Effects of methionine hydroxyl analog chelated zinc on laying performance, eggshell quality, eggshell mineral deposition, and activities of Zn-containing enzymes in aged laying hens

Effects of methionine hydroxyl analog chelated zinc on laying performance, eggshell quality,... ABSTRACT The study aimed to determine the effects of methionine hydroxyl analog chelated zinc (MHA-Zn) on laying performance, eggshell quality and mineral deposits, and the activities of Zn-containing enzymes on aged laying hens. A total of 960 layers (Hy-Line Grey, 57 wk old) were fed a basal diet (Zn: 35.08 mg/kg) without extra zinc for 2 wk. During the ensuing 14 wk, birds were randomly divided into 4 groups according to body weight and laying rate, with 8 replicates per treatment, and each group had 8 replicates of 30 hens. Four levels of Zn (ZnSO4: 80 mg/kg; MHA-Zn: 20, 40, 80 mg/kg) were added to the diet, respectively. The results shown that dietary Zn did not affect laying rate, average egg weight, average daily feed intake, or feed conversion ratio (P > 0.05); however, compared to the inorganic group, dietary supplementation with 40 or 80 mg/kg MHA-Zn decreased broken egg rate significantly in the whole period (P < 0.05), while significantly increased eggshell weight in week 62 to 72, eggshell thickness and eggshell strength in wk 66 to 72, eggshell weight percent and eggshell density in week 62 to 72 (P < 0.05). Besides, dietary supplementation with different sources and levels of Zn did not affect ash concentration of eggshell (P > 0.05), whereas dietary supplementation with 80 mg/kg MHA-Zn improved the Zn and Ca concentrations of eggshells and carbonic anhydrase (CA) activity of liver, and 40 mg/kg MHA-Zn increased Zn concentration of liver (P < 0.05). Moreover, no significant differences in alkaline phosphatase activity were observed among the treatment groups (P > 0.05). Therefore, dietary supplementation with 40 mg/kg MHA-Zn can improve eggshell quality by promoting Ca deposition and CA activity. INTRODUCTION Zinc (Zn) acts as a necessary micromineral for layer, and it is a component of more than 300 enzymes, mainly involved in the metabolism of cells, immune, and antioxides (Yan and Waldroup, 2006; Yi et al., 2007). Functions of Zn can be categorized as catalytic (metalloenzymes), structural (e.g., Zn finger domains of proteins), and regulatory (e.g., metal response elements of gene promoters). For the later period of laying, there are some changes in the layers, characterized by the decreasing of reproductive system and antioxide states, bigger and heavier eggs, and poor eggshell quality. It was reported there was 8% egg production losses resulted by the poor eggshell quality, which bring a great economic losses for poultry industry (Nys, 2001; Abdallan et al., 2009). To improve eggshell quality, researchers have taken great efforts in the genetics, environment, and nutrition, especially in the micromineral nutrition (Vallee and Falchuk, 1993; Batal et al., 2001; Berg et al., 2004; Gheisari et al., 2010). For example, zinc is a component of carbonic anhydrase (CA) enzyme, which involved in supplying carbonate ions during eggshell formation. Is it possible for microminerals such as Zn, Mn, and Cu to change the formation of CaCO3 calcite crystals and the construction of the eggshell, and finally affect the physical property of eggshell (Nys et al., 1999; Creech et al., 2004; Holm et al., 2006; Ao et al., 2009; Pierce et al., 2009; Yildiz et al., 2010). However, Zn deficiency or excess can cause metabolic disorders or imbalance and other symptoms, affecting normal production performance (Tabatabaie et al., 2007). Mabe et al. (2003) reported that dietary supplementation with 30 or 60 mg/kg Zn could improve the eggshell strength of aged layers. Zamani et al. (2005) observed that a diet containing 50, 100, or 150 mg/kg Zn could significantly improve eggshell thickness, and supplementation with 150 mg/kg Zn resulted in the thickest eggshells. Inorganic mineral elements are wildly used for its cheaper price. But there are existing antagonistic effect among different minerals and easily getting the formation of complex with other ingredients such as phytate, tannin in the diets in the intestinal, which result in the decreasing of the utilization (Swiatkiewicz and Koreleski, 2008). However, organic microminerals are characterized by reducing the chelating effects in the intestinal, decreasing metal ions interaction, increasing mineral steability in the lower pH value, more abosoption channel, higher abosorption and lower pollution, it is more and more used in the layer diets (Wedekind et al., 1992; Cao et al., 2000). It is generally accepted that organic trace elements (amino acid complex) have beneficial effects on eggshell quality (Henry et al., 1989; Neto et al., 2011). Cheng and Guo (2004) found that 60 mg/kg amino acid zinc (AA-Zn) decreased the broken egg rate. Abbas et al. (2010) reported that organic minerals could be utilized at a lower concentration in the diet than inorganic minerals, without having a negative impact on production performance. Similarly, Swiatkiewicz and Koreleski (2008) demonstrated that amino-acid-chelated trace elements could reduce adverse effects on the eggshell quality of aged layers. Trace elements may improve eggshell quality by promoting Ca deposition and the activities of key enzymes involved in eggshell formation (Zamani et al., 2005). Zn is found in the active center of CA and plays an important role in the conversion of carbon dioxide to bicarbonate (Bakst and Holm, 2003). Sun