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Mn related enzymes and fecal mineral excretion in broiler chickens
Animal Feed Science and Technology 168 (2011) 72–79
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Effect of dietary Mintrex-Zn/Mn on performance, gene expression of Zn transfer proteins, activities of Zn/Mn related enzymes and fecal mineral excretion in broiler chickens Jianmin Yuan ∗ , Zhihong Xu, Chunxi Huang, Shuliang Zhou, Yuming Guo State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China
a r t i c l e
i n f o
Article history: Received 23 August 2010 Received in revised form 15 March 2011 Accepted 18 March 2011
Keywords: Trace mineral Mintrex Excretion Mineral status Zn transfer proteins Broilers
a b s t r a c t This study was to evaluate dietary Mintrex-Zn/Mn on growth performance, enzyme activities, and trace mineral absorption and utilization in 0–3-wk broilers. One hundred sixty day-old male broiler chickens were allotted to 4 dietary treatments with 5 replicate cages of 8 birds. Broilers were fed maize-soybean basal diets containing zinc (Zn, 100 mg/kg) and manganese (Mn, 120 mg/kg) from sulphate salts, while three other treatments included 100%, 80%, and 60% of the basal dietary mineral (actually 70%, 56% and 42% of sulphate salts based on 70% of sulphates would be available) from Mintrex-Zn/Mn, respectively. At the end of 3 weeks of age, the growth performance, mRNA abundance of Zn/Mn transfer proteins in jejunum mucosa, enzyme activities of liver lactate dehydrogenase (LDH), glutamic oxalacetic transaminase (GOT) and glutamate-pyruvate transaminase (GPT), activities of serum alkaline phosphatase (ALP), Mn-superoxide dismutase (SOD), total superoxide dismutase (T-SOD) and CuZn-SOD and bone mineralization were measured. Total collection procedure in the balance trial of excreta was done from the age of 20 to 23 days. The results showed that supplementation with 100% Mintrex-Zn/Mn significantly improved the average daily gain (ADG) (P<0.01) and decreased fecal mineral excretion (P<0.05) compared to inorganic supplementation, but did not affect Zn/Mn transfer proteins mRNA abundance (P > 0.05). Supplementation with 80% Mintrex-Zn/Mn lowered serum ALP activity (P<0.05), however, it did not affect the growth performance, mRNA abundance of Zn/Mn transfer proteins and bone mineralization in comparison to inorganic supplementation (P > 0.05). The decrease of excretion is mainly due to the reduction of dietary supplemental level of minerals. Supplementation with 80% Mintrex-Zn/Mn decreased Mn and Zn excretion in the feces (mg/kg diet) by 39.79% and 30.13% (P<0.001), respectively. Supplementation with 60% Mintrex-Zn/Mn significantly decreased ADG (P<0.01), the length of metatarsus, serum ALP activities and mRNA expression of Zn transfer proteins but increased mortality and culling rate (P<0.05). There were no differences in activities of serum T-SOD, CuZn-SoD and Mn-SOD, and liver metabolic enzyme (LDH, GOT, and GPT) among inorganic versus Mintrex Zn/Mn supplementations. This study suggested that using approximately 30–40% Mintrex Zn/Mn could not substitute for the inorganic trace mineral. LDH, GOT, GPT may not be sensitive variables to reflect the states of the Zn and Mn in the body. Supplementation with 80% Mintrex-Zn/Mn (56% of inorganic mineral level) had a better effect in reducing the fecal mineral excretion without compromise in the growth performance. Using relative mineral concentration in fecal material may overestimate the values of mineral reduction, and it would be more accurate to use trace mineral concentration per chick day or per kg diet intake. © 2011 Elsevier B.V. All rights reserved.
∗ Corresponding author. Tel.: +86 01062732337; fax: +86 01062732712. E-mail address: [email protected] (J. Yuan). 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.03.011
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1. Introduction Essential trace minerals are important for a wide variety of physiological processes in all animals. Several hundred enzymes require the presence of minerals for their activity. Practicality, it is crucial to use an optimal level of trace minerals to allow animals to reach their genetic potential for performance. The levels of trace minerals supplemented in the diet are mostly based on National Research Council (NRC) recommendations. Some values of NRC (1994) for trace minerals in poultry, however, were mainly estimated based on extrapolation from other species and early studies in old bred of chickens (Leeson and Caston, 2007). With the advancement of genetic selection, the growth performance of commercial broiler strains is continually improved. Much higher than NRC levels of Zn and Mn supplements have been suggested in commercial diets to meet the growth of the modern commercial broilers (Leeson, 2005). For example, 100 mg Zn (or Mn)/kg of diet to the Cobb 500 and 120 mg Mn and 100 mg Zn/kg of diet to Ross 308 and AA plus are recommended. Traditionally, trace minerals have been supplemented in animal diets using inorganic salts such as Zinc oxide (Zn) and manganese (Mn) oxide or sulphate salts. However, in recent years there are increased concerns about the usage of inorganic trace minerals due to their poor bioavailability and potential to cause ingredient antagonism, which impairs absorption and increases environment pollution. Therefore, it is necessary to use organic trace minerals to substitute for inorganic trace minerals in the diet. Due to the higher bioavailability compared to inorganic forms (Leeson, 2005; Predieri et al., 2005; Ao et al., 2006; Yan and Waldroup, 2006), organic trace minerals (OTMs) are widely used in animal diets today. Organic trace minerals have been shown to enhance mineral uptake (Leeson, 2005; Predieri et al., 2005; Yan and Waldroup, 2006), improve the body weight gain (Ao et al., 2006; Nollet et al., 2008; Bao et al., 2009, 2010), and reduce mineral excretion compared to inorganic counterparts (Predieri et al., 2005; Yan and Waldroup, 2006; Nollet et al., 2007, 2008). However, some studies indicate that the absorption and bioavailability of organic trace elements were largely affected by their instabilities at low pH (Cao et al., 2000; Guo et al., 2001; Cao et al., 2002), and the formation of complexes with amino acids (Yu et al., 2008; Huang et al., 2009). Mintrex, a chelate of two 2-hydroxy-4 (methylthio) butanoic acid (HMTBa) ligands to one atom of trace mineral, i.e. zinc, copper or manganese, has been attracting much attention in recent years. Studies in broilers showed that Mintrex Zn (Yi et al., 2007), Mintrex Mn (Yan and Waldroup, 2006) and Mintrex Cu (Wang et al., 2007) had greater bioavailability than sulphate minerals. Predieri et al. (2005) suggested using MHA chelates could be a valuable tool to increase the bioavailability of trace minerals and to reduce the environmental pollution of animal manure. Although various authors (Yan and Waldroup, 2006; Wang et al., 2007) have considered the effect of the individual Mintrex Zn or Mintrex Mn on the performance and mineral excretion of broilers, little attention has been given to the effect of the combination of Mintrex Zn and Mn on growth performance, mineral absorption, metabolic enzyme activity and bone mineralization. The current study was conducted to determine the effects of dietary supplementing different concentrations of Mintrex-Zn/Mn on growth performance, serum and liver mineral-related enzyme activities, gene expression of Zn transfer proteins, bone mineralization and fecal mineral excretion in chickens. It aims to determine the minimal amount of Mintrex-Zn/Mn supplementing to 0–3 wk-old chickens without comprising on growth performance and environmental pollution.
2. Materials and methods 2.1. Animals and diets One hundred sixty day-old male broiler chickens (Arbor Acres plus, AA+ ) were randomly allotted to 4 dietary treatments (5 cages/diet, 8 birds/cage). A maize-soybean meal basal diet (Table 1) was used and formulated to be adequate in all nutrients according to Chinese Chicken Feeding Standard Requirement, (NY/T 33-2004) except for Zn and Mn. The nutrient values of diet were calculated based on the tables of feed composition and nutritive values in china. The Zn and Mn concentration in the basal diet was 29.94 mg/kg and 28.14 mg/kg, respectively. Trace minerals were provided as inorganic sulphate salts or as organic minerals (MINTREX® Zn/Mn). Mintrex Zn/Mn was supplied by Novus International. Mintrex Zn delivers 16% Zn as Zn methionine hydroxy analogue chelate and 80% methionine value, and Mintrex Mn delivers 13% Mn as Mn methionine hydroxy analogue chelate and 76% methionine value. In the Treatment 1, 100 mg Zn as sulphate/kg of diet and 120 mg Mn as sulphate/kg of diet was formulated in the basal diet according to NY/T 33-2004. It was arbitrarily assumed that 70% of these sulphates would be available (Leeson and Summers, 2001) and therefore the available minerals were Zn 70 mg/kg diet, Mn 84 mg/kg diet, respectively. In the Treatment 2, the supplement of Mintrex Zn/Mn was adjusted to the bioavailable level of Zn and Mn as in the Treatment 1 (70 mg/kg Zn, 84 mg/kg Mn), when the bioavailablity Mintrex Zn/Mn was arbitrarily assumed to be 100%. 80% and 60% of Mintrex Zn/Mn in the Treatment 2 was formulated in the diet of Treatments 3 (44.8 mg/kg and 67.2 mg/kg) and 4 (33.6 mg/kg and 50.4 mg/kg), respectively. The assayed mineral content of diet is in Table 2. The Met content from HMTBA of Mintrex in Treatments 2–4 was calculated and adjusted in the diet accordingly. All birds were raised in battery cages. Each pen was equipped with an individual feeder and water supply. All birds were allowed to consume mash feed and water ad libitum. Environmental temperature was initially set at 33 ◦ C and then gradually reduced to normal brooding practice. Birds had 24 h light. Hatchery body weight (BW) and body weight at day 20 posthatch, after 6 h fast, and feed intake from 0 to 20 d of age, was measured for the determination of average daily gain (ADG), average daily feed intake (ADFI) and gain/feed (G:F). Daily mortality and culling were recorded for each pen. The present study was approved by the China Agricultural University and carried out in accordance with the Guidelines for Experimental Animals.
