electrophoresis. Relative quantities of mRNA were calculated using the [2.sup.-[DELTA][DELTA]Ct] method , and values were normalized by [beta]-actin as an internal control.
On d 22, two birds per pen were randomly selected (14 ducks per treatment, 70 ducks in total) and transferred to metabolic cages (two ducks per cage) and fed with the original starter diets mixed with chromic oxide (0.3%). After a three-day adaptive period (d 22, 23, and 24), the total excreta samples from each cage were collected for 72 hours (d 25, 26, and 27). Excreta were weighed and then stored at -20[degrees]C immediately These excreta were dried at 65[degrees]C [+ or -] 5[degrees]C for 24 h, weighed and crushed to pass through a 40-mesh sieve for dry matter (DM), Cr, N, ether extract and energy availability according to Adeola  and Zeng et al .
All data were analyzed using SAS statistical software (version 9.2, SAS Institute Inc., Cary, NC, USA), and data were analyzed based on methods described by Seo et al . The experimental unit was the replicate pen (n = 7) for growth performance, and an individual duck (n = 7) for gizzard and intestinal characteristics measurements at 14 and 21 d of age, respectively. The effect of dietary nPP levels was determined by performing a one-way analysis of variance using the general linear model procedure in SAS software (SAS Institute Inc., USA). When the dietary effect was significant (p<0.05), polynomial contrasts and the linearity of the response to analyzed dietary nPP levels were examined using linear and quadratic regression. The [R.sup.2] value was provided to compare these regressions when the linear or quadratic effect was significant (p<0.05) . Probability values <0.05 were considered significant, and those <0.1 but >0.05 were considered to show a tendency towards approaching significance.
The chemical values of Ca and TP in diets were close to expected values, confirming that the ingredients were mixed correctly.
Dietary nPP levels linearly and quadratically increased (p< 0.05, Table 2) BW (d 14 and 21), as well as BWG and FI (all intervals), but they had no effect on F:G. Ducks fed the diet with 0.22% nPP had the lowest (p<0.05) BW, BWG, and FI in all intervals compared to ducks fed the other four diets. In addition, when compared to the 0.46% nPP group, birds fed the diet containing 0.34% nPP had lower (p<0.05) BW at 14 d of age and BWG during 1 to 14 d of age. From 1 to 21 d of age, F:G showed a linear (p = 0.079) and quadratic (p = 0.061) decrease as dietary nPP increased.
As shown Table 3, dietary nPP levels linearly and quadratically decreased (p<0.05) the serum Ca content (d 14 and 21), and linearly and quadratically increased (p<0.05) the serum P content (d 14). With the increase of dietary nPP levels, serum ALP activity at 14 d of age presented a linear and quadratic decrease (p<0.05), but at 21 d of age, it showed a linear decrease (p<0.05). Ducks fed the diet containing 0.22% nPP had a higher (p<0.05) serum Ca content and ALP activity and lower (p<0.05) serum P content.
Intestinal pH value, digestive enzyme activity and morphology
Data for the gizzard and intestinal pH value are presented in Table 4. In general, dietary nPP levels had no effect on the pH value in the gizzard, duodenum (d 21), jejunum (d 21), ileum (d 14) and cecum. With the increase in dietary nPP levels, the pH value linearly increased (p<0.05) in the jejunum (d 14), showed a tendency towards increasing (p = 0.071) in the duodenum (d 14), and showed a tendency towards quadratically increasing (p = 0.072) in the ileum (d 21).
With an increase in dietary nPP levels, jejunal [Na.sup.+]-[K.sup.+]-ATPase activity linearly decreased (p<0.05, Table 5) at 14 d of age and showed a tendency towards decreasing (p = 0.058) at 21 d of age. However, dietary nPP levels had no effect on jejunal ALP activity.
Dietary nPP levels presented a linear effect (p<0.05, Table 6) on the duodenal muscle layer thickness (d 14) and showed a tendency towards increasing the duodenal villus height (p = 0.077). Dietary nPP levels had no effect on the other morphologic duodenal parameters and jejunal morphology.
Excreta nutrient utilization
The effects of dietary nPP levels on excreta nutrient utilization are shown in Table 7. The increase of dietary nPP levels linearly increased (p<0.05) the excreta DM, crude protein, energy, Ca and TP utilization. In general, the diet containing 0.58% nPP had better excreta nutrient utilization and higher (p<0.05) DM, energy, Ca, and TP availability compared with the diets containing 0.22%, 0.34%, and 0.40% nPP.