et al. (2012) reported that organic trace elements could improve CA activity in the liver. Alkaline phosphatase (AKP) is a Zn-containing enzyme, and its activity of serum or liver has been used as indicator of Zn status. Qin et al. (2017) showed that dietary supplementation with 60 mg/kg Zn significantly increased serum AKP activity. Previous studies have shown that organic Zn has more advantages than inorganic Zn in improving eggshell quality. However, the optimum supplementation amount has not been fully studied. The present study was designed to evaluate the effect of organic Zn on productivity, eggshell quality and mineral deposits, as well as activities of Zn-containing enzymes in late-phase laying hens. MATERIALS AND METHODS Animal Management and Diet Treatment A total of 960 layers (Hy-Line Grey, 57 wk old) were fed a basal diet (Zn = 35.08) without extra Zn to exhaust Zn that had previously accumulated. After 2 wk, the birds were randomly divided into 4 groups according to body weight and laying egg rate, with 8 replicates per treatment, and each group had 8 replicates of 30 hens. During the following 14 wk, the 4 groups were fed diets with 4 different levels of Zn added to the basal diet (ZnSO4: 80 mg/kg; MHA-Zn: 20, 40, 80 mg/kg). The experimental protocol used in the study was approved by the Animal Care and Use Committee of Northwest A & F University, Yangling, China. Research on live animals met the guidelines approved by the Institutional Animal Care and Use Committee. The basal diet was formulated to provide all other nutrients except Zn in accordance with the nutrient recommendations from NRC (1994). The dietary composition and nutrient levels are listed in Table 1. Table 1. Composition and nutrient levels of the basal diet (air-dry basis). Ingredients  %  Nutrients  %  Corn  58.50  ME (MJ/kg)  12.29  Soybean meal  23.00  Crude protein2  15.47  Ground limestone  8.74  Calcium2  3.60  CaHPO4  1.10  Total phosphorus  0.62  Wheat bran  7.50  Available phosphorus  0.32  Soybean oil  0.50  Lys  0.94  NaCl  0.24  Met  0.44  Met  0.16  Thr  0.70  Thr  0.03  Zinc2 (mg/kg)  35.08  Lys  0.05      Premix1  0.18      Total  100.00      Ingredients  %  Nutrients  %  Corn  58.50  ME (MJ/kg)  12.29  Soybean meal  23.00  Crude protein2  15.47  Ground limestone  8.74  Calcium2  3.60  CaHPO4  1.10  Total phosphorus  0.62  Wheat bran  7.50  Available phosphorus  0.32  Soybean oil  0.50  Lys  0.94  NaCl  0.24  Met  0.44  Met  0.16  Thr  0.70  Thr  0.03  Zinc2 (mg/kg)  35.08  Lys  0.05      Premix1  0.18      Total  100.00      1Supplied the following per kilogram of diet: Vitamin A 10,000 IU, VD3 31, 800 IU, VE 10 IU, VK 10 mg, VB125 μg, thiamine l mg, riboflavin 4.5 mg, calcium pantothenate 50 mg, niacin 24.5 mg, pyridoxine 5 mg, biotin 1 mg, folic acid 1 mg, choline 500 mg, Mn (as manganese sulfate) 60 mg, I (as calcium iodate) 0.4 mg, Fe (as ferrous sulfate) 60 mg, Cu (as copper sulfate) 8 mg, Se (sodium selenite) 0.30 mg. 2Analyzed values, each value based on triplicate determinations. View Large Table 1. Composition and nutrient levels of the basal diet (air-dry basis). Ingredients  %  Nutrients  %  Corn  58.50  ME (MJ/kg)  12.29  Soybean meal  23.00  Crude protein2  15.47  Ground limestone  8.74  Calcium2  3.60  CaHPO4  1.10  Total phosphorus  0.62  Wheat bran  7.50  Available phosphorus  0.32  Soybean oil  0.50  Lys  0.94  NaCl  0.24  Met  0.44  Met  0.16  Thr  0.70  Thr  0.03  Zinc2 (mg/kg)  35.08  Lys  0.05      Premix1  0.18      Total  100.00      Ingredients  %  Nutrients  %  Corn  58.50  ME (MJ/kg)  12.29  Soybean meal  23.00  Crude protein2  15.47  Ground limestone  8.74  Calcium2  3.60  CaHPO4  1.10  Total phosphorus  0.62  Wheat bran  7.50  Available phosphorus  0.32  Soybean oil  0.50  Lys  0.94  NaCl  0.24  Met  0.44  Met  0.16  Thr  0.70  Thr  0.03  Zinc2 (mg/kg)  35.08  Lys  0.05      Premix1  0.18      Total  100.00      1Supplied the following per kilogram of diet: Vitamin A 10,000 IU, VD3 31, 800 IU, VE 10 IU, VK 10 mg, VB125 μg, thiamine l mg, riboflavin 4.5 mg, calcium pantothenate 50 mg, niacin 24.5 mg, pyridoxine 5 mg, biotin 1 mg, folic acid 1 mg, choline 500 mg, Mn (as manganese sulfate) 60 mg, I (as calcium iodate) 0.4 mg, Fe (as ferrous sulfate) 60 mg, Cu (as copper sulfate) 8 mg, Se (sodium selenite) 0.30 mg. 2Analyzed values, each value based on triplicate determinations. View Large The inorganic trace elements in the diet were ZnSO4 (Zn ≥ 34.5%), CuSO4 (Cu ≥ 25.0%), FeSO4 (Fe ≥ 30.0%), MnSO4 (Mn ≥ 32.0%), Na2SeO3 (S ≥ 1.0%), and Ca (IO3)2 (I ≥ 1.0%). The organic Zn was MHA-Zn (MINTREX), purchased from Novus International Trading (Shanghai) Company. The actual concentrations of the total Zn analyzed in the 4 treatment diets are shown in Table 2. Table 2. Concentrations of Zn in experimental treatment diets (mg/kg).1 Dietary zinc supplementation  Calculated  Analyzed1  80 mg/kg ZnSO4  115.08  115.53  20 mg/kg MHA-Zn2  55.08  55.38  40 mg/kg MHA-Zn  75.08  75.97  80 mg/kg MHA-Zn  115.08  114.18  Dietary zinc supplementation  Calculated  Analyzed1  80 mg/kg ZnSO4  115.08  115.53  20 mg/kg MHA-Zn2  55.08  55.38  40 mg/kg MHA-Zn  75.08  75.97  80 mg/kg MHA-Zn  115.08  114.18  1Value based on triplicate determinations. 2MHA-Zn: methionine hydroxy analog chelate zinc, methionine yielded 80%. View Large Table 2. Concentrations of Zn in experimental treatment diets (mg/kg).1 Dietary zinc supplementation  Calculated  Analyzed1  80 mg/kg ZnSO4  115.08  115.53  20 mg/kg MHA-Zn2  55.08  55.38  40 mg/kg MHA-Zn  75.08  75.97  80 mg/kg MHA-Zn  115.08  114.18  Dietary zinc supplementation  Calculated  Analyzed1  80 mg/kg ZnSO4  115.08  115.53  20 mg/kg MHA-Zn2  55.08  55.38  40 mg/kg MHA-Zn  75.08  75.97  80 mg/kg MHA-Zn  115.08  114.18  1Value based on triplicate determinations. 