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Table 1 Composition (g/kg) and calculated analysis of experimental basal diets. Ingredients
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
Maize Soybean meal Corn gluten Soybean oil Dicalcium phosphate Limestone Table salts l-Lysine HCl dl-methionine Choline chloride (50%) Topomicin (15%) Washed sand Oxethyl quinoline (33%) Vitamin premixa Mineral premixb Zinc sulphate (ZnSO4 ·H2 O) Manganese sulphate (MnSO4 ·H2 O) Mintrex Zn, 16% Mintrex Mn,13% Total ME (MJ/kg) CPc Lysinec Methioninec Cac Nonphytate Pc
552.70 327.40 50.00 29.70 17.30 11.10 3.50 0.90 1.40 1.60 0.80 1.67 0.35 0.25 1.00 0.14 0.19 1000.00 12.54 217 110 4.9 9.2 4.2
552.70 327.40 50.00 29.70 17.30 11.10 3.50 0.90 0.56 1.60 0.80 2.30 0.35 0.25 1.00 0.00 0.00 0.22 0.32 1000.00 12.54 217 110 4.9 9.2 4.2
552.70 327.40 50.00 29.70 17.30 11.10 3.50 0.90 0.73 1.60 0.80 2.23 0.35 0.25 1.00 0.00 0.00 0.18 0.26 1000.00 12.54 217 110 4.9 9.2 4.2
552.70 327.40 50.00 29.70 17.30 11.10 3.50 0.90 0.90 1.60 0.80 2.18 0.35 0.25 1.00 0.00 0.00 0.13 0.19 1000.00 12.54 217 110 4.9 9.2 4.2
a Provides per kg diet: Vitamin A, 12,5000 IU; Vitamin D3, 2500 IU; Vitamin E, 18.75 IU; Vitamin K, 2.65 mg; niacin, 36.8 mg; pantothenic acid, 12 mg; folic acid, 1.25 mg; thiamin, 2.5 mg; riboflavin, 6.6 mg; pyridoxine, 4.9 mg; Vitamin B12, 0.025 mg; biotin, 0.013 mg. b Provides per kg diet: Ferrous, 100 mg/kg (FeSO4 ·H2 O); Cu, 10 mg/kg (CuSO4 ·5H2 O); selenium, 0.3 mg/kg (Na2 SeO3 ); I, 0.7 mg/kg (Ca(IO3 )·H2 O. c Values calculated based on the Table of Feed Composition and Nutritive Values (2009) in China.
2.2. Tissue sampling and preparation At the end of the trial, blood samples of 10 chicks from each treatment were drawn from wing vein and centrifuged at 3600 × g for 10 min, serum was removed and stored at –30 ◦ C until assayed. The mucosa of jejunum near the Meckel’s diverticulum was removed by gentle scraping with a clean microscope slide, washed with cold PBS, frozen in liquid nitrogen, and stored at –80◦ C for later determination of the mRNA levels of metallothionein (MT), Zn transfer protein 1 (ZnT 1) and Zn transfer protein 5 (ZnT 5) using real-time PCR. The liver was removed, frozen in liquid nitrogen, and then stored at –30 ◦ C for later measurement of metabolic enzyme activity. The left metatarsus was removed and the length was measured. The metatarsus was cleaned of the muscle before the bone was dried for a minimum of 24 h at 100 ◦ C. After 72 h-extraction in the diethyl ether, the bone was weighed and dried for another 6 h at 100 ◦ C. Ash content of metatarsus was then determined gravimetrically after being ashed in a muffle furnace for 10 h at 550 ◦ C. 2.3. Liver and blood metabolic enzyme activity determination The activity of serum enzymes including alkaline phosphatase (ALP), Mn superoxide dismutase (Mn-SOD), CuZn superoxide dismutase (CuZn-SOD), total superoxide dismutase (T- SOD), and liver metabolic enzyme including lactate dehydrogenase (LDH), glutamic oxalacetic transaminase (GOT), glutamate-pyruvate transaminase (GPT) was measured using commercial kits (Institute of Nanjing Jiancheng Bioengineering).
Table 2 Mineral content of diet (mg/kg)a . Mineral
Content
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
Zn
Supplemented Calculated Assayed Supplemented Calculated Assayed
100 129.94 99.64 120 148.14 144.46
70 99.94 88.66 84 112.14 108.39
44.8 74.74 77.68 67.2 95.34 87.91
33.6 63.54 69.96 50.4 78.54 107.68
Mn
a
The Zn and Mn content in the basal diet was 29.94 mg/kg and 28.14 mg/kg, respectively.
J. Yuan et al. / Animal Feed Science and Technology 168 (2011) 72–79
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Table 3 Sequences of the primers used in real-time PCR assays. Gene name
Genebank accession number
Primer sequence (5 → 3 )
Predicted size (bp)
Metallothionein
NM 205275
163
Zn transfer protein 1
AJ619980
Zn transfer protein 5
NM 001031419
-actin
NM 205518
F 5 -AAG GGC TGT GTC TGC AAG GA- 3 R 5 -CTT CAT CGG TAT GGA AGG TAC AAA-3 F 5 -TGC GAG TGC CTT CTT CCT-3 R 5 -AAG GAG CTG TCA GGT CTG TAA T-3 F 5 -ATG CTG TTG TGG GAT GTA-3 R 5 -TTG TCT TGG CTG GTC CTC-3 F 5 -GAG AAA TTG TGC GTG ACA TCA-3 R 5 -CCT GAA CCT CTC ATT GCC A-3
131 159 152
2.4. Quantification of Zn transfer proteins mRNA using real-time PCR 2.4.1. Total RNA isolation and reverse transcription Total RNA was isolated from jejunum mucosa using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacture’s instruction. The integrity of total RNA was assessed via agarose gel electrophoresis and the RNA concentration and purity were spectrophotometrically determined using A260 and A280 measurements. Reverse transcription (RT) reactions (20 L) consisted of 16 L total RNA, 1 L of RNAse inhibitor (Promega), 1 L dNTPs (sigma), 2 L of 5 × M-MLV RT reaction buffer (Promega). The RT procedure was 20 ◦ C, 5 min, followed by 42 ◦ C, 60 min, and then 70 ◦ C, 5 min. The reaction was stopped by putting on ice. The RT products (cDNA) were stored at −20 ◦ C for PCR 2.4.2. Real-time PCR for quantification of mRNA abundance According to the sequence of the gene published in GenBank (Table 3), primers for MT, ZnT-1, ZnT-5 and -actin gene were designed with Primer Express Software (Applied Biosystems Incorporation, Foster, CA) and sequences for each primer was provided in Table 3. All PCR was performed with the ABI PRISM 7700 sequence detection system (ABI Biosystems) according to optimized PCR protocols. SYBR Green qPCR kit was used for the fluorescent detection (ABI Biosystems). A total volume of 20 L PCR reaction system was used which contained 10 L SYBR Green PCR Master Mix, and 2.0 L primer (1.0 L forward and 1.0 L reverse), and 2.0 L cDNA template, and 6 L sterile super-stilled water. For the PCR reaction, the experimental protocol was: denature at 95 ◦ C for 3 min, 42 cycles of amplification (94 ◦ C for 30 s, 51 ◦ C for 30 s, 72 ◦ C for 60 s) with a single fluorescence measurement at 72 ◦ C extension, and finally extension at 72 ◦ C for 7 min. The Ct value was determined and was used to calculate the relative expression level 2−Ct . 2.5. Fecal mineral excretion measurement Fecal mineral excretion was evaluated by the total collection procedure in the balance trial at the age of 20–23 days. After fasted for 17 h, chicks were fed with experimental diets for 55 h and starved again for 17 h. All excreta were collected for the last 72 h. Each 24-h output was collected and stored at −20 ◦ C until the end of the balance trial. At the end of the collection period, feed intake was recorded precisely. In the meantime, excreta output for each cage was weighed, commixed and sampled. The fecal samples were dried at 65 ◦ C, ashed and then assayed for minerals by atomic absorption spectrophotometer. Feed samples were also assayed for minerals following the same procedure. 2.6. Statistical analysis Data were subjected to analysis of variance (ANOVA), general linear models (SAS Institute, 1991). Duncan’s multiple range tests was used to assess differences in means among all treatments. The percentage values were transformed with arcsin function before statistical evaluation. Differences among means with P<0.05 were accepted as statistically significant differences. Difference among means 0.05 < P<0.10 were accepted as tendencies to have differences. For comparison the relationship of performance of broiler and the levels of Mintrex Zn/Mn in diets, linear regression analysis was conducted. 3. Results Table 2 shows the assayed mineral content of the diets together with the level of supplemental minerals, and calculated mineral content. The assayed mineral content was close to the calculated content. 3.1. Performance The growth performance of broilers was significantly affected by the supplementation of Mintrex Zn/Mn (Table 4). Compared to the supplementation of Zn/Mn sulphates (Treatment 1), organic supplementation with similar level of Zn/Mn (Treatment 2) significantly improved ADG (P<0.01). Supplementation with 60% mintrex Zn/Mn significantly decreased ADG
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Table 4 Performance of broilers fed various levels and sources of trace minerals from 0 to 3 wk of agea . Items
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
ADG (g/d) ADFI (g) F/G Mortality plus culls (%) European production indexb
25.72b 38.30 1.49 0.00b 86.42a , b
26.74a 39.05 1.46 0.00b 92.06a
25.30b ,c 38.63 1.52 0.00b 83.13b
24.44c 37.36 1.53 5.00a 79.94b
0.245 1.517 0.015 0.860 2.342
0.001 0.594 0.295 <0.001 0.006
a
Data are means of 5 replications with 8 male broilers per replicate pen, and experiment lasted from 0 to 20 d of age. European production index = [(BW, kg × livability, %)/(FCR × age, days)] × 100. a-c Means in the same rows with different superscript are significant difference. b
Table 5 Serum and liver enzymes activity of broiler at 3wk of age fed various levels and sources of trace minerals.a Items Serum
liver
ALP(U/100ml) T- SOD (U/ml) CuZn-SOD (U/ml) Mn-SOD (U/ml) LDH(U/g protein) GOT(U/mg protein) GPT(U/mg protein)
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
69.62a 163.17 111.45 35.74 2624.50 89.06 2.38
55.04ab 164.08 131.97 32.11 2715.63 92.94 3.34
29.58c 156.78 118.31 38.47 2423.01 86.62 2.87
36.29b 168.49 137.67 32.01 2748.17 90.51 2.68
4.400 2.484 3.865 2.333 65.464 1.562 0.150
<0.001 0.402 0.740 0.553 0.282 0.615 0.167
a a-c
Data are means of 10 broilers per treatment. Means in the same rows with different superscript are significant difference.