Intestinal NaPi-IIb gene expression
As the dietary nPP levels increased, a linear (p<0.05, Figure 1) decrease was observed for the NaPi-IIb mRNA level in the duodenum at 21 d of age, and a tendency towards decreasing (p = 0.077, Figure 2) was observed for the NaPi-IIb mRNA level in the jejunum at 14 d of age.
In the current study, a 0.34% level of nPP in the diet reduced BW (14 d) and BWG (d 1 to 14), and a lower dietary nPP level (0.22%) further decreased the growth performance in ducks aged 1 to 21 d. These results suggest that the nPP requirement of ducks decreases as the ducks' age increases. Ducks fed diet with 0.40% nPP presented a normal performance. The dietary 0.40% nPP level is in line with the NRC  recommended, and which agreed with Xie et al  who studied the interaction between dietary Ca and nPP on growth performance and bone ash in Pekin ducklings, and they used a quadratic regression to predict that the requirements of Ca and nPP for maximum weight gain and minimum feed/gain for ducks aged from 1 to 14 d were 0.806% and 0.403%, 0.796% and 0.379%, respectively.
One reason for the decrease of FI and growth performance that occurs when ducks are fed a diet with a deficiency in P, and in the present experiment those that were fed the diets with 0.22% and 0.34% nPP was that these two diets had a higher Ca:nPP (4.09:1 and 2.65:1). Hulan et al  found that the dietary Ca:nPP above 2.81:1 could depress the growth performance and FI in broilers. And Rama Rao et al  also observed that broilers fed diets with 2.3-3:1 Ca:nPP had poor performance. Because the two minerals tend to form calcium phosphate, an insoluble complex in the chicken gut resulting in reduced absorption, which may be the reason for growth retardation at higher Ca:nPP . Another reason might be due to differences in dietary Ca and metabolizable energy levels, as well as the types of basal diet. Zeng et al  reported that dietary metabolizable energy concentration could affect the dietary nPP level by regulating the FI of ducks. A further reason for the decrease of FI and growth performance that occurs when ducks are fed a diet with a deficiency in P, is that their gut microbial communities need to obtain P from the degradation of phytate , requiring that more energy is spent on the maintenance of the basic metabolism, and less energy is available to spend on nutrient utilization and growth.
The important finding of the present study was that as the dietary nPP level increased, the serum ALP activity, jejunal [Na.sup.+]-[K.sup.+]-ATPase activity, and NaPi-IIb mRNA expression linearly decreased, while the intestinal morphology and nutrient utilization linearly increased, especially at 14 d of age, indicating that dietary nPP deficiency impaired the intestinal digestion and absorption ability of ducks. Because Manzanilla et al  and Hou et al  reported that the intestinal villus height and the crypt depth were closely related to intestinal absorption ability, the shallower crypt depth and the higher villus height indicated that intestinal tract had a better absorption ability. In this study, the villus height of duodenum and jejunum in 0.46% nPP group increased by 14.19% and 5.52%, and 9.50% and 1.02% compared with 0.22% nPP group at 14 and 21 d of age, respectively. Emami et al  also stated that birds fed a P deficient diet had a shorter villus height, a higher crypt depth, and a lower villus height/crypt depth ratio than birds fed the adequate amount of available phosphorus diet in broiler. In the current study, we found ducks fed diet with 0.46% and 0.58% nPP had the best nutrient utilization when compared to the other 3 diets, which suggested that duck need more nPP to maintain a better intestinal digestion and absorption function. This result agreed with Dai et al , who found ducks fed diets with 0.46% and 0.58% nPP had a better cecal microbiota diversity and composition.