2MHA-Zn: methionine hydroxy analog chelate zinc, methionine yielded 80%. View Large Chemical Analysis To analyze the dietary Zn concentration, a sample from each treatment was prepared according to Mabe et al. (2003). The Zn concentrations in diets were measured using flame atomic absorption spectrophotometry (No. 968.08). DM (No. 934.01), CP (No. 976.05), calcium (927.02), and phosphorus (No. 965.17) were determined using standard procedures from Association of Official Analytical Chemists (AOAC). Laying Performance and Eggshell Quality The number of eggs produced and the number of broken eggs were recorded every day. The amount of feed provided and residual feed were measured weekly. The laying rate, average egg weight, average daily feed intake(DFI), feed conversion rate (FCR), and the broken egg rate were calculated based on the records every 7 wk. At the end of 60, 62, 64, 66, 68, 70, and 72 wk, 24 eggs from each treatment group (3 eggs/replicate, 8 replicates) were obtained for the measurement of egg weight, eggshell weight, eggshell strength, and eggshell thickness. Egg weight and eggshell weight were measured using electronic balance (accuracy, 0.01 g). Eggshell strength and eggshell thickness were measured using eggshell strength tester (EFG-0503, Robotmation, Japan) and eggshell thickness tester (ETG-1601A, Robotmation, Japan), respectively. Eggshell weight percent and eggshell density were calculated using the following formulas: The eggshell weight percent was calculated as eggshell weight/egg weight × 100; eggshell density (g/100 cm2) was calculated as I = (C/S) × 100, where C is eggshell weight (g) and S (cm2) is shell surface (S = 4.68 × P2/3, where P is egg weight [g]) (Gheisar et al., 2010). Eggshell Mineral Deposit At the end of week 62, 66, 70, and 72, 3 eggs from each replicate with 8 replicates per treatment were taken. Washing eggshell membrane, dry at 80°C overnight, weighed the eggshell. The Zn concentrations in eggshell were measured using flame atomic absorption spectrophotometry (No. 968.08). Calcium (927.02) and phosphorus (No. 965.17) were determined using standard procedures from AOAC. Zn-containing Enzyme Activity At the end of the trial (72 wk), 1 bird from each replicate was chosen according to body weight. Those birds were sacrificed. The liver was taken and frozen in liquid nitrogen and then stored at –80°C. Frozen livers were homogenized and then centrifuged at 4000 r/min for 10 min at 4°C, and the supernatant was stored at –80°C until analysis. All samples were measured in triplicate, at the appropriate dilutions, to obtain enzyme activities in the linear range of standard curves constructed with pure enzymes. The protein concentration of supernatants was determined using a Coomassie Brilliant Blue kit assay with BSA as the standard. Meanwhile, CA and AKP activities were measured using CA assay kits and AKP assay kits. These 3 kits were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. The CA and AKP activities were assayed spectrophotometrically using colorimetric methods (Bio-Tek). Statistical Analysis Data were analyzed by one-way ANOVA using SPSS 21.0 (SPSS software for Windows, release 22.0, SPSS Inc., Chicago, IL). Duncan's test was used for multiple comparisons, and a P-value of 0.05 or less was declared significant. RESULTS Production Performance Production performance data are listed in Table 3. During the entire trial, neither dietary Zn levels nor sources affected the laying rate, average egg weight, average DFI, and FCR (P > 0.05). In addition, layers fed 40 mg/kg MHA-Zn exhibited the lowest FCR, but no significant differences were observed between the treatments. Moreover, the results indicated that the broken egg rate decreased with the increasing of MHA-Zn supplementation. In addition, at the same Zn level, the broken egg rate was significantly lower in the organic group than in the inorganic group (P < 0.05). Table 3. Effects of Zn on production performance of aged laying hens at the end of 66 and 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Laying rate (%)  Week 59 to 66  85.88 ± 0.82  86.10 ± 0.85  86.15 ± 0.78  86.13 ± 1.28  0.934  Week 67 to 72  82.31 ± 1.18  82.65 ± 1.47  82.97 ± 1.43  82.64 ± 1.24  0.804  Average egg weight (g)  Week 59 to 66  64.94 ± 0.36  64.89 ± 0.38  65.01 ± 0.58  64.91 ± 0.43  0.954  Week 67 to 72  66.19 ± 0.49  66.10 ± 0.52  66.17 ± 0.53  66.26 ± 0.59  0.941  Average daily feed intake (g)  Week 59 to 66  121.18 ± 0.48  121.34 ± 0.43  121.22 ± 0.50  121.25 ± 0.48  0.918  Week 67 to 72  121.15 ± 1.21  120.71 ± 1.28  120.53 ± 0.83  121.03 ± 1.34  0.716  Feed conversion rate  Week 59 to 66  2.17 ± 0.02  2.17 ± 0.03  2.16 ± 0.03  2.17 ± 0.04  0.942  Week 67 to 72  2.22 ± 0.04  2.21 ± 0.04  2.20 ± 0.05  2.21 ± 0.04  0.604  Broken egg rate (%)  Week 59 to 66  2.77 ± 0.18a  2.83 ± 0.18a  2.17 ± 0.27b  2.14 ± 0.30b  <0.001  Week 67 to 72  3.75 ± 0.40a  3.87 ± 0.38a  2.88 ± 0.40b  2.86 ± 0.23b  <0.001  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Laying rate (%)  Week 59 to 66  85.88 ± 0.82  86.10 ± 0.85  86.15 ± 0.78  86.13 ± 1.28  0.934  Week 67 to 72  82.31 ± 1.18  82.65 ± 1.47  82.97 ± 1.43  82.64 ± 1.24  0.804  Average egg weight (g)  Week 59 to 66  64.94 ± 0.36  64.89 ± 0.38  65.01 ± 0.58  64.91 ± 0.43  0.954  Week 67 to 72  66.19 ± 0.49  66.10 ± 0.52  66.17 ± 0.53  66.26 ± 0.59  0.941  Average daily feed