(P<0.01) but increased mortality and culling rate than other groups. However, supplementation of 80% mintrex Zn/Mn in the diet did not influence the growth performance when compared to inorganic Zn/Mn supplementation (P > 0.05). There was no effect of organic Zn/Mn supplementation on ADFI and feed conversion compared to sulphates (P > 0.05). European production index of birds fed with diets containing 100% Mintrex Zn/Mn was significantly greater than those fed with diet containing only 80% and 60% Mintrex Zn/Mn (P<0.05), however, none of the organic Zn/Mn supplementation groups significantly differed in the index with inorganic Zn/Mn supplement group (P > 0.05). 3.2. Serum and liver enzyme activity The ALP activity of serum was significantly affected by the supplementation of Mintrex Zn/Mn. Compared to the inorganic group, supplementation of 80% and 60% Mintrex Zn/Mn significantly decreased the serum ALP activity (P<0.05). There were no differences in activities of serum T-SOD, CuZn-SoD and Mn-SOD, and liver metabolic enzyme (LDH, GOT, and GPT) among inorganic and Mintrex Zn/Mn supplementations (Table 5). 3.3. The mRNA abundance of Zn transfer proteins Gene expression of MT and ZnT 5 in jejunum mucosa did not differ among all 4 treatments (P > 0.05) (Table 6), however, supplementation 60% Mintrex Zn/Mn significantly decreased the mRNA abundance of ZnT 1 when compared to the inorganic Zn/Mn supplementation (P<0.05). 3.4. Bone mineralization Supplementation of Zn/Mn as organic chelate did not affect dry weight, ash weight and ash percentage of metatarsus compared to the supplementation of Zn/Mn as inorganic (Table 7). With the reduction of dietary Mintrex Zn/Mn level, length of the metatarsus also reduced. Compared to Zn/Mn sulphates, supplementation of 60% Mintrex Zn/Mn significantly decreased the length of the metatarsus (P<0.01), and tended to reduce ash percentage in the metatarsus (P<0.10). Table 6 The abundance(relative to -actin)of Zn transfer proteins mRNA of jejunum mucosa of broiler at 3 wk of age fed various levels and sources of trace minerals.a Items
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
Metallothionein Zn transfer protein 1 Zn transfer protein 5
1.02 0.59a 0.55
0.60 0.55ab 0.62
1.28 0.39ab 0.36
0.67 0.35b 0.59
0.129 0.037 0.044
0.118 0.067 0.129
a a-b
Data are means of 8 broilers per treatment. Means in the same rows with different superscript are significant difference.
J. Yuan et al. / Animal Feed Science and Technology 168 (2011) 72–79
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Table 7 Metatarsus of broilers at 3 wk of age fed various levels and sources of trace minerals.a Items
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
Dry weight (g) Length (cm) Ash weight (g) Ash percentage (%)
1.14 5.54ab 0.58 50.63
1.16 5.56a 0.58 50.42
1.16 5.40bc 0.58 49.75
1.13 5.32c 0.56 49.62
0.021 0.032 0.010 0.168
0.911 0.006 0.827 0.086
a a-c
Data are means of 10 broilers per treatment. Means in the same rows with different superscript are significant difference.
3.5. Fecal mineral excretion Supplementation with different levels of Mintrex Zn/Mn significantly affected the mineral excretion (Table 8). Supplementation with 100% Mintrex Zn/Mn significantly decreased mineral excretion compared to the inorganic supplementation. Supplementation of 80% Mintrex Zn/Mn decreased the Mn output by 41.10%, 39.33% and 39.79% (P<0.001), respectively (based on concentration of dry manure, and per chicken day or per kg feed intake); and decreased Zn output by 35.58%, 28.68% and 30.13% (P<0.001), respectively. Supplementation of 60% Mintrex Zn/Mn significantly decreased Mn output in the dry manure by 38.89%, 37.80% and 36.68% (P<0.001) and Zn output by 32.59%, 31.47% and 30.16% (P<0.001), respectively. There was no difference for mineral excretion between 80% and 60% Mintrex Zn/Mn supplementation. 4. Discussion Required for the normal function of numerous structural proteins, enzymes, and cellular proteins, Zn and Mn are essential for the growth and development of broilers. Insufficiency of Zn and Mn in the diet depresses feed intake and reduces body weight gain, however, it could be reversed by supplementation of Zn and Mn (Ao et al., 2006, 2009; Nollet et al., 2008; Bao et al., 2009, 2010). Previous studies (Leeson and Caston, 2007; Nollet et al., 2007) indicated that organic minerals could produce the best performance in broilers at much lower concentrations than those recommended by the NRC (1994). However, Bao et al. (2007) and Nollet et al. (2008) found supplementation with organic minerals (Bioplex) Mn, Zn, Fe lower than 22.5 mg/kg decreased the growth performance. These inconsistent results might be due to different levels of control. Some studies used the NRC-recommended levels of trace minerals (1994) as the control, while other studies used levels of trace minerals suggested by a commercial company. There is great difference in the levels of required trace minerals between the standard of NRC (1994) and commercial broiler breeding companies. For example, the recommended Mn supplement is about 20 mg/kg greater in the Ross 308 and AA+ (120 mg/kg of diet) than in the Cobb 500 (100 mg/kg of diet). In this study, supplemental levels of inorganic Zn and Mn in the Treatment 1 met the commercial recommendation for AA+ . Furthermore, there was a dose-dependent decrease in the ADG when supplementing different levels of Mintrex Zn/Mn in the broiler diets (y = 0.058X + 20.85, R2 = 0.980). When the supplementation of Mintrex Zn/Mn was lowered to 60% (Zn 33.6 mg/kg, Mn 50.4 mg/kg), it significantly decreased the ADG of broilers. European production index (EPI) is a composite indicator to evaluate the effect of additives. Although the EPI of 80% or 60% Mintrex Zn/Mn supplementation was significantly lower than that of 100% Mintrex Zn/Mn supplementation, there was no significant difference between 80%, or 60% Mintrex Zn/Mn supplementation and the inorganic control. Trace minerals are required for the normal function of all biochemical processes in animal body. As a cofactor or component of more than 240 enzymes, Zn participates in protein and carbohydrate metabolism. Manganese is required as a catalytic cofactor for arginase, pyruvate carboxylase, and also an activator of glycosyltransferases and glutamine synthetase. Bao et al. (2009) indicated that reduced growth of broilers with lower dietary levels of Zn and Mn was due mainly to decreased feed intake, and they explained that supplementation of Mn and Zn had no effect on AME contents and apparent digestibility of Ca and P, but significantly improved apparent protein digestibility. Therefore, the reduction in feed intake associated with a lack of Zn/Mn may be related to the reduced feed digestibility in chicks. In the current study, ADFI and FCR of broilers were Table 8 Fecal mineral excretion of broilers at 3wk of age fed various levels and sources of trace mineralsa . Items Mn output in manure Dry manure, mg/g Mg/d, chick Mg/kg diet Zn output in manure Dry manure mg/g Mg/d, chick Mg/kg diet a a-c
Inorganic salts
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
108.15a 12.05a 150.69a
93.47b (−13.57%) 11.37b (−5.89%) 137.38b (−8.83%)
63.70c (−41.10%) 7.31c (−39.33%) 90.73c (−39.79%)
66.08c (−38.89%) 7.50c (−37.80%) 95.41c (−36.68%)
4.618 0.549 6.517
<0.001 <0.001 <0.001
34.64a 7.72a 96.53a
28.85b (−16.70%) 7.00b (−9.38%) 84.82b (−12.13%)
22.32c (−35.58%) 5.51c (−28.68%) 67.44c (−30.13%)
23.35c (−32.59%) 5.29c (−31.47%) 67.41c (−30.16%)
1.237 0.277 3.278
<0.001 <0.001 <0.001
Data are means of 5 replications per treatment. Means in the same rows with different superscript are significant difference.