Rhoads et al  reported that the activity of [Na.sup.+]-[K.sup.+]-ATPase indirectly reflected the absorption function of intestinal mucosa, and ATPase was required to provide energy in active absorption. It is reported that the activity of [Na.sup.+]-[K.sup.+]-ATPase in the lower intestine was significantly reduced in Wistar rats fed diets with added phytic acid compared with controls . The decrease in activity of [Na.sup.+]-[K.sup.+]-ATPase may be related with the increase of pH value in the intestine. In this current study, we observed that when dietary inorganic P supplementation increased, the pH values in the jejunum, duodenum and ileum tended to increase. Intestinal pH values may be considered when using inorganic P sources to increase dietary nPP levels because gastrointestinal tract acidity is considered an important factor that affects the digestive environment . Capuano et al  reported that the manipulation of dietary pH to produce a lower gastric and intestinal pH may be beneficial for digestion and for the maintenance of more desirable microflora.
Moreover, some researchers have observed that NaPi-IIb expression was up-regulated under conditions of dietary P deprivation . When dietary nPP was increased, the NaPi-IIb expression levels were reduced in our study and in studies with broilers [10,11], laying hens  and mice ; this outcome indicates that the duodenal P absorption capacity could be decreased with increasing serum P levels. In the present study, we also observed consistently that serum P levels increased as dietary nPP levels increased. Therefore, both the present experiment and the previous researches indicate that ducks might regulate to increase P absorption in the small intestine when dietary nPP is lower than the nPP requirement. Increasing active absorption of P under a lower level of dietary nPP, means that more energy was consumed, which leads to the decrease of nutrient utilization and growth performance.
In summary, a 0.22% level of nPP in the diet of ducks aged from 1 to 21 d reduced growth performance and nutrient utilization, damaged the intestinal morphology, and increased intestinal [Na.sup.+]-[K.sup.+]-ATPase activity and NaPi-IIb mRNA levels, which indicated a poor intestinal digestion and absorption function. Increasing dietary nPP levels could increase the growth performance of meat ducks via improving the intestinal digestion and absorption ability, and ducks aged from 1 to 21 d fed diets with 0.40%, 0.46%, and 0.58% nPP presented a better growth performance, and a better intestinal digestion and absorption ability.
CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. https://doi.org/10.5713/ajas.18.0683
Submitted Sept 11, 2018; Revised Nov 20, 2018;
Accepted Mar 7, 2019
This research was supported by grants from the National Key R & D Program of China (2017YFD0502004), and Sichuan Agricultural University 211 Foundation of China.
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Huimin Xu (1), Shujun Dai (1), Keying Zhang (1), Xuemei Ding (1), Shiping Bai (1), Jianping Wang (1), Huanwei Peng (1), and Qiufeng Zeng (1)*
* Corresponding Author: Qiufeng Zeng Tel: +86-13778765040, Fax: +86-028-86290922, E-mail: firstname.lastname@example.org
(1) Institute of Animal Nutrition, Key Laboratory for Animal Disease-Resistance Nutrition of China, Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China
Caption: Figure 1. Effect of dietaty non-phytate phosphorous levels on NaPI-llb gene expression in duodenum of ducks at 14 d and 21 d of age. NaPi-IIb, type IIb sodium-phosphate cotransporter; npp, non-phytate phosphorous.
Caption: Figure 2. Effect of dietary non-phytate phosphorous levels on NaPi-IIb gene expression in jejunum of ducks at 14 d and 21 d of age. NaPi-IIb, type IIb sodium-phosphate cotransporter; npp, non-phytate phosphorous.