intake (g)  Week 59 to 66  121.18 ± 0.48  121.34 ± 0.43  121.22 ± 0.50  121.25 ± 0.48  0.918  Week 67 to 72  121.15 ± 1.21  120.71 ± 1.28  120.53 ± 0.83  121.03 ± 1.34  0.716  Feed conversion rate  Week 59 to 66  2.17 ± 0.02  2.17 ± 0.03  2.16 ± 0.03  2.17 ± 0.04  0.942  Week 67 to 72  2.22 ± 0.04  2.21 ± 0.04  2.20 ± 0.05  2.21 ± 0.04  0.604  Broken egg rate (%)  Week 59 to 66  2.77 ± 0.18a  2.83 ± 0.18a  2.17 ± 0.27b  2.14 ± 0.30b  <0.001  Week 67 to 72  3.75 ± 0.40a  3.87 ± 0.38a  2.88 ± 0.40b  2.86 ± 0.23b  <0.001  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a,bMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc. View Large Table 3. Effects of Zn on production performance of aged laying hens at the end of 66 and 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Laying rate (%)  Week 59 to 66  85.88 ± 0.82  86.10 ± 0.85  86.15 ± 0.78  86.13 ± 1.28  0.934  Week 67 to 72  82.31 ± 1.18  82.65 ± 1.47  82.97 ± 1.43  82.64 ± 1.24  0.804  Average egg weight (g)  Week 59 to 66  64.94 ± 0.36  64.89 ± 0.38  65.01 ± 0.58  64.91 ± 0.43  0.954  Week 67 to 72  66.19 ± 0.49  66.10 ± 0.52  66.17 ± 0.53  66.26 ± 0.59  0.941  Average daily feed intake (g)  Week 59 to 66  121.18 ± 0.48  121.34 ± 0.43  121.22 ± 0.50  121.25 ± 0.48  0.918  Week 67 to 72  121.15 ± 1.21  120.71 ± 1.28  120.53 ± 0.83  121.03 ± 1.34  0.716  Feed conversion rate  Week 59 to 66  2.17 ± 0.02  2.17 ± 0.03  2.16 ± 0.03  2.17 ± 0.04  0.942  Week 67 to 72  2.22 ± 0.04  2.21 ± 0.04  2.20 ± 0.05  2.21 ± 0.04  0.604  Broken egg rate (%)  Week 59 to 66  2.77 ± 0.18a  2.83 ± 0.18a  2.17 ± 0.27b  2.14 ± 0.30b  <0.001  Week 67 to 72  3.75 ± 0.40a  3.87 ± 0.38a  2.88 ± 0.40b  2.86 ± 0.23b  <0.001  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Laying rate (%)  Week 59 to 66  85.88 ± 0.82  86.10 ± 0.85  86.15 ± 0.78  86.13 ± 1.28  0.934  Week 67 to 72  82.31 ± 1.18  82.65 ± 1.47  82.97 ± 1.43  82.64 ± 1.24  0.804  Average egg weight (g)  Week 59 to 66  64.94 ± 0.36  64.89 ± 0.38  65.01 ± 0.58  64.91 ± 0.43  0.954  Week 67 to 72  66.19 ± 0.49  66.10 ± 0.52  66.17 ± 0.53  66.26 ± 0.59  0.941  Average daily feed intake (g)  Week 59 to 66  121.18 ± 0.48  121.34 ± 0.43  121.22 ± 0.50  121.25 ± 0.48  0.918  Week 67 to 72  121.15 ± 1.21  120.71 ± 1.28  120.53 ± 0.83  121.03 ± 1.34  0.716  Feed conversion rate  Week 59 to 66  2.17 ± 0.02  2.17 ± 0.03  2.16 ± 0.03  2.17 ± 0.04  0.942  Week 67 to 72  2.22 ± 0.04  2.21 ± 0.04  2.20 ± 0.05  2.21 ± 0.04  0.604  Broken egg rate (%)  Week 59 to 66  2.77 ± 0.18a  2.83 ± 0.18a  2.17 ± 0.27b  2.14 ± 0.30b  <0.001  Week 67 to 72  3.75 ± 0.40a  3.87 ± 0.38a  2.88 ± 0.40b  2.86 ± 0.23b  <0.001  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a,bMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc. View Large Eggshell Quality Eggshell quality was shown in Table 4. Compared to inorganic group, groups with organic Zn did not influenced eggshell weight at the end of 60 wk; however, dietary supplementation with 40 or 80 mg/kg MHA-Zn significantly improved eggshell weight in week 62 to 72. Besides, the results showed that eggshell strength and eggshell thickness were not significantly affected by different levels and sources in week 60 to 64, whereas dietary supplementation with 40 or 80 mg/kg MHA-Zn exhibited higher eggshell strength in week 66 to 72. Dietary supplementation with 20 mg/kg MHA-Zn got the lowest eggshell thickness and eggshell strength, but the difference was not remarkable. Moreover, dietary supplementation with 40 or 80 mg/kg MHA-Zn significantly improved the eggshell weight percent and eggshell density (P < 0.05) compared with the control group in week 62 to 72. However, dietary supplementation with 20 mg/kg MHA-Zn did not exhibit a substantial difference. Table 4. Effects of Zn on the eggshell quality of aged laying hens at the end of 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Eggshell weight (g)  Week 60  7.82 ± 0.27a,b  7.75 ± 0.21b  8.04 ± 0.38a,b  8.13 ± 0.45a  0.081  Week 62  7.64 ± 0.38b  7.66 ± 0.29b  8.15 ± 0.25a  8.07 ± 0.32a  0.003  Week 64  7.41 ± 0.50b  7.40 ± 0.30b  7.91 ± 0.26a  7.92 ± 0.25a  0.003  Week 66  7.36 ± 0.40b  7.29 ± 0.23b  7.84 ± 0.40a  7.79 ± 0.40a  0.007  Week 68  7.28 ± 0.30b  7.29 ± 0.45b  7.78 ± 0.38a  7.81 ± 0.39a  0.009  Week 70  7.12 ± 0.35b  7.11 ± 0.40b  7.70 ± 0.48a  7.71 ± 0.64a  0.017  Week 72  6.89 ± 0.29b  6.90 ± 0.27b  7.58 ± 0.38a  7.54 ± 0.63a  0.001  Eggshell thickness (mm)  Week 60  0.402 ± 0.019  0.392 ± 0.005  0.401 ± 0.016  0.402 ± 0.018  0.484  Week 62  0.377 ± 0.016  0.373 ± 0.010  0.381 ± 0.010  0.385 ± 0.011  0.249  Week 64  0.362 ± 0.010  0.360 ± 0.013  0.372 ± 0.014  0.374 ± 0.012  0.080  Week 66  0.353 ± 0.014b  0.351 ± 0.014b  0.370 ± 0.021a  0.373 ± 0.019a  0.025  Week 68  0.336 ± 0.014b  0.335 ± 0.013b  0.362 ± 0.008a  0.364 ± 0.017a  <0.001  Week 70  0.324 ± 0.008b  0.321 ± 0.016b  0.365 ± 0.012a  0.363 ± 0.016a  <0.001  Week 72  0.308 ± 0.012b  0.306 ± 0.011b  0.352 ± 0.012a  0.354 ± 0.011a  <0.001  Eggshell strength (N)  Week 60  4.25 ± 0.19  4.21 ± 0.26  4.25 ± 0.22  4.24 ± 0.10  0.979  Week 62  3.96 ± 0.20  3.91 ± 0.13  4.02 ± 0.28  4.05 ± 0.31  0.634  Week 64  3.65 ± 0.24a,b  3.63 ± 0.30b  3.99 ± 0.30a  3.98 ± 0.18a,b  0.010  Week 66  3.50 ± 0.23b  3.50 ± 0.13b  3.83 ± 0.23a  3.80 ± 0.27a  0.003  Week 68  3.43 ± 0.22b  3.44 ± 0.30b  3.79 ± 0.21a,b  3.83 ± 0.31a  0.005  Week 70  3.32 ± 0.27b  3.26 ± 0.24b  3.66 ± 0.16a  3.63 ± 0.20a  0.001  Week 72  3.27 ± 0.12b  3.29 ± 0.29b  3.57 ± 0.17a  3.57 ± 0.15a  0.001  Eggshell weight percent (%)  Week 60  12.11 ± 0.41a,b  12.01 ± 0.36b  12.45 ± 0.59a,b  12.64 ± 