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not significantly affected by different levels of Mintrex Zn/Mn, however; a linear, negative regression was found between Mintrex Zn/Mn supplementation and ADFI and FCR. This indicated that supplementation with significantly lower levels of Mintrex Zn/Mn could eventually influence the feed intake. In this study, we did not measure the apparent protein digestibility, but found there were no significant differences in the enzyme activities of LDH, GOT and GPT among all treatments. This suggested that 60% supplementation of Mintrex Zn/Mn was enough to maintain the enzyme activities of LDH, GOT and GPT. Zinc and Mn are the key component of Mn-SOD, CuZn-SOD, which have the antioxidant properties able to reduce cellular injury. Zn can improve immune response in broilers and high levels of Zn could increase the productions of IgM and IgG antibodies (Bartlett and Smith, 2003). In this study, we did not check the immune response of broilers and we did not find differences in T-SOD, CuZn-SOD and Mn-SOD activities among all 4 treatments. Huang et al. (2007) found that various levels of inorganic Zn supplementation, (0, 20, 40, 60, 80, 100, 120, or 140 mg/kg) did not affected the liver CuZnSOD activity, suggesting that the liver CuZnSOD activity was not a useful criterion for Zn requirement estimation. Luo et al. (2007) showed that heart MnSOD and MnSOD mRNA were not affected by Mn supplementation regardless of the organic or inorganic sources. In the current study, however, we found that supplementation of 60% Mintrex Zn/Mn significantly decreased the activity of ALP and increased of 5% mortality and culling rate of broilers. ALP is a pivotal part of growth and development. It affects the bone mineralization and the depositions of crystals of calcium salt on the structure and architecture of the bone, improving the strength of the bone. The decreased serum ALP activity may be due to the Zn deficiency. Bao et al. (2009) showed that a deficiency of Mn and Zn strongly inhibited tibia bone growth, while supplementation of Zn and Mn linearly increased tibia bone length as well as tibia Zn and Mn content but had no effect on tibia bone width, strength, and Ca and P concentrations. This study showed that supplementation of 60% Mintrex Zn/Mn significantly decreased the length of the metatarsus compared to supplementation with Zn/Mn sulphates. It confirmed that Zn had a strong effect on tibia length growth (Scott et al., 1982; Bao et al., 2009). Previous studies indicated the mineral concentration of bone was significantly affected by dietary levels (Huang et al., 2009) and sources of trace minerals (Ao et al., 2009). The current study showed that supplementing 80% and 60% Mintrex Zn/Mn tended to decrease the metatarsus ash percentage, and 60% Mintrex Zn/Mn significantly decreased length of metatarsus compared to inorganic supplementation. However, there were no significantly difference between 80% Mintrex Zn/Mn and inorganic supplementation. It suggested that using 80% Mintrex Zn/Mn had no effect on bone mineralization, and 60% Mintrex Zn/Mn, might not meet the requirement for normal bone mineralization. To evaluate mineral bioavailability, expression of mineral-responsive biomarkers is widely used, including several transfer proteins (Huang et al., 2007, 2009) and mineral-related enzyme activities (Luo et al., 2007). Previous study showed Zn supplementation increased ZnT 5, ZnT 1, and MT expression (Yu et al., 2008). In the current study, we found that the ZnT 1 mRNA decreased along with Mintrex Zn/Mn supplement levels. In the Treatment 4 with 60% Mintrex Zn/Mn supplement, the uptake of Zn seems to be affected because of the low availability of Zn transfer protein ZnT 1 and consequently reflected by the decreased activity of ALP and the poor growth performance in this treatment group. We did not find any difference in MT and ZnT5 mRNA abundance between inorganic supplementation and any level of Mintrex Zn/Mn supplementation. It suggests the absorption of Zn/Mn is much improved even with low supplemental level of Mintrex Zn/Mn compared to inorganic supplementation. Unlike the significantly decreased expression of ZnT 1 mRNA, MT and ZnT 5 mRNA expression was comparable among all three levels of Mintrex Zn/Mn, indicating that gene expression of MT ZnT5 is not as sensitive as ZnT1 gene to changes in level of Zn/Mn. It is thought to be better way to reduce mineral emission and protect the environment by substituting inorganic salts with organic counterparts. Many researchers have indicated that 100% substitution of inorganic trace minerals with organic ones did not reduce mineral emission (Bao et al., 2007; Nollet et al., 2008), except a lower level of substitution being used (Leeson and Caston, 2007; Nollet et al., 2008). It is critical to balance the animal’s requirement to maintain growth performance and the low level of mineral emission because too low levels of organic trace minerals in the diet significantly reduce growth performance (Nollet et al., 2008). This study suggested that supplemental 80% Mintrex Zn/Mn could effectively reduce mineral emission as compared to inorganic Zn/Mn supplementation without comprising the growth performance. Most of studies used relative mineral concentration to dry fecal material (Yan and Waldroup, 2006; Nollet et al., 2007, 2008) to determine mineral excretion or trace mineral concentration per chick day (Bao et al., 2007). However, mineral concentration relative to dry fecal material is usually affected by feed intake and feces excretion, and difficult to quantify and compare. In the current study, the mineral excretion was determined using total collection procedure and feed intake was also monitored. Fecal mineral excretion was corrected with total fecal excretion (mg/d/bird) or corrected with feed intake (mg/kg diet). It allows us to assess the actual amount of total mineral excretion compared to the relative mineral concentration in dry fecal material (mg/g). We noticed that the first two methods gave closer estimations (mg/d/bird and mg/kg diet) and the relative concentration method (dry manure, mg/g) overestimated mineral reduction. Therefore it would be more accurate to use trace mineral concentration per chick day or per kg diet intake to assess fecal minerals excretion 5. Conclusions Supplementation of different levels of Mintrex Zn/Mn affected broiler performance, function enzyme and transfer protein mRNA expression, bone growth and feces minerals excretion. Supplemental 80% Mintrex Zn/Mn can maximize the reduction in Zn/Mn excretion without affecting the growth performance and the bone growth compared to the inorganic supplementation. Therefore 80% Mintrex Zn/Mn (Zn 44.8 mg/kg and Mn 67.2 mg/kg, approximately 56% inorganic mineral level) was suggested as the suitable level to substitute inorganic Zn/Mn supplementation for 0–3-wk-old broilers.
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Acknowledgement This research was supported by “11th five-year” plan projects national science and technology support of China ministry (2008BADA7B04). The review of this manuscript and suggestions for improvement by Jing Jing Xie and Kim Kuenzel is gratefully appreciated. References Chinese Chicken Feeding Standard Requirement (NY/T 33-2004). Agricultural standard Council, China Agricultural Ministry 2004, Beijing. Ao, T., Pierce, J.L., Power, R., Dawson, K.A., Pescatore, A.J., Cantor, A.H., Ford, M.J., 2006. Evaluation of bioplex Zn® as an organic zinc source for chicks. Int. J. Poult. Sci. 5, 808–811. Ao, T., Pierce, J.L., Power, R., Pescatore, A.J., Cantor, A.H., Dawson, K.A., Ford, M.J., 2009. Effects of feeding different forms of zinc and copper on the performance and tissue mineral content of chicks. Poult. Sci. 88, 2171–2175, Aviagen, Newbridge, Midlothian, UK. Bao, Y.M., Choct, M., IJI, P.A., Bruerton, K., 2007. Effect of organically complexed copper, iron, manganese and zinc on broiler performance, mineral excretion and accumulation in tissues. J. Appl. Poult. Res. 16, 448–455. Bao, Y.M., Choct, M., IJI, P.A., Bruerton, K., 2009. Optimal dietary inclusion of organically complexed zinc for broiler chickens. Brit. Poult. Sci. 50, 95–102. Bao, Y.M., Choct, M., IJI, P.A., Bruerton, K., 2010. Trace mineral interactions in broiler chicken diets. Brit. Poult. Sci. 51, 109–117. Bartlett, J.R., Smith, M.O., 2003. Effects of different levels of zinc on the performance and immunocompetence of broilers under heat stress. Poult. Sci. 82, 1580–1588. Cao, J., Henry, P.R., Davis, S.R., Cousins, R.J., Miles, R.D., Littell, R.C., Ammerman, C.B., 2002. Realtive bioavailability of organic zinc sources based on tissue zinc and metallothionein in chicks fed conventional dietary zinc concentrations. Anim. Feed Sci. Technol. 101, 161–170. Cao, J., Henry, P.R., Guo, R., 2000. Chemical characteristics and relative bioavailability of supplemental organic zinc sources for poultry and ruminants. J. Anim. Sci. 78, 2039–2054. Guo, R., Henry, P.R., Holwerda, R.A., Cao, J., Littell, R.C., Miles, R.D., Ammerman, C.B., 2001. Chemical characteristics and relative bioavailability of supplemental organic copper sources for poultry. J. Anim. Sci. 79, 1132–1141. Huang, Y.L., Lu, L., Luo, X.G., Liu, B., 2007. An optimal dietary zinc level of broiler chicks fed a corn-soybean meal diet. Poult. Sci. 86, 2582–2589. Huang, Y.L., Lu, L., Li, S.F., Luo, X.G., Liu, B., 2009. Relative bioavailabilities of organic zinc sources with different chelation strengths for broilers fed a conventional corn-soybean meal diet. J. Anim. Sci. 87, 2038–2046. Leeson, S., 2005. Trace mineral requirements of poultry—validity of the NRC recommendations. In: Taylor-Pickard, J.A., Tucker, L.A. (Eds.), Redefining Mineral Nutrition. Nottingham Univ. Press, UK, pp. 107–117. Leeson, S., Caston, L., 2007. Using minimal supplements of trace minerals as a method of reducing trace mineral content of poultry manure. Anim. Feed Sci. Technol., doi:10.1016/j.anifeedsci.2007.08.004. Leeson, S., Summers, J., 2001. Scott’s Nutrition of the Chicken. Univ. Books, Guelph, Ontario, Canada. Luo, X.G., Li, S.F., Lu, L., Liu, B., Kuang, X., Shao, G.Z., Yu, S.X., 2007. Gene expression of manganese-containing superoxide dismutase as a biomarker of manganese bioavailability for manganese sources in broilers. Poult. Sci. 86, 888–894. Nollet, L., Huyghebaert, G., Spring, P., 2008. Effect of different levels of dietary organic (Bioplex) trace minerals on live performance of broiler chickens by growth phases. J. Appl. Poult. Res. 17, 109–115. Nollet, L., van der Klis, J.D., Lensing, M., Spring, P., 2007. The effect of replacing inorganic with organic trace minerals in broiler diets on productive performance and mineral excretion. J. Appl. Poult. Res. 16, 592–597. Predieri, G., Elviri, L., Tegoni, M., Zagnoni, I., Cinti, E., Biagi, G., Ferruzza, S., Leonardi, G., 2005. Metal chelates of 2-hydroxy-4-methylthiobutanoic acid in animal feeding. Part 2: further characterizations, in vitro and in vivo investigations. J. Inorg. Biochem. 99, 627–636. Scott, M.L., Nesheim, M.C., Yang, R.J., 1982. Essential inorganic elements. In: Nutrition of the Chicken, New York, USA, pp. 277–382. Tables of Feed Composition and Nutritive Values (2009) in China. China Feedstuff Information Center, 2009. Beijing, China. Wang, Z., Cerrate, S., Coto, C., Yan, F., Waldroup, P.W., 2007. Evaluation of Mintrex® copper as a source of copper in broiler diets. Int. J. Poult. Sci. 6, 308–313. Yan, F., Waldroup, P.W., 2006. Evaluation of Mintrex® manganese as a source of manganese for young broilers. Int. J. Poult. Sci. 5, 708-703. Yi, G.F., Atwell, C.A., Hume, J.A., Dibner, J.J., Knight, C.D., Richards, J.D., 2007. Determining the methionine activity of mintrex organic trace minerals in broiler chicks by using radiolabel tracing or growth assay. Poult. Sci. 86, 877–887. Yu, Y., Lu, L., Luo, X., Liu, B., 2008. Studies on characterizations of organic zinc absorption in different intestinal segments in broilers. Acta Nutrimenta Sin. 30, 148–152.