Table 1. Ingredients and compositions of the experimental diets (%, dry matter basis) Ingredients Diet 1 Diet 2 Diet 3 Corn 59.50 59.50 59.50 Soybean oil 1.87 1.87 1.87 Soybean meal 33.85 33.85 33.85 L-lysine-HCL 0.024 0.024 0.024 DL-methionine 0.197 0.197 0.197 Limestone 1.905 1.710 1.613 Monocalcium phosphate 0.395 0.928 1.196 Tryptophan 0.061 0.061 0.061 Bentonite 1.169 0.831 0.660 Sodium chloride 0.35 0.35 0.35 Choline chloride 0.15 0.15 0.15 Vitamin premi x (1) 0.03 0.03 0.03 Mineral premix (2) 0.50 0.50 0.50 Total 100.00 100.00 100.00 Calculated nutrients levels (%) Metabolizable energy 12.12 12.12 12.12 (MJ/kg) Crude protein 19.5 19.5 19.5 Calcium 0.90 0.90 0.90 Non-phytate phosphorus 0.22 0.34 0.40 Digestible lysine 0.96 0.96 0.96 Digestible methionine 0.46 0.46 0.46 Digestible threonine 0.66 0.66 0.66 Digestible tryptophan 0.26 0.26 0.26 Analyzed nutrients concentration Total phosphorus (%) 0.54 0.72 0.79 Calcium (%) 0.93 0.94 0.92 Ingredients Diet 4 Diet 5 Corn 59.50 59.50 Soybean oil 1.87 1.87 Soybean meal 33.85 33.85 L-lysine-HCL 0.024 0.024 DL-methionine 0.197 0.197 Limestone 1.516 1.320 Monocalcium phosphate 1.465 2.001 Tryptophan 0.061 0.061 Bentonite 0.488 0.150 Sodium chloride 0.35 0.35 Choline chloride 0.15 0.15 Vitamin premix (1) 0.03 0.03 Mineral premix (2) 0.50 0.50 Total 100.00 100.00 Calculated nutrients levels (%) Metabolizable energy 12.12 12.12 (MJ/kg) Crude protein 19.5 19.5 Calcium 0.90 0.90 Non-phytate phosphorus 0.46 0.58 Digestible lysine 0.96 0.96 Digestible methionine 0.46 0.46 Digestible threonine 0.66 0.66 Digestible tryptophan 0.26 0.26 Analyzed nutrients concentration Total phosphorus (%) 0.84 0.92 Calcium (%) 0.94 0.95 (1) Provided per kilogram of diet vitamin A, 8,000 IU; cholecalciferol, 2,000 IU; vitamin E, 5 IU; vitamin K, 1 mg; thiamine, 0.4 mg; riboflavin, 3.2 mg; pyridoxine, 1.2 mg; vitamin [B.sub.12], 6 [micro]g; folic acid, 100 [micro]g; niacin, 7 mg; calcium pantothenate, 5 mg. (2) Provided per kilogram of diet: Fe (FeS[O.sub.4] x [H.sub.2]O) 80 mg, Cu (CuS[O.sub.4] x 5[H.sub.2]O) 8 mg, Mn (MnS[O.sub.4] x [H.sub.2]O) 70 mg, Zn (ZnS[O.sub.4] x [H.sub.2]O) 90 mg, I (KI) 0.4 mg, Se ([Na.sub.2]Se[O.sub.3]) 0.3 mg. Table 2. Effect of dietary non-phytate phosphorus levels on growth performance of ducks Index Dietary non-phytate phosphorus level (%) 0.22 0.34 0.40 Body weight (g) 1 d (1) 57.4 57.4 57.4 14 d (1) 745(c) 852(b) 875(ab) 21 d (2) 1,310(b) 1,498(a) 1,505(a) Body weight gain (g) 1-14 d 687(c) 795(b) 818(ab) 15-21 d 566(b) 646(a) 630(a) 1-21 d 1,253(b) 1,441(a) 1,448(a) Feed intake (g) 1-14 d 849(b) 965(a) 1,002(a) 15-21 d 999(b) 1,108(a) 1,093(a) 1-21 d 2,158(b) 2,405(a) 2,448(a) Feed to gain ratio 1-14 d 1.23 1.21 1.23 15-21 d 1.77 1.72 1.74 1-21 d 1.72 1.67 1.69 Index Dietary non-phytate SEM phosphorus level (%) 0.46 0.58 Body weight (g) 1 d (1) 57.4 57.3 0.05 14 d (1) 887(a) 865(ab) 11.94 21 d (2) 1,540(a) 1,505(a) 17.90 Body weight gain (g) 1-14 d 830(a) 808(ab) 11.95 15-21 d 653(a) 640(a) 12.73 1-21 d 1,483(a) 1,448(a) 