0.60a  0.071  Week 62  11.78 ± 0.52b  11.83 ± 0.41b  12.53 ± 0.31a  12.41 ± 0.48a  0.002  Week 64  11.37 ± 0.80b  11.35 ± 0.49b  12.14 ± 0.48a  12.12 ± 0.46a  0.008  Week 66  11.17 ± 0.60b  11.16 ± 0.50b  11.91 ± 0.51a  11.87 ± 0.39a  0.004  Week 68  10.98 ± 0.42b  10.99 ± 0.66b  11.71 ± 0.62a  11.78 ± 0.73a  0.016  Week 70  10.67 ± 0.56b  10.67 ± 0.73b  11.55 ± 0.56a  11.59 ± 0.98a  0.016  Week 72  10.35 ± 0.33b  10.37 ± 0.52b  11.39 ± 0.64a  11.26 ± 0.58a  <0.001  Eggshell density (%)  Week 60  10.38 ± 0.35a,b  10.29 ± 0.30b  10.67 ± 0.50a,b  10.82 ± 0.52a  0.076  Week 62  10.11 ± 0.50b  10.15 ± 0.36b  10.77 ± 0.28a  10.66 ± 0.40a  0.003  Week 64  9.77 ± 0.68b  9.76 ± 0.40b  10.44 ± 0.38a  10.43 ± 0.35a  0.005  Week 66  9.64 ± 0.50b  9.60 ± 0.37b  10.28 ± 0.45a  10.23 ± 0.38a  0.003  Week 68  9.49 ± 0.37b  9.51 ± 0.57b  10.13 ± 0.52a  10.19 ± 0.58a  0.012  Week 70  9.24 ± 0.45b  9.24 ± 0.59b  10.01 ± 0.52a  10.03 ± 0.83a  0.014  Week 72  8.96 ± 0.31b  8.98 ± 0.41b  9.86 ± 0.52a  9.76 ± 0.61a  <0.001  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Eggshell weight (g)  Week 60  7.82 ± 0.27a,b  7.75 ± 0.21b  8.04 ± 0.38a,b  8.13 ± 0.45a  0.081  Week 62  7.64 ± 0.38b  7.66 ± 0.29b  8.15 ± 0.25a  8.07 ± 0.32a  0.003  Week 64  7.41 ± 0.50b  7.40 ± 0.30b  7.91 ± 0.26a  7.92 ± 0.25a  0.003  Week 66  7.36 ± 0.40b  7.29 ± 0.23b  7.84 ± 0.40a  7.79 ± 0.40a  0.007  Week 68  7.28 ± 0.30b  7.29 ± 0.45b  7.78 ± 0.38a  7.81 ± 0.39a  0.009  Week 70  7.12 ± 0.35b  7.11 ± 0.40b  7.70 ± 0.48a  7.71 ± 0.64a  0.017  Week 72  6.89 ± 0.29b  6.90 ± 0.27b  7.58 ± 0.38a  7.54 ± 0.63a  0.001  Eggshell thickness (mm)  Week 60  0.402 ± 0.019  0.392 ± 0.005  0.401 ± 0.016  0.402 ± 0.018  0.484  Week 62  0.377 ± 0.016  0.373 ± 0.010  0.381 ± 0.010  0.385 ± 0.011  0.249  Week 64  0.362 ± 0.010  0.360 ± 0.013  0.372 ± 0.014  0.374 ± 0.012  0.080  Week 66  0.353 ± 0.014b  0.351 ± 0.014b  0.370 ± 0.021a  0.373 ± 0.019a  0.025  Week 68  0.336 ± 0.014b  0.335 ± 0.013b  0.362 ± 0.008a  0.364 ± 0.017a  <0.001  Week 70  0.324 ± 0.008b  0.321 ± 0.016b  0.365 ± 0.012a  0.363 ± 0.016a  <0.001  Week 72  0.308 ± 0.012b  0.306 ± 0.011b  0.352 ± 0.012a  0.354 ± 0.011a  <0.001  Eggshell strength (N)  Week 60  4.25 ± 0.19  4.21 ± 0.26  4.25 ± 0.22  4.24 ± 0.10  0.979  Week 62  3.96 ± 0.20  3.91 ± 0.13  4.02 ± 0.28  4.05 ± 0.31  0.634  Week 64  3.65 ± 0.24a,b  3.63 ± 0.30b  3.99 ± 0.30a  3.98 ± 0.18a,b  0.010  Week 66  3.50 ± 0.23b  3.50 ± 0.13b  3.83 ± 0.23a  3.80 ± 0.27a  0.003  Week 68  3.43 ± 0.22b  3.44 ± 0.30b  3.79 ± 0.21a,b  3.83 ± 0.31a  0.005  Week 70  3.32 ± 0.27b  3.26 ± 0.24b  3.66 ± 0.16a  3.63 ± 0.20a  0.001  Week 72  3.27 ± 0.12b  3.29 ± 0.29b  3.57 ± 0.17a  3.57 ± 0.15a  0.001  Eggshell weight percent (%)  Week 60  12.11 ± 0.41a,b  12.01 ± 0.36b  12.45 ± 0.59a,b  12.64 ± 0.60a  0.071  Week 62  11.78 ± 0.52b  11.83 ± 0.41b  12.53 ± 0.31a  12.41 ± 0.48a  0.002  Week 64  11.37 ± 0.80b  11.35 ± 0.49b  12.14 ± 0.48a  12.12 ± 0.46a  0.008  Week 66  11.17 ± 0.60b  11.16 ± 0.50b  11.91 ± 0.51a  11.87 ± 0.39a  0.004  Week 68  10.98 ± 0.42b  10.99 ± 0.66b  11.71 ± 0.62a  11.78 ± 0.73a  0.016  Week 70  10.67 ± 0.56b  10.67 ± 0.73b  11.55 ± 0.56a  11.59 ± 0.98a  0.016  Week 72  10.35 ± 0.33b  10.37 ± 0.52b  11.39 ± 0.64a  11.26 ± 0.58a  <0.001  Eggshell density (%)  Week 60  10.38 ± 0.35a,b  10.29 ± 0.30b  10.67 ± 0.50a,b  10.82 ± 0.52a  0.076  Week 62  10.11 ± 0.50b  10.15 ± 0.36b  10.77 ± 0.28a  10.66 ± 0.40a  0.003  Week 64  9.77 ± 0.68b  9.76 ± 0.40b  10.44 ± 0.38a  10.43 ± 0.35a  0.005  Week 66  9.64 ± 0.50b  9.60 ± 0.37b  10.28 ± 0.45a  10.23 ± 0.38a  0.003  Week 68  9.49 ± 0.37b  9.51 ± 0.57b  10.13 ± 0.52a  10.19 ± 0.58a  0.012  Week 70  9.24 ± 0.45b  9.24 ± 0.59b  10.01 ± 0.52a  10.03 ± 0.83a  0.014  Week 72  8.96 ± 0.31b  8.98 ± 0.41b  9.86 ± 0.52a  9.76 ± 0.61a  <0.001  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a,bMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc. View Large Table 4. Effects of Zn on the eggshell quality of aged laying hens at the end of 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Eggshell weight (g)  Week 60  7.82 ± 0.27a,b  7.75 ± 0.21b  8.04 ± 0.38a,b  8.13 ± 0.45a  0.081  Week 62  7.64 ± 0.38b  7.66 ± 0.29b  8.15 ± 0.25a  8.07 ± 0.32a  0.003  Week 64  7.41 ± 0.50b  7.40 ± 0.30b  7.91 ± 0.26a  7.92 ± 0.25a  0.003  Week 66  7.36 ± 0.40b  7.29 ± 0.23b  7.84 ± 0.40a  7.79 ± 0.40a  0.007  Week 68  7.28 ± 0.30b  7.29 ± 0.45b  7.78 ± 0.38a  7.81 ± 0.39a  0.009  Week 70  7.12 ± 0.35b  7.11 ± 0.40b  7.70 ± 0.48a  7.71 ± 0.64a  0.017  Week 72  6.89 ± 0.29b  6.90 ± 0.27b  7.58 ± 0.38a  7.54 ± 0.63a  0.001  Eggshell thickness (mm)  Week 60  0.402 ± 0.019  0.392 ± 0.005  0.401 ± 0.016  0.402 ± 0.018  0.484  Week 62  0.377 ± 0.016  0.373 ± 0.010  0.381 ± 0.010  0.385 ± 0.011  0.249  Week 64  0.362 ± 0.010  0.360 ± 0.013  0.372 ± 0.014  0.374 ± 0.012  0.080  Week 66  0.353 ± 0.014b  0.351 ± 0.014b  0.370 ± 0.021a  0.373 ± 0.019a  0.025  Week 68  0.336 ± 0.014b  0.335 ± 0.013b  0.362 ± 0.008a  0.364 ± 0.017a  <0.001  Week 70  0.324 ± 0.008b  0.321 ± 0.016b  0.365 ± 0.012a  0.363 ± 0.016a  <0.001  Week 72  0.308 ± 0.012b  0.306 ± 0.011b  0.352 ± 0.012a  0.354 ± 0.011a  <0.001  Eggshell strength (N)  Week 60  4.25 ± 0.19  4.21 ± 0.26  4.25 ± 0.22  4.24 ± 0.10  0.979  Week 62  3.96 ± 0.20  3.91 ± 0.13  4.02 ± 0.28  4.05 ± 0.31  0.634  Week 64  3.65 ± 0.24a,b  3.63 ± 0.30b  3.99 ± 0.30a  3.98 ± 0.18a,b  0.010  Week 66  3.50 ± 0.23b  3.50 ± 0.13b  3.83 ± 0.23a  3.80 ± 0.27a  0.003  Week 68  3.43 ± 0.22b  3.44 ± 0.30b  3.79 ± 0.21a,b  3.83 ± 0.31a  0.005  Week 70  3.32 ± 0.27b  3.26 ± 0.24b  3.66 ± 0.16a  