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Effect of dietary Mintrex-Zn/Mn on performance, gene expression of Zn transfer proteins, activities of Zn/Mn related enzymes and fecal mineral excretion in broiler chickens Jianmin Yuan ∗ , Zhihong Xu, Chunxi Huang, Shuliang Zhou, Yuming Guo State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China
a r t i c l e
i n f o
Article history: Received 23 August 2010 Received in revised form 15 March 2011 Accepted 18 March 2011
Keywords: Trace mineral Mintrex Excretion Mineral status Zn transfer proteins Broilers
a b s t r a c t This study was to evaluate dietary Mintrex-Zn/Mn on growth performance, enzyme activities, and trace mineral absorption and utilization in 0–3-wk broilers. One hundred sixty day-old male broiler chickens were allotted to 4 dietary treatments with 5 replicate cages of 8 birds. Broilers were fed maize-soybean basal diets containing zinc (Zn, 100 mg/kg) and manganese (Mn, 120 mg/kg) from sulphate salts, while three other treatments included 100%, 80%, and 60% of the basal dietary mineral (actually 70%, 56% and 42% of sulphate salts based on 70% of sulphates would be available) from Mintrex-Zn/Mn, respectively. At the end of 3 weeks of age, the growth performance, mRNA abundance of Zn/Mn transfer proteins in jejunum mucosa, enzyme activities of liver lactate dehydrogenase (LDH), glutamic oxalacetic transaminase (GOT) and glutamate-pyruvate transaminase (GPT), activities of serum alkaline phosphatase (ALP), Mn-superoxide dismutase (SOD), total superoxide dismutase (T-SOD) and CuZn-SOD and bone mineralization were measured. Total collection procedure in the balance trial of excreta was done from the age of 20 to 23 days. The results showed that supplementation with 100% Mintrex-Zn/Mn significantly improved the average daily gain (ADG) (P<0.01) and decreased fecal mineral excretion (P<0.05) compared to inorganic supplementation, but did not affect Zn/Mn transfer proteins mRNA abundance (P > 0.05). Supplementation with 80% Mintrex-Zn/Mn lowered serum ALP activity (P<0.05), however, it did not affect the growth performance, mRNA abundance of Zn/Mn transfer proteins and bone mineralization in comparison to inorganic supplementation (P > 0.05). The decrease of excretion is mainly due to the reduction of dietary supplemental level of minerals. Supplementation with 80% Mintrex-Zn/Mn decreased Mn and Zn excretion in the feces (mg/kg diet) by 39.79% and 30.13% (P<0.001), respectively. Supplementation with 60% Mintrex-Zn/Mn significantly decreased ADG (P<0.01), the length of metatarsus, serum ALP activities and mRNA expression of Zn transfer proteins but increased mortality and culling rate (P<0.05). There were no differences in activities of serum T-SOD, CuZn-SoD and Mn-SOD, and liver metabolic enzyme (LDH, GOT, and GPT) among inorganic versus Mintrex Zn/Mn supplementations. This study suggested that using approximately 30–40% Mintrex Zn/Mn could not substitute for the inorganic trace mineral. LDH, GOT, GPT may not be sensitive variables to reflect the states of the Zn and Mn in the body. Supplementation with 80% Mintrex-Zn/Mn (56% of inorganic mineral level) had a better effect in reducing the fecal mineral excretion without compromise in the growth performance. Using relative mineral concentration in fecal material may overestimate the values of mineral reduction, and it would be more accurate to use trace mineral concentration per chick day or per kg diet intake. © 2011 Elsevier B.V. All rights reserved.
∗ Corresponding author. Tel.: +86 01062732337; fax: +86 01062732712. E-mail address: [email protected] (J. Yuan). 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.03.011
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1. Introduction Essential trace minerals are important for a wide variety of physiological processes in all animals. Several hundred enzymes require the presence of minerals for their activity. Practicality, it is crucial to use an optimal level of trace minerals to allow animals to reach their genetic potential for performance. The levels of trace minerals supplemented in the diet are mostly based on National Research Council (NRC) recommendations. Some values of NRC (1994) for trace minerals in poultry, however, were mainly estimated based on extrapolation from other species and early studies in old bred of chickens (Leeson and Caston, 2007). With the advancement of genetic selection, the growth performance of commercial broiler strains is continually improved. Much higher than NRC levels of Zn and Mn supplements have been suggested in commercial diets to meet the growth of the modern commercial broilers (Leeson, 2005). For example, 100 mg Zn (or Mn)/kg of diet to the Cobb 500 and 120 mg Mn and 100 mg Zn/kg of diet to Ross 308 and AA plus are recommended. Traditionally, trace minerals have been supplemented in animal diets using inorganic salts such as Zinc oxide (Zn) and manganese (Mn) oxide or sulphate salts. However, in recent years there are increased concerns about the usage of inorganic trace minerals due to their poor bioavailability and potential to cause ingredient antagonism, which impairs absorption and increases environment pollution. Therefore, it is necessary to use organic trace minerals to substitute for inorganic trace minerals in the diet. Due to the higher bioavailability compared to inorganic forms (Leeson, 2005; Predieri et al., 2005; Ao et al., 2006; Yan and Waldroup, 2006), organic trace minerals (OTMs) are widely used in animal diets today. Organic trace minerals have been shown to enhance mineral uptake (Leeson, 2005; Predieri et al., 2005; Yan and Waldroup, 2006), improve the body weight gain (Ao et al., 2006; Nollet et al., 2008; Bao et al., 2009, 2010), and reduce mineral excretion compared to inorganic counterparts (Predieri et al., 2005; Yan and Waldroup, 2006; Nollet et al., 2007, 2008). However, some studies indicate that the absorption and bioavailability of organic trace elements were largely affected by their instabilities at low pH (Cao et al., 2000; Guo et al., 2001; Cao et al., 2002), and the formation of complexes with amino acids (Yu et al., 2008; Huang et al., 2009). Mintrex, a chelate of two 2-hydroxy-4 (methylthio) butanoic acid (HMTBa) ligands to one atom of trace mineral, i.e. zinc, copper or manganese, has been attracting much attention in recent years. Studies in broilers showed that Mintrex Zn (Yi et al., 2007), Mintrex Mn (Yan and Waldroup, 2006) and Mintrex Cu (Wang et al., 2007) had greater bioavailability than sulphate minerals. Predieri et al. (2005) suggested using MHA chelates could be a valuable tool to increase the bioavailability of trace minerals and to reduce the environmental pollution of animal manure. Although various authors (Yan and Waldroup, 2006; Wang et al., 2007) have considered the effect of the individual Mintrex Zn or Mintrex Mn on the performance and mineral excretion of broilers, little attention has been given to the effect of the combination of Mintrex Zn and Mn on growth performance, mineral absorption, metabolic enzyme activity and bone mineralization. The current study was conducted to determine the effects of dietary supplementing different concentrations of Mintrex-Zn/Mn on growth performance, serum and liver mineral-related enzyme activities, gene expression of Zn transfer proteins, bone mineralization and fecal mineral excretion in chickens. It aims to determine the minimal amount of Mintrex-Zn/Mn supplementing to 0–3 wk-old chickens without comprising on growth performance and environmental pollution.