17.90 Feed intake (g) 1-14 d 996(a) 988(a) 21.69 15-21 d 1,117(a) 1,106(a) 19.94 1-21 d 2,454(a) 2,435(a) 27.59 Feed to gain ratio 1-14 d 1.20 1.22 0.02 15-21 d 1.71 1.73 0.04 1-21 d 1.65 1.68 0.02 Index p-value ANOVA Linear Quadratic Body weight (g) 1 d (1) 0.949 0.562 0.716 14 d (1) <0.01 <0.01 <0.01 21 d (2) <0.01 <0.01 <0.01 Body weight gain (g) 1-14 d <0.01 <0.01 <0.01 15-21 d 0.00 0.00 0.002 1-21 d <0.01 <0.01 <0.01 Feed intake (g) 1-14 d <0.01 <0.01 0.001 15-21 d 0.001 0.001 0.008 1-21 d <0.01 <0.01 <0.01 Feed to gain ratio 1-14 d 0.803 0.637 0.447 15-21 d 0.865 0.499 0.502 1-21 d 0.066 0.079 0.061 SEM, standard error of the mean; ANOVA, analysis of variance. 1) Means represent 7 pens of 15 ducks per pen. 2) Means represent 7 pens of 14 ducks per pen. (a-c) Means in the same row with no common superscript are significantly different (p< 0.05). Table 3. Effect of dietary non-phytate phosphorus levels on the serum parameters of ducks Dietary non-phytate phosphorus level (%) 0.22 0.34 0.40 Serum calcium (mmol/L) 14 d (1) 2.68(a) 1.86(b) 1.94(b) 21 d 2.92(a) 2.30(b) 2.28(b) Serum phosphorus (mmol/L) 14 d 1.26(b) 1.98(a) 1.87(a) 21 d 1.56 2.13 2.10 Alkaline phosphatase (K/100 mL) 14 d 1,326(a) 792(bc) 898(b) 21 d 1,211(a) 872(b) 770(b) Dietary non-phytate SEM phosphorus level (%) 0.46 0.58 Serum calcium (mmol/L) 14 d (1) 1.94(b) 1.98(b) 0.06 21 d 2.28(b) 2.20(b) 0.13 Serum phosphorus (mmol/L) 14 d 1.87(a) 1.84(a) 0.08 21 d 2.00 1.98 0.19 Alkaline phosphatase (K/100 mL) 14 d 707(c) 734(c) 42.7 21 d 774(b) 752(b) 105 p-value ANOVA Linear Quadratic Serum calcium (mmol/L) 14 d (1) <0.01 <0.01 <0.01 21 d 0.002 0.001 0.025 Serum phosphorus (mmol/L) 14 d <0.01 0.000 <0.01 21 d 0.240 0.193 0.086 Alkaline phosphatase (K/100 mL) 14 d <0.01 <0.01 <0.01 21 d 0.021 0.004 0.071 SEM, standard error of the mean; ANOVA, analysis of variance. 1) Means represent 7 pens of 1 duck per pen. (a-c) Means in the same row with no common superscript are significantly different (p< 0.05). Table 4. Effect of dietary non-phytate phosphorus levels on gastrointestinal pH of ducks Index Dietary non-phytate phosphorus SEM level (%) 0.22 0.34 0.40 0.46 0.58 Gizzard 14 d (1) 3.74 3.96 4.17 3.27 3.53 0.29 21 d 3.25 2.95 3.05 3.38 3.31 0.26 Duodenum 14 d 6.20 6.50 6.42 6.41 6.47 0.09 21 d 6.51 6.19 6.41 6.30 6.33 0.10 Jejunum 14 d 6.36 6.43 6.48 6.45 6.60 0.07 21 d 6.37 6.20 6.35 6.30 6.16 0.10 Ileum 14 d 7.08 6.94 6.96 7.03 7.05 0.14 21 d 6.73 7.04 7.24 6.76 6.73 0.18 Cecum 14 d 6.38 6.55 6.82 6.51 6.53 0.22 21 d 5.78 5.58 5.79 5.92 5.81 0.18 Index p-value ANOVA Linear Quadratic Gizzard 14 d (1) 0.222 0.312 0.441 21 d 0.737 0.605 0.495 Duodenum 14 d 0.145 0.071 0.199 21 d 0.280 0.357 0.244 Jejunum 14 d 0.238 0.029 0.760 21 d 0.478 0.238 0.765 Ileum 14 d 0.943 0.971 0.482 21 d 0.192 0.721 0.072 Cecum 14 d 0.711 0.675 0.353 21 d 0.763 0.596 0.878 SEM, standard error of the mean; ANOVA, analysis of variance. (1) Means represent 7 pens of 1 duck per pen. Table 5. Effect of dietary non-phytate phosphorus