3.63 ± 0.20a  0.001  Week 72  3.27 ± 0.12b  3.29 ± 0.29b  3.57 ± 0.17a  3.57 ± 0.15a  0.001  Eggshell weight percent (%)  Week 60  12.11 ± 0.41a,b  12.01 ± 0.36b  12.45 ± 0.59a,b  12.64 ± 0.60a  0.071  Week 62  11.78 ± 0.52b  11.83 ± 0.41b  12.53 ± 0.31a  12.41 ± 0.48a  0.002  Week 64  11.37 ± 0.80b  11.35 ± 0.49b  12.14 ± 0.48a  12.12 ± 0.46a  0.008  Week 66  11.17 ± 0.60b  11.16 ± 0.50b  11.91 ± 0.51a  11.87 ± 0.39a  0.004  Week 68  10.98 ± 0.42b  10.99 ± 0.66b  11.71 ± 0.62a  11.78 ± 0.73a  0.016  Week 70  10.67 ± 0.56b  10.67 ± 0.73b  11.55 ± 0.56a  11.59 ± 0.98a  0.016  Week 72  10.35 ± 0.33b  10.37 ± 0.52b  11.39 ± 0.64a  11.26 ± 0.58a  <0.001  Eggshell density (%)  Week 60  10.38 ± 0.35a,b  10.29 ± 0.30b  10.67 ± 0.50a,b  10.82 ± 0.52a  0.076  Week 62  10.11 ± 0.50b  10.15 ± 0.36b  10.77 ± 0.28a  10.66 ± 0.40a  0.003  Week 64  9.77 ± 0.68b  9.76 ± 0.40b  10.44 ± 0.38a  10.43 ± 0.35a  0.005  Week 66  9.64 ± 0.50b  9.60 ± 0.37b  10.28 ± 0.45a  10.23 ± 0.38a  0.003  Week 68  9.49 ± 0.37b  9.51 ± 0.57b  10.13 ± 0.52a  10.19 ± 0.58a  0.012  Week 70  9.24 ± 0.45b  9.24 ± 0.59b  10.01 ± 0.52a  10.03 ± 0.83a  0.014  Week 72  8.96 ± 0.31b  8.98 ± 0.41b  9.86 ± 0.52a  9.76 ± 0.61a  <0.001  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Eggshell weight (g)  Week 60  7.82 ± 0.27a,b  7.75 ± 0.21b  8.04 ± 0.38a,b  8.13 ± 0.45a  0.081  Week 62  7.64 ± 0.38b  7.66 ± 0.29b  8.15 ± 0.25a  8.07 ± 0.32a  0.003  Week 64  7.41 ± 0.50b  7.40 ± 0.30b  7.91 ± 0.26a  7.92 ± 0.25a  0.003  Week 66  7.36 ± 0.40b  7.29 ± 0.23b  7.84 ± 0.40a  7.79 ± 0.40a  0.007  Week 68  7.28 ± 0.30b  7.29 ± 0.45b  7.78 ± 0.38a  7.81 ± 0.39a  0.009  Week 70  7.12 ± 0.35b  7.11 ± 0.40b  7.70 ± 0.48a  7.71 ± 0.64a  0.017  Week 72  6.89 ± 0.29b  6.90 ± 0.27b  7.58 ± 0.38a  7.54 ± 0.63a  0.001  Eggshell thickness (mm)  Week 60  0.402 ± 0.019  0.392 ± 0.005  0.401 ± 0.016  0.402 ± 0.018  0.484  Week 62  0.377 ± 0.016  0.373 ± 0.010  0.381 ± 0.010  0.385 ± 0.011  0.249  Week 64  0.362 ± 0.010  0.360 ± 0.013  0.372 ± 0.014  0.374 ± 0.012  0.080  Week 66  0.353 ± 0.014b  0.351 ± 0.014b  0.370 ± 0.021a  0.373 ± 0.019a  0.025  Week 68  0.336 ± 0.014b  0.335 ± 0.013b  0.362 ± 0.008a  0.364 ± 0.017a  <0.001  Week 70  0.324 ± 0.008b  0.321 ± 0.016b  0.365 ± 0.012a  0.363 ± 0.016a  <0.001  Week 72  0.308 ± 0.012b  0.306 ± 0.011b  0.352 ± 0.012a  0.354 ± 0.011a  <0.001  Eggshell strength (N)  Week 60  4.25 ± 0.19  4.21 ± 0.26  4.25 ± 0.22  4.24 ± 0.10  0.979  Week 62  3.96 ± 0.20  3.91 ± 0.13  4.02 ± 0.28  4.05 ± 0.31  0.634  Week 64  3.65 ± 0.24a,b  3.63 ± 0.30b  3.99 ± 0.30a  3.98 ± 0.18a,b  0.010  Week 66  3.50 ± 0.23b  3.50 ± 0.13b  3.83 ± 0.23a  3.80 ± 0.27a  0.003  Week 68  3.43 ± 0.22b  3.44 ± 0.30b  3.79 ± 0.21a,b  3.83 ± 0.31a  0.005  Week 70  3.32 ± 0.27b  3.26 ± 0.24b  3.66 ± 0.16a  3.63 ± 0.20a  0.001  Week 72  3.27 ± 0.12b  3.29 ± 0.29b  3.57 ± 0.17a  3.57 ± 0.15a  0.001  Eggshell weight percent (%)  Week 60  12.11 ± 0.41a,b  12.01 ± 0.36b  12.45 ± 0.59a,b  12.64 ± 0.60a  0.071  Week 62  11.78 ± 0.52b  11.83 ± 0.41b  12.53 ± 0.31a  12.41 ± 0.48a  0.002  Week 64  11.37 ± 0.80b  11.35 ± 0.49b  12.14 ± 0.48a  12.12 ± 0.46a  0.008  Week 66  11.17 ± 0.60b  11.16 ± 0.50b  11.91 ± 0.51a  11.87 ± 0.39a  0.004  Week 68  10.98 ± 0.42b  10.99 ± 0.66b  11.71 ± 0.62a  11.78 ± 0.73a  0.016  Week 70  10.67 ± 0.56b  10.67 ± 0.73b  11.55 ± 0.56a  11.59 ± 0.98a  0.016  Week 72  10.35 ± 0.33b  10.37 ± 0.52b  11.39 ± 0.64a  11.26 ± 0.58a  <0.001  Eggshell density (%)  Week 60  10.38 ± 0.35a,b  10.29 ± 0.30b  10.67 ± 0.50a,b  10.82 ± 0.52a  0.076  Week 62  10.11 ± 0.50b  10.15 ± 0.36b  10.77 ± 0.28a  10.66 ± 0.40a  0.003  Week 64  9.77 ± 0.68b  9.76 ± 0.40b  10.44 ± 0.38a  10.43 ± 0.35a  0.005  Week 66  9.64 ± 0.50b  9.60 ± 0.37b  10.28 ± 0.45a  10.23 ± 0.38a  0.003  Week 68  9.49 ± 0.37b  9.51 ± 0.57b  10.13 ± 0.52a  10.19 ± 0.58a  0.012  Week 70  9.24 ± 0.45b  9.24 ± 0.59b  10.01 ± 0.52a  10.03 ± 0.83a  0.014  Week 72  8.96 ± 0.31b  8.98 ± 0.41b  9.86 ± 0.52a  9.76 ± 0.61a  <0.001  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a,bMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc. View Large Eggshell Mineral Deposit The results of eggshell mineral deposit were shown in Table 5. The concentration of ash in eggshells was not affected by dietary supplementation or the source of Zn in the whole period. Compared with inorganic group, dietary supplementation with 80 mg/kg MHA-Zn improved the Ca concentration in eggshells at week 62, 70, and 72, and Zn concentration in eggshell at week 66, 70, and 72. On the other hand, dietary supplementation with 40 mg/kg MHA-Zn increased Zn and Ca concentration in liver, whereas group with 20 mg/kg MHA-Zn decreased Zn and Ca concentration but the difference was not significant. Moreover, P concentration in liver was not influenced by Zn level and source during the entire trial. Table 5. Effects of Zn on the mineral content in eggshell of aged laying hens.