2. Materials and methods 2.1. Animals and diets One hundred sixty day-old male broiler chickens (Arbor Acres plus, AA+ ) were randomly allotted to 4 dietary treatments (5 cages/diet, 8 birds/cage). A maize-soybean meal basal diet (Table 1) was used and formulated to be adequate in all nutrients according to Chinese Chicken Feeding Standard Requirement, (NY/T 33-2004) except for Zn and Mn. The nutrient values of diet were calculated based on the tables of feed composition and nutritive values in china. The Zn and Mn concentration in the basal diet was 29.94 mg/kg and 28.14 mg/kg, respectively. Trace minerals were provided as inorganic sulphate salts or as organic minerals (MINTREX® Zn/Mn). Mintrex Zn/Mn was supplied by Novus International. Mintrex Zn delivers 16% Zn as Zn methionine hydroxy analogue chelate and 80% methionine value, and Mintrex Mn delivers 13% Mn as Mn methionine hydroxy analogue chelate and 76% methionine value. In the Treatment 1, 100 mg Zn as sulphate/kg of diet and 120 mg Mn as sulphate/kg of diet was formulated in the basal diet according to NY/T 33-2004. It was arbitrarily assumed that 70% of these sulphates would be available (Leeson and Summers, 2001) and therefore the available minerals were Zn 70 mg/kg diet, Mn 84 mg/kg diet, respectively. In the Treatment 2, the supplement of Mintrex Zn/Mn was adjusted to the bioavailable level of Zn and Mn as in the Treatment 1 (70 mg/kg Zn, 84 mg/kg Mn), when the bioavailablity Mintrex Zn/Mn was arbitrarily assumed to be 100%. 80% and 60% of Mintrex Zn/Mn in the Treatment 2 was formulated in the diet of Treatments 3 (44.8 mg/kg and 67.2 mg/kg) and 4 (33.6 mg/kg and 50.4 mg/kg), respectively. The assayed mineral content of diet is in Table 2. The Met content from HMTBA of Mintrex in Treatments 2–4 was calculated and adjusted in the diet accordingly. All birds were raised in battery cages. Each pen was equipped with an individual feeder and water supply. All birds were allowed to consume mash feed and water ad libitum. Environmental temperature was initially set at 33 ◦ C and then gradually reduced to normal brooding practice. Birds had 24 h light. Hatchery body weight (BW) and body weight at day 20 posthatch, after 6 h fast, and feed intake from 0 to 20 d of age, was measured for the determination of average daily gain (ADG), average daily feed intake (ADFI) and gain/feed (G:F). Daily mortality and culling were recorded for each pen. The present study was approved by the China Agricultural University and carried out in accordance with the Guidelines for Experimental Animals.
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Table 1 Composition (g/kg) and calculated analysis of experimental basal diets. Ingredients
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
Maize Soybean meal Corn gluten Soybean oil Dicalcium phosphate Limestone Table salts l-Lysine HCl dl-methionine Choline chloride (50%) Topomicin (15%) Washed sand Oxethyl quinoline (33%) Vitamin premixa Mineral premixb Zinc sulphate (ZnSO4 ·H2 O) Manganese sulphate (MnSO4 ·H2 O) Mintrex Zn, 16% Mintrex Mn,13% Total ME (MJ/kg) CPc Lysinec Methioninec Cac Nonphytate Pc
552.70 327.40 50.00 29.70 17.30 11.10 3.50 0.90 1.40 1.60 0.80 1.67 0.35 0.25 1.00 0.14 0.19 1000.00 12.54 217 110 4.9 9.2 4.2
552.70 327.40 50.00 29.70 17.30 11.10 3.50 0.90 0.56 1.60 0.80 2.30 0.35 0.25 1.00 0.00 0.00 0.22 0.32 1000.00 12.54 217 110 4.9 9.2 4.2
552.70 327.40 50.00 29.70 17.30 11.10 3.50 0.90 0.73 1.60 0.80 2.23 0.35 0.25 1.00 0.00 0.00 0.18 0.26 1000.00 12.54 217 110 4.9 9.2 4.2
552.70 327.40 50.00 29.70 17.30 11.10 3.50 0.90 0.90 1.60 0.80 2.18 0.35 0.25 1.00 0.00 0.00 0.13 0.19 1000.00 12.54 217 110 4.9 9.2 4.2
a Provides per kg diet: Vitamin A, 12,5000 IU; Vitamin D3, 2500 IU; Vitamin E, 18.75 IU; Vitamin K, 2.65 mg; niacin, 36.8 mg; pantothenic acid, 12 mg; folic acid, 1.25 mg; thiamin, 2.5 mg; riboflavin, 6.6 mg; pyridoxine, 4.9 mg; Vitamin B12, 0.025 mg; biotin, 0.013 mg. b Provides per kg diet: Ferrous, 100 mg/kg (FeSO4 ·H2 O); Cu, 10 mg/kg (CuSO4 ·5H2 O); selenium, 0.3 mg/kg (Na2 SeO3 ); I, 0.7 mg/kg (Ca(IO3 )·H2 O. c Values calculated based on the Table of Feed Composition and Nutritive Values (2009) in China.
2.2. Tissue sampling and preparation At the end of the trial, blood samples of 10 chicks from each treatment were drawn from wing vein and centrifuged at 3600 × g for 10 min, serum was removed and stored at –30 ◦ C until assayed. The mucosa of jejunum near the Meckel’s diverticulum was removed by gentle scraping with a clean microscope slide, washed with cold PBS, frozen in liquid nitrogen, and stored at –80◦ C for later determination of the mRNA levels of metallothionein (MT), Zn transfer protein 1 (ZnT 1) and Zn transfer protein 5 (ZnT 5) using real-time PCR. The liver was removed, frozen in liquid nitrogen, and then stored at –30 ◦ C for later measurement of metabolic enzyme activity. The left metatarsus was removed and the length was measured. The metatarsus was cleaned of the muscle before the bone was dried for a minimum of 24 h at 100 ◦ C. After 72 h-extraction in the diethyl ether, the bone was weighed and dried for another 6 h at 100 ◦ C. Ash content of metatarsus was then determined gravimetrically after being ashed in a muffle furnace for 10 h at 550 ◦ C. 2.3. Liver and blood metabolic enzyme activity determination The activity of serum enzymes including alkaline phosphatase (ALP), Mn superoxide dismutase (Mn-SOD), CuZn superoxide dismutase (CuZn-SOD), total superoxide dismutase (T- SOD), and liver metabolic enzyme including lactate dehydrogenase (LDH), glutamic oxalacetic transaminase (GOT), glutamate-pyruvate transaminase (GPT) was measured using commercial kits (Institute of Nanjing Jiancheng Bioengineering).
Table 2 Mineral content of diet (mg/kg)a . Mineral
Content
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
Zn
Supplemented Calculated Assayed Supplemented Calculated Assayed
100 129.94 99.64 120 148.14 144.46
70 99.94 88.66 84 112.14 108.39
44.8 74.74 77.68 67.2 95.34 87.91
33.6 63.54 69.96 50.4 78.54 107.68
Mn
a
The Zn and Mn content in the basal diet was 29.94 mg/kg and 28.14 mg/kg, respectively.