levels on jejunal enzyme activity of ducks Dietary non-phytate phosphorus SEM level (%) 0.22 0.34 0.40 0.46 0.58 Alkaline phosphatase (U/mgprot) 14 d (1) 930 931 899 901 894 18.57 21 d 933 936 921 923 921 14.68 Na+-K+-ATPase (U/mgprot) 14 d 42 42 41 40 39 0.86 21 d 43 42 40 39 40 1.53 p-value ANOVA Linear Quadratic Alkaline phosphatase (U/mgprot) 14 d (1) 0.464 0.113 0.887 21 d 0.921 0.467 0.928 Na+-K+-ATPase (U/mgprot) 14 d 0.265 0.035 0.750 21 d 0.300 0.058 0.486 SEM, standard error of the mean; ANOVA, analysis of variance. 1) Means represent 7 pens of 1 duck per pen. Table 6. Effect of dietary non-phytate phosphorus levels onintestinal morphology of ducks Dietary non-phytate phosphorus SEM level (%) Index 0.22 0.34 0.40 0.46 0.58 Duodenum Villus height ([micro]m) 14 d (1) 628 635 682 717 732 48.93 21 d 594 599 602 627 617 51.19 Crypt depth ([micro]m) 14 d 136 149 154 149 153 9.42 21 d 148 135 156 150 142 8.61 Villus height/ crypt depth 14 d 4.72 4.48 4.65 4.85 4.87 0.37 21 d 4.16 4.48 3.98 4.48 4.57 0.44 Muscle layer thickness ([micro]m) 14 d 261 290 302 356 335 25.72 21 d 357 320 313 325 333 16.28 Jejunum Villus height ([micro]m) 14 d 516 587 604 565 552 37.67 21 d 530 578 511 535 554 29.71 Crypt depth ([micro]m) 14 d 138 142 125 133 133 7.87 21 d 127 128 136 136 126 9.11 Villus height/ crypt depth 14 d 3.83 4.26 4.97 4.38 4.43 0.39 21 d 4.56 4.75 3.84 4.19 4.78 0.45 Muscle layer thickness ([micro]m) 14 d 271 284 272 298 280 18.17 21 d 265 287 291 308 292 15.49 p-value Index ANOVA Linear Quadratic Duodenum Villus height ([micro]m) 14 d (1) 0.459 0.077 0.973 21 d 0.990 0.671 0.953 Crypt depth ([micro]m) 14 d 0.670 0.229 0.444 21 d 0.504 0.991 0.745 Villus height/ crypt depth 14 d 0.948 0.632 0.696 21 d 0.856 0.527 0.840 Muscle layer thickness ([micro]m) 14 d 0.091 0.015 0.484 21 d 0.374 0.346 0.086 Jejunum Villus height ([micro]m) 14 d 0.537 0.607 0.133 21 d 0.569 0.834 0.904 Crypt depth ([micro]m) 14 d 0.645 0.519 0.656 21 d 0.886 0.928 0.411 Villus height/ crypt depth 14 d 0.353 0.266 0.208 21 d 0.540 0.968 0.284 Muscle layer thickness ([micro]m) 14 d 0.825 0.600 0.652 21 d 0.420 0.148 0.258 SEM, standard error of the mean; ANOVA, analysis of variance. (1) Means represent 7 pens of 1 duck per pen. Table 7. Effect of dietary non-phytate phosphorous levels on excreta nutrient utilization of ducks Index Dietary non-phytate phosphorus level (%) SEM 0.22 0.34 0.40 0.46 0.58 DM (%) 67(c) 68(c) 72(b) 73(ab) 74(a) 0.72 CP (%) 65(b) 56(c) 66(ab) 64(b) 68(a) 1.02 Energy (%) 73(c) 73(c) 76(b) 77(ab) 78(a) 0.61 Ca (%) 51(c) 49(c) 55(b) 66(a) 65(a) 1.48 TP (%) 35(d) 46(bc) 51(b) 45(c) 59(a) 2.39 Index p-value ANOVA Linear Quadratic DM (%) <0.01 <0.01 0.516 CP (%) <0.01 0.001 0.000 Energy (%) <0.01 <0.01 0.935 Ca (%) <0.01 <0.01 0.410 TP (%) <0.01 <0.01 0.775 SEM, standard error of the mean; ANOVA, analysis of variance; DM, dry matter; CP, crude protein; Ca, calcium; TP, totalphosphorus. (a-d) Means in the same row with no common superscript are significantly different (p < 0.05).