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Ash content of eggshell (%)  Week 62  94.39 ± 1.28  94.16 ± 1.55  93.04 ± 3.88  92.21 ± 2.86  0.325  Week 66  93.78 ± 1.14  92.65 ± 2.43  93.85 ± 0.95  93.26 ± 0.61  0.340  Week 70  93.21 ± 1.26  93.33 ± 0.99  93.05 ± 1.66  93.14 ± 2.05  0.986  Week 72  93.31 ± 0.84  93.04 ± 0.96  92.99 ± 0.99  93.64 ± 0.79  0.473  Ca content of eggshell (%)  Week 62  37.28 ± 1.70b  37.42 ± 2.36b  38.86 ± 0.95a,b  39.21 ± 0.96a  0.046  Week 66  37.14 ± 2.18  36.68 ± 2.50  38.32 ± 1.98  39.02 ± 0.89  0.098  Week 70  37.42 ± 1.06a,b  36.70 ± 1.65b  37.97 ± 1.07a,b  38.74 ± 1.47a  0.039  Week 72  37.17 ± 1.58b  36.71 ± 1.03b  37.18 ± 1.58a  38.70 ± 1.11a  0.048  P content of eggshell (%)  Week 62  1.06 ± 0.07  1.03 ± 0.09  1.05 ± 0.03  1.04 ± 0.09  0.817  Week 66  1.01 ± 0.05  0.96 ± 0.08  1.01 ± 0.03  1.02 ± 0.06  0.214  Week 70  0.99 ± 0.03  0.99 ± 0.03  1.00 ± 0.04  0.99 ± 0.05  0.988  Week 72  0.99 ± 0.04a  0.95 ± 0.02b  0.98 ± 0.01a,b  1.00 ± 0.05a  0.011  Zn content of eggshell (mg/kg)  Week 62  81.29 ± 7.22  80.00 ± 7.58  81.71 ± 6.35  82.23 ± 5.29  0.933  Week 66  70.26 ± 7.04b,c  68.98 ± 8.92c  76.95 ± 3.47a,b  80.91 ± 5.56a  0.006  Week 70  69.90 ± 8.88b,c  68.07 ± 7.81c  76.33 ± 3.70a,b  77.94 ± 3.66a  0.030  Week 72  65.56 ± 7.49b  64.50 ± 6.43b  75.23 ± 3.33a  73.25 ± 6.15a  0.005  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Ash content of eggshell (%)  Week 62  94.39 ± 1.28  94.16 ± 1.55  93.04 ± 3.88  92.21 ± 2.86  0.325  Week 66  93.78 ± 1.14  92.65 ± 2.43  93.85 ± 0.95  93.26 ± 0.61  0.340  Week 70  93.21 ± 1.26  93.33 ± 0.99  93.05 ± 1.66  93.14 ± 2.05  0.986  Week 72  93.31 ± 0.84  93.04 ± 0.96  92.99 ± 0.99  93.64 ± 0.79  0.473  Ca content of eggshell (%)  Week 62  37.28 ± 1.70b  37.42 ± 2.36b  38.86 ± 0.95a,b  39.21 ± 0.96a  0.046  Week 66  37.14 ± 2.18  36.68 ± 2.50  38.32 ± 1.98  39.02 ± 0.89  0.098  Week 70  37.42 ± 1.06a,b  36.70 ± 1.65b  37.97 ± 1.07a,b  38.74 ± 1.47a  0.039  Week 72  37.17 ± 1.58b  36.71 ± 1.03b  37.18 ± 1.58a  38.70 ± 1.11a  0.048  P content of eggshell (%)  Week 62  1.06 ± 0.07  1.03 ± 0.09  1.05 ± 0.03  1.04 ± 0.09  0.817  Week 66  1.01 ± 0.05  0.96 ± 0.08  1.01 ± 0.03  1.02 ± 0.06  0.214  Week 70  0.99 ± 0.03  0.99 ± 0.03  1.00 ± 0.04  0.99 ± 0.05  0.988  Week 72  0.99 ± 0.04a  0.95 ± 0.02b  0.98 ± 0.01a,b  1.00 ± 0.05a  0.011  Zn content of eggshell (mg/kg)  Week 62  81.29 ± 7.22  80.00 ± 7.58  81.71 ± 6.35  82.23 ± 5.29  0.933  Week 66  70.26 ± 7.04b,c  68.98 ± 8.92c  76.95 ± 3.47a,b  80.91 ± 5.56a  0.006  Week 70  69.90 ± 8.88b,c  68.07 ± 7.81c  76.33 ± 3.70a,b  77.94 ± 3.66a  0.030  Week 72  65.56 ± 7.49b  64.50 ± 6.43b  75.23 ± 3.33a  73.25 ± 6.15a  0.005  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a–cMeans with different superscripts within the same row are significantly different (P < 0.05). View Large Table 5. Effects of Zn on the mineral content in eggshell of aged laying hens.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Ash content of eggshell (%)  Week 62  94.39 ± 1.28  94.16 ± 1.55  93.04 ± 3.88  92.21 ± 2.86  0.325  Week 66  93.78 ± 1.14  92.65 ± 2.43  93.85 ± 0.95  93.26 ± 0.61  0.340  Week 70  93.21 ± 1.26  93.33 ± 0.99  93.05 ± 1.66  93.14 ± 2.05  0.986  Week 72  93.31 ± 0.84  93.04 ± 0.96  92.99 ± 0.99  93.64 ± 0.79  0.473  Ca content of eggshell (%)  Week 62  37.28 ± 1.70b  37.42 ± 2.36b  38.86 ± 0.95a,b  39.21 ± 0.96a  0.046  Week 66  37.14 ± 2.18  36.68 ± 2.50  38.32 ± 1.98  39.02 ± 0.89  0.098  Week 70  37.42 ± 1.06a,b  36.70 ± 1.65b  37.97 ± 1.07a,b  38.74 ± 1.47a  0.039  Week 72  37.17 ± 1.58b  36.71 ± 1.03b  37.18 ± 1.58a  38.70 ± 1.11a  0.048  P content of eggshell (%)  Week 62  1.06 ± 0.07  1.03 ± 0.09  1.05 ± 0.03  1.04 ± 0.09  0.817  Week 66  1.01 ± 0.05  0.96 ± 0.08  1.01 ± 0.03  1.02 ± 0.06  0.214  Week 70  0.99 ± 0.03  0.99 ± 0.03  1.00 ± 0.04  0.99 ± 0.05  0.988  Week 72  0.99 ± 0.04a  0.95 ± 0.02b  0.98 ± 0.01a,b  1.00 ± 0.05a  0.011  Zn content of eggshell (mg/kg)  Week 62  81.29 ± 7.22  80.00 ± 7.58  81.71 ± 6.35  82.23 ± 5.29  0.933  Week 66  70.26 ± 7.04b,c  68.98 ± 8.92c  76.95 ± 3.47a,b  80.91 ± 5.56a  0.006  Week 70  69.90 ± 8.88b,c  68.07 ± 7.81c  76.33 ± 3.70a,b  77.94 ± 3.66a  0.030  Week 72  65.56 ± 7.49b  64.50 ± 6.43b  75.23 ± 3.33a  73.25 ± 6.15a  0.005  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Ash content of eggshell (%)  Week 62  94.39 ± 1.28  94.16 ± 1.55  93.04 ± 3.88  92.21 ± 2.86  0.325  Week 66  93.78 ± 1.14  92.65 ± 2.43  93.85 ± 0.95  93.26 ± 0.61  0.340  Week 70  93.21 ± 1.26  93.33 ± 0.99  93.05 ± 1.66  93.14 ± 2.05  0.986  Week 72  93.31 ± 0.84  93.04 ± 0.96  92.99 ± 0.99  93.64 ± 0.79  0.473  Ca content of eggshell (%)  Week 62  37.28 ± 1.70b  37.42 ± 2.36b  38.86 ± 0.95a,b  39.21 ± 0.96a  0.046  Week 66  37.14 ± 2.18  36.68 ± 2.50  38.32 ± 1.98  39.02 ± 0.89  0.098  Week 70  37.42 ± 1.06a,b  36.70 ± 1.65b  37.97 ± 1.07a,b  38.74 ± 1.47a  0.039  Week 72  37.17 ± 1.58b  36.71 ± 1.03b  37.18 ± 1.58a  38.70 ± 1.11a  0.048  P content of eggshell (%)  Week 62  1.06 ± 0.07  1.03 ± 0.09  1.05 ± 0.03  1.04 ± 0.09  0.817  Week 66  1.01 ± 0.05  0.96 ± 0.08  1.01 ± 0.03  1.02 ± 0.06  0.214  Week 70  0.99 ± 0.03  0.99 ± 0.03  1.00 ± 0.04  0.99 ± 0.05  0.988  Week 72  0.99 ± 0.04a  0.95 ± 0.02b  0.98 ± 0.01a,b  1.00 ± 0.05a  0.011  Zn content of eggshell (mg/kg)  Week 62  81.29 ± 7.22  80.00 ± 7.58  81.71 ± 6.35  82.23 ± 5.29  0.933  Week 66  70.26 ± 7.04b,c  68.98 ± 8.92c  76.95 ± 3.47a,b  80.91 ± 5.56a  0.006  Week 70  69.90 ± 8.88b,c  68.07 ± 7.81c  76.33 ± 3.70a,b  77.94 ± 3.66a  0.030  Week 72  65.56 ± 7.49b  64.50 ± 6.43b  75.23 ± 3.33a  73.25 ± 6.15a  0.005  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a–cMeans with different superscripts within the same row are significantly different (P < 0.05). View Large Activities of Zn-containing Enzymes As shown in Table 6, both the dietary supplementation levels and sources of Zn significantly influenced CA activity (P < 0.05). With the increasing of MHA-Zn supplementation, the activity of CA increased accordingly. At equal levels of Zn supplementation, the group with organic Zn showed significantly improved CA activity. In addition, 40 mg/kg MHA-Zn also greatly increased CA activity. Moreover, we found that AKP activity increased