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Table 3 Sequences of the primers used in real-time PCR assays. Gene name
Genebank accession number
Primer sequence (5 → 3 )
Predicted size (bp)
Metallothionein
NM 205275
163
Zn transfer protein 1
AJ619980
Zn transfer protein 5
NM 001031419
-actin
NM 205518
F 5 -AAG GGC TGT GTC TGC AAG GA- 3 R 5 -CTT CAT CGG TAT GGA AGG TAC AAA-3 F 5 -TGC GAG TGC CTT CTT CCT-3 R 5 -AAG GAG CTG TCA GGT CTG TAA T-3 F 5 -ATG CTG TTG TGG GAT GTA-3 R 5 -TTG TCT TGG CTG GTC CTC-3 F 5 -GAG AAA TTG TGC GTG ACA TCA-3 R 5 -CCT GAA CCT CTC ATT GCC A-3
131 159 152
2.4. Quantification of Zn transfer proteins mRNA using real-time PCR 2.4.1. Total RNA isolation and reverse transcription Total RNA was isolated from jejunum mucosa using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacture’s instruction. The integrity of total RNA was assessed via agarose gel electrophoresis and the RNA concentration and purity were spectrophotometrically determined using A260 and A280 measurements. Reverse transcription (RT) reactions (20 L) consisted of 16 L total RNA, 1 L of RNAse inhibitor (Promega), 1 L dNTPs (sigma), 2 L of 5 × M-MLV RT reaction buffer (Promega). The RT procedure was 20 ◦ C, 5 min, followed by 42 ◦ C, 60 min, and then 70 ◦ C, 5 min. The reaction was stopped by putting on ice. The RT products (cDNA) were stored at −20 ◦ C for PCR 2.4.2. Real-time PCR for quantification of mRNA abundance According to the sequence of the gene published in GenBank (Table 3), primers for MT, ZnT-1, ZnT-5 and -actin gene were designed with Primer Express Software (Applied Biosystems Incorporation, Foster, CA) and sequences for each primer was provided in Table 3. All PCR was performed with the ABI PRISM 7700 sequence detection system (ABI Biosystems) according to optimized PCR protocols. SYBR Green qPCR kit was used for the fluorescent detection (ABI Biosystems). A total volume of 20 L PCR reaction system was used which contained 10 L SYBR Green PCR Master Mix, and 2.0 L primer (1.0 L forward and 1.0 L reverse), and 2.0 L cDNA template, and 6 L sterile super-stilled water. For the PCR reaction, the experimental protocol was: denature at 95 ◦ C for 3 min, 42 cycles of amplification (94 ◦ C for 30 s, 51 ◦ C for 30 s, 72 ◦ C for 60 s) with a single fluorescence measurement at 72 ◦ C extension, and finally extension at 72 ◦ C for 7 min. The Ct value was determined and was used to calculate the relative expression level 2−Ct . 2.5. Fecal mineral excretion measurement Fecal mineral excretion was evaluated by the total collection procedure in the balance trial at the age of 20–23 days. After fasted for 17 h, chicks were fed with experimental diets for 55 h and starved again for 17 h. All excreta were collected for the last 72 h. Each 24-h output was collected and stored at −20 ◦ C until the end of the balance trial. At the end of the collection period, feed intake was recorded precisely. In the meantime, excreta output for each cage was weighed, commixed and sampled. The fecal samples were dried at 65 ◦ C, ashed and then assayed for minerals by atomic absorption spectrophotometer. Feed samples were also assayed for minerals following the same procedure. 2.6. Statistical analysis Data were subjected to analysis of variance (ANOVA), general linear models (SAS Institute, 1991). Duncan’s multiple range tests was used to assess differences in means among all treatments. The percentage values were transformed with arcsin function before statistical evaluation. Differences among means with P<0.05 were accepted as statistically significant differences. Difference among means 0.05 < P<0.10 were accepted as tendencies to have differences. For comparison the relationship of performance of broiler and the levels of Mintrex Zn/Mn in diets, linear regression analysis was conducted. 3. Results Table 2 shows the assayed mineral content of the diets together with the level of supplemental minerals, and calculated mineral content. The assayed mineral content was close to the calculated content. 3.1. Performance The growth performance of broilers was significantly affected by the supplementation of Mintrex Zn/Mn (Table 4). Compared to the supplementation of Zn/Mn sulphates (Treatment 1), organic supplementation with similar level of Zn/Mn (Treatment 2) significantly improved ADG (P<0.01). Supplementation with 60% mintrex Zn/Mn significantly decreased ADG
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J. Yuan et al. / Animal Feed Science and Technology 168 (2011) 72–79
Table 4 Performance of broilers fed various levels and sources of trace minerals from 0 to 3 wk of agea . Items
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
ADG (g/d) ADFI (g) F/G Mortality plus culls (%) European production indexb
25.72b 38.30 1.49 0.00b 86.42a , b
26.74a 39.05 1.46 0.00b 92.06a
25.30b ,c 38.63 1.52 0.00b 83.13b
24.44c 37.36 1.53 5.00a 79.94b
0.245 1.517 0.015 0.860 2.342
0.001 0.594 0.295 <0.001 0.006
a
Data are means of 5 replications with 8 male broilers per replicate pen, and experiment lasted from 0 to 20 d of age. European production index = [(BW, kg × livability, %)/(FCR × age, days)] × 100. a-c Means in the same rows with different superscript are significant difference. b
Table 5 Serum and liver enzymes activity of broiler at 3wk of age fed various levels and sources of trace minerals.a Items Serum
liver
ALP(U/100ml) T- SOD (U/ml) CuZn-SOD (U/ml) Mn-SOD (U/ml) LDH(U/g protein) GOT(U/mg protein) GPT(U/mg protein)
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
69.62a 163.17 111.45 35.74 2624.50 89.06 2.38
55.04ab 164.08 131.97 32.11 2715.63 92.94 3.34
29.58c 156.78 118.31 38.47 2423.01 86.62 2.87
36.29b 168.49 137.67 32.01 2748.17 90.51 2.68
4.400 2.484 3.865 2.333 65.464 1.562 0.150
<0.001 0.402 0.740 0.553 0.282 0.615 0.167
a a-c
Data are means of 10 broilers per treatment. Means in the same rows with different superscript are significant difference.
(P<0.01) but increased mortality and culling rate than other groups. However, supplementation of 80% mintrex Zn/Mn in the diet did not influence the growth performance when compared to inorganic Zn/Mn supplementation (P > 0.05). There was no effect of organic Zn/Mn supplementation on ADFI and feed conversion compared to sulphates (P > 0.05). European production index of birds fed with diets containing 100% Mintrex Zn/Mn was significantly greater than those fed with diet containing only 80% and 60% Mintrex Zn/Mn (P<0.05), however, none of the organic Zn/Mn supplementation groups significantly differed in the index with inorganic Zn/Mn supplement group (P > 0.05). 3.2. Serum and liver enzyme activity The ALP activity of serum was significantly affected by the supplementation of Mintrex Zn/Mn. Compared to the inorganic group, supplementation of 80% and 60% Mintrex Zn/Mn significantly decreased the serum ALP activity (P<0.05). There were no differences in activities of serum T-SOD, CuZn-SoD and Mn-SOD, and liver metabolic enzyme (LDH, GOT, and GPT) among inorganic and Mintrex Zn/Mn supplementations (Table 5). 3.3. The mRNA abundance of Zn transfer proteins Gene expression of MT and ZnT 5 in jejunum mucosa did not differ among all 4 treatments (P > 0.05) (Table 6), however, supplementation 60% Mintrex Zn/Mn significantly decreased the mRNA abundance of ZnT 1 when compared to the inorganic Zn/Mn supplementation (P<0.05). 3.4. Bone mineralization Supplementation of Zn/Mn as organic chelate did not affect dry weight, ash weight and ash percentage of metatarsus compared to the supplementation of Zn/Mn as inorganic (Table 7). With the reduction of dietary Mintrex Zn/Mn level, length of the metatarsus also reduced. Compared to Zn/Mn sulphates, supplementation of 60% Mintrex Zn/Mn significantly decreased the length of the metatarsus (P<0.01), and tended to reduce ash percentage in the metatarsus (P<0.10). Table 6 The abundance(relative to -actin)of Zn transfer proteins mRNA of jejunum mucosa of broiler at 3 wk of age fed various levels and sources of trace minerals.a Items
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
Metallothionein Zn transfer protein 1 Zn transfer protein 5
1.02 0.59a 0.55
0.60 0.55ab 0.62
1.28 0.39ab 0.36
0.67 0.35b 0.59
0.129 0.037 0.044
0.118 0.067 0.129
a a-b
Data are means of 8 broilers per treatment. Means in the same rows with different superscript are significant difference.
J. Yuan et al. / Animal Feed Science and Technology 168 (2011) 72–79
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Table 7 Metatarsus of broilers at 3 wk of age fed various levels and sources of trace minerals.a Items
Zn/Mn sulphates
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
Dry weight (g) Length (cm) Ash weight (g) Ash percentage (%)
1.14 5.54ab 0.58 50.63
1.16 5.56a 0.58 50.42
1.16 5.40bc 0.58 49.75
1.13 5.32c 0.56 49.62
0.021 0.032 0.010 0.168
0.911 0.006 0.827 0.086
a a-c
Data are means of 10 broilers per treatment. Means in the same rows with different superscript are significant difference.
3.5. Fecal mineral excretion Supplementation with different levels of Mintrex Zn/Mn significantly affected the mineral excretion (Table 8). Supplementation with 100% Mintrex Zn/Mn significantly decreased mineral excretion compared to the inorganic supplementation. Supplementation of 80% Mintrex Zn/Mn decreased the Mn output by 41.10%, 39.33% and 39.79% (P<0.001), respectively (based on concentration of dry manure, and per chicken day or per kg feed intake); and decreased Zn output by 35.58%, 28.68% and 30.13% (P<0.001), respectively. Supplementation of 60% Mintrex Zn/Mn significantly decreased Mn output in the dry manure by 38.89%, 37.80% and 36.68% (P<0.001) and Zn output by 32.59%, 31.47% and 30.16% (P<0.001), respectively. There was no difference for mineral excretion between 80% and 60% Mintrex Zn/Mn supplementation. 4. Discussion Required for the normal function of numerous structural proteins, enzymes, and cellular proteins, Zn and Mn are essential for the growth and development of broilers. Insufficiency of Zn and Mn in the diet depresses feed intake and reduces body weight gain, however, it could be reversed by supplementation of Zn and Mn (Ao et al., 2006, 2009; Nollet et al., 2008; Bao et al., 2009, 2010). Previous studies (Leeson and Caston, 2007; Nollet et al., 2007) indicated that organic minerals could produce the best performance in broilers at much lower concentrations than those recommended by the NRC (1994). However, Bao et al. (2007) and Nollet et al. (2008) found supplementation with organic minerals (Bioplex) Mn, Zn, Fe lower than 22.5 mg/kg decreased the growth performance. These inconsistent results might be due to different levels of control. Some studies used the NRC-recommended levels of trace minerals (1994) as the control, while other studies used levels of trace minerals suggested by a commercial company. There is great difference in the levels of required trace minerals between the standard of NRC (1994) and commercial broiler breeding companies. For example, the recommended Mn supplement is about 20 mg/kg greater in the Ross 308 and AA+ (120 mg/kg of diet) than in the Cobb 500 (100 mg/kg of diet). In this study, supplemental levels of inorganic Zn and Mn in the Treatment 1 met the commercial recommendation for AA+ . Furthermore, there was a dose-dependent decrease in the ADG when supplementing different levels of Mintrex Zn/Mn in the broiler diets (y = 0.058X + 20.85, R2 = 0.980). When the supplementation of Mintrex Zn/Mn was lowered to 60% (Zn 33.6 mg/kg, Mn 50.4 mg/kg), it significantly decreased the ADG of broilers. European production index (EPI) is a composite indicator to evaluate the effect of additives. Although the EPI of 80% or 60% Mintrex Zn/Mn supplementation was significantly lower than that of 100% Mintrex Zn/Mn supplementation, there was no significant difference between 80%, or 60% Mintrex Zn/Mn supplementation and the inorganic control. Trace minerals are required for the normal function of all biochemical processes in animal body. As a cofactor or component of more than 240 enzymes, Zn participates in protein and carbohydrate metabolism. Manganese is required as a catalytic cofactor for arginase, pyruvate carboxylase, and also an activator of glycosyltransferases and glutamine synthetase. Bao et al. (2009) indicated that reduced growth of broilers with lower dietary levels of Zn and Mn was due mainly to decreased feed intake, and they explained that supplementation of Mn and Zn had no effect on AME contents and apparent digestibility of Ca and P, but significantly improved apparent protein digestibility. Therefore, the reduction in feed intake associated with a lack of Zn/Mn may be related to the reduced feed digestibility in chicks. In the current study, ADFI and FCR of broilers were Table 8 Fecal mineral excretion of broilers at 3wk of age fed various levels and sources of trace mineralsa . Items Mn output in manure Dry manure, mg/g Mg/d, chick Mg/kg diet Zn output in manure Dry manure mg/g Mg/d, chick Mg/kg diet a a-c
Inorganic salts
100%Mintrex
80%Mintrex
60%Mintrex
SEM
P value
108.15a 12.05a 150.69a
93.47b (−13.57%) 11.37b (−5.89%) 137.38b (−8.83%)
63.70c (−41.10%) 7.31c (−39.33%) 90.73c (−39.79%)
66.08c (−38.89%) 7.50c (−37.80%) 95.41c (−36.68%)
4.618 0.549 6.517
<0.001 <0.001 <0.001
34.64a 7.72a 96.53a
28.85b (−16.70%) 7.00b (−9.38%) 84.82b (−12.13%)
22.32c (−35.58%) 5.51c (−28.68%) 67.44c (−30.13%)
23.35c (−32.59%) 5.29c (−31.47%) 67.41c (−30.16%)
1.237 0.277 3.278
<0.001 <0.001 <0.001
Data are means of 5 replications per treatment. Means in the same rows with different superscript are significant difference.
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J. Yuan et al. / Animal Feed Science and Technology 168 (2011) 72–79
not significantly affected by different levels of Mintrex Zn/Mn, however; a linear, negative regression was found between Mintrex Zn/Mn supplementation and ADFI and FCR. This indicated that supplementation with significantly lower levels of Mintrex Zn/Mn could eventually influence the feed intake. In this study, we did not measure the apparent protein digestibility, but found there were no significant differences in the enzyme activities of LDH, GOT and GPT among all treatments. This suggested that 60% supplementation of Mintrex Zn/Mn was enough to maintain the enzyme activities of LDH, GOT and GPT. Zinc and Mn are the key component of Mn-SOD, CuZn-SOD, which have the antioxidant properties able to reduce cellular injury. Zn can improve immune response in broilers and high levels of Zn could increase the productions of IgM and IgG antibodies (Bartlett and Smith, 2003). In this study, we did not check the immune response of broilers and we did not find differences in T-SOD, CuZn-SOD and Mn-SOD activities among all 4 treatments. Huang et al. (2007) found that various levels of inorganic Zn supplementation, (0, 20, 40, 60, 80, 100, 120, or 140 mg/kg) did not affected the liver CuZnSOD activity, suggesting that the liver CuZnSOD activity was not a useful criterion for Zn requirement estimation. Luo et al. (2007) showed that heart MnSOD and MnSOD mRNA were not affected by Mn supplementation regardless of the organic or inorganic sources. In the current study, however, we found that supplementation of 60% Mintrex Zn/Mn significantly decreased the activity of ALP and increased of 5% mortality and culling rate of broilers. ALP is a pivotal part of growth and development. It affects the bone mineralization and the depositions of crystals of calcium salt on the structure and architecture of the bone, improving the strength of the bone. The decreased serum ALP activity may be due to the Zn deficiency. Bao et al. (2009) showed that a deficiency of Mn and Zn strongly inhibited tibia bone growth, while supplementation of Zn and Mn linearly increased tibia bone length as well as tibia Zn and Mn content but had no effect on tibia bone width, strength, and Ca and P concentrations. This study showed that supplementation of 60% Mintrex Zn/Mn significantly decreased the length of the metatarsus compared to supplementation with Zn/Mn sulphates. It confirmed that Zn had a strong effect on tibia length growth (Scott et al., 1982; Bao et al., 2009). Previous studies indicated the mineral concentration of bone was significantly affected by dietary levels (Huang et al., 2009) and sources of trace minerals (Ao et al., 2009). The current study showed that supplementing 80% and 60% Mintrex Zn/Mn tended to decrease the metatarsus ash percentage, and 60% Mintrex Zn/Mn significantly decreased length of metatarsus compared to inorganic supplementation. However, there were no significantly difference between 80% Mintrex Zn/Mn and inorganic supplementation. It suggested that using 80% Mintrex Zn/Mn had no effect on bone mineralization, and 60% Mintrex Zn/Mn, might not meet the requirement for normal bone mineralization. To evaluate mineral bioavailability, expression of mineral-responsive biomarkers is widely used, including several transfer proteins (Huang et al., 2007, 2009) and mineral-related enzyme activities (Luo et al., 2007). Previous study showed Zn supplementation increased ZnT 5, ZnT 1, and MT expression (Yu et al., 2008). In the current study, we found that the ZnT 1 mRNA decreased along with Mintrex Zn/Mn supplement levels. In the Treatment 4 with 60% Mintrex Zn/Mn supplement, the uptake of Zn seems to be affected because of the low availability of Zn transfer protein ZnT 1 and consequently reflected by the decreased activity of ALP and the poor growth performance in this treatment group. We did not find any difference in MT and ZnT5 mRNA abundance between inorganic supplementation and any level of Mintrex Zn/Mn supplementation. It suggests the absorption of Zn/Mn is much improved even with low supplemental level of Mintrex Zn/Mn compared to inorganic supplementation. Unlike the significantly decreased expression of ZnT 1 mRNA, MT and ZnT 5 mRNA expression was comparable among all three levels of Mintrex Zn/Mn, indicating that gene expression of MT ZnT5 is not as sensitive as ZnT1 gene to changes in level of Zn/Mn. It is thought to be better way to reduce mineral emission and protect the environment by substituting inorganic salts with organic counterparts. Many researchers have indicated that 100% substitution of inorganic trace minerals with organic ones did not reduce mineral emission (Bao et al., 2007; Nollet et al., 2008), except a lower level of substitution being used (Leeson and Caston, 2007; Nollet et al., 2008). It is critical to balance the animal’s requirement to maintain growth performance and the low level of mineral emission because too low levels of organic trace minerals in the diet significantly reduce growth performance (Nollet et al., 2008). This study suggested that supplemental 80% Mintrex Zn/Mn could effectively reduce mineral emission as compared to inorganic Zn/Mn supplementation without comprising the growth performance. Most of studies used relative mineral concentration to dry fecal material (Yan and Waldroup, 2006; Nollet et al., 2007, 2008) to determine mineral excretion or trace mineral concentration per chick day (Bao et al., 2007). However, mineral concentration relative to dry fecal material is usually affected by feed intake and feces excretion, and difficult to quantify and compare. In the current study, the mineral excretion was determined using total collection procedure and feed intake was also monitored. Fecal mineral excretion was corrected with total fecal excretion (mg/d/bird) or corrected with feed intake (mg/kg diet). It allows us to assess the actual amount of total mineral excretion compared to the relative mineral concentration in dry fecal material (mg/g). We noticed that the first two methods gave closer estimations (mg/d/bird and mg/kg diet) and the relative concentration method (dry manure, mg/g) overestimated mineral reduction. Therefore it would be more accurate to use trace mineral concentration per chick day or per kg diet intake to assess fecal minerals excretion 5. Conclusions Supplementation of different levels of Mintrex Zn/Mn affected broiler performance, function enzyme and transfer protein mRNA expression, bone growth and feces minerals excretion. Supplemental 80% Mintrex Zn/Mn can maximize the reduction in Zn/Mn excretion without affecting the growth performance and the bone growth compared to the inorganic supplementation. Therefore 80% Mintrex Zn/Mn (Zn 44.8 mg/kg and Mn 67.2 mg/kg, approximately 56% inorganic mineral level) was suggested as the suitable level to substitute inorganic Zn/Mn supplementation for 0–3-wk-old broilers.
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Acknowledgement This research was supported by “11th five-year” plan projects national science and technology support of China ministry (2008BADA7B04). The review of this manuscript and suggestions for improvement by Jing Jing Xie and Kim Kuenzel is gratefully appreciated. References Chinese Chicken Feeding Standard Requirement (NY/T 33-2004). Agricultural standard Council, China Agricultural Ministry 2004, Beijing. Ao, T., Pierce, J.L., Power, R., Dawson, K.A., Pescatore, A.J., Cantor, A.H., Ford, M.J., 2006. Evaluation of bioplex Zn® as an organic zinc source for chicks. Int. J. Poult. Sci. 5, 808–811. Ao, T., Pierce, J.L., Power, R., Pescatore, A.J., Cantor, A.H., Dawson, K.A., Ford, M.J., 2009. Effects of feeding different forms of zinc and copper on the performance and tissue mineral content of chicks. Poult. Sci. 88, 2171–2175, Aviagen, Newbridge, Midlothian, UK. Bao, Y.M., Choct, M., IJI, P.A., Bruerton, K., 2007. 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