with the increasing of Zn supplementation and dietary supplementation with 40 or 80 mg/kg MHA-Zn, the activity was higher than inorganic group. Table 6. Effects of Zn on CA and AKP activity in the livers of aged hens at the end of 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    CA (ng/mL)2  76.4 ± 1.35c  71.44 ± 3.10b  79.52 ± 1.20b  82.54 ± 2.47a  <0.001  AKP(U/gprot)2  43.70 ± 2.98a,b  40.89 ± 4.23b  43.94 ± 2.30a,b  46.22 ± 2.40a  0.064  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    CA (ng/mL)2  76.4 ± 1.35c  71.44 ± 3.10b  79.52 ± 1.20b  82.54 ± 2.47a  <0.001  AKP(U/gprot)2  43.70 ± 2.98a,b  40.89 ± 4.23b  43.94 ± 2.30a,b  46.22 ± 2.40a  0.064  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a–cMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc; CA: carbonic anhydrase; AKP: alkaline phosphatase. View Large Table 6. Effects of Zn on CA and AKP activity in the livers of aged hens at the end of 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    CA (ng/mL)2  76.4 ± 1.35c  71.44 ± 3.10b  79.52 ± 1.20b  82.54 ± 2.47a  <0.001  AKP(U/gprot)2  43.70 ± 2.98a,b  40.89 ± 4.23b  43.94 ± 2.30a,b  46.22 ± 2.40a  0.064  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    CA (ng/mL)2  76.4 ± 1.35c  71.44 ± 3.10b  79.52 ± 1.20b  82.54 ± 2.47a  <0.001  AKP(U/gprot)2  43.70 ± 2.98a,b  40.89 ± 4.23b  43.94 ± 2.30a,b  46.22 ± 2.40a  0.064  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a–cMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc; CA: carbonic anhydrase; AKP: alkaline phosphatase. View Large DISCUSSION Production Performance The present study showed that 40 mg/kg MHA-Zn can be used in diets for aged laying hens with no negative effects on production performance and can reduce the broken egg rate compared with dietary supplementation with 80 mg/kg ZnSO4. Similarly, Niyomdecha et al. (2008) found that the source of Zn did not affect laying rate, egg weight, DFI, and FCR. In addition, when the level of MHA-Zn was increased, the production performance did not improve. It seems that laying performance was not a sensitive criterion for assessing the dietary Zn requirement of laying hens fed a normal diet. We inferred that the main reason for this is that the Zn supplementation in the diet had met the layers’ requirement, so the production performance did not improve with increased Zn. However, some studies shown that organic Zn has more advantages than inorganic Zn in improving the production performance of hens (Skrivan et al., 2006; Bao et al., 2007; Jahanian et al., 2008; Sunder et al., 2008). The above inconsistencies might be due to differences of Zn level, experimental phase, or type of hens. Eggshell Quality Eggshell quality is an important indicator of the production efficiency of laying hens (Lien et al., 2004; Yousaf et al., 2009). The current study found that supplementation with MHA-Zn improved eggshell quality during the late phase (66 to 72 wk), including eggshell weight, eggshell thickness, eggshell strength, eggshell weight percent, and eggshell density. These results indicate that the effects of MHA-Zn on eggshell quality developed over time. Previous studies have reported that organic Zn has beneficial effects on eggshell quality (Klecker et al. 2002; Sahin et al., 2002; Xiao et al., 2014). Zhang et al. (2017) indicated that eggshell thickness was higher in organic treatment group than inorganic treatment group, which is consistent with our findings. This may be due to the higher efficiency of mineral deposition and calcium utilization of organic Zn compared with inorganic Zn. However, previous studies indicated that Zn sources did not affect eggshell quality (Lim and Paik, 2003; Mabe et al., 2003; Gheisari et al., 2011). It seems that some of these contradictory results arose from hen age, dietary composition, the concentration of examined trace elements in the basal diets, and the source of organic minerals (amino acid complex, proteinate, etc.). Eggshell Mineral Deposit The concentration of Ca in eggshells is closely related to eggshell quality, and Ca deposition is related to Ca level in the diet and Ca metabolism as well as the metabolism of trace elements such as Zn. Our study found that the concentration of Ca in eggshells increased with the increase in zinc supplementation, and at the same Zn level, the effect of organic Zn was better than that of inorganic Zn. This may be due to the higher bioavailability of the organic form. On one hand, the higher bioavailability of MHA-Zn may be linked to the shielding of the positive charge of the mineral during chelation. This allows the mineral avoid binding to the negatively charged mucin layer and results in lower competition between minerals of similar charge in their resorption from the gut and transfer to enterocytes (Power 2006). On the other hand, the use of inorganic salts can result in poor bioavailability of the mineral, primarily because of the numerous nutrient and ingredient antagonisms that impair absorption (Abdallah et al., 2009). Activities of Zn-containing Enzymes To date, few studies have focused on how Zn affects eggshell quality (Zhang et al., 2017). Zn is found in the active site of CA, which can promote the hydrolysis of H2CO3, increase the concentration of HCO3−, and promote the deposition of calcium carbonate. Supplementation of Zn in the diet can improve CA activity, reduce the broken egg rate and increase eggshell strength (Balnave and Zhang, 1993). Zhang et al. (2017) found that dietary supplementation with Zn could increase the CA activity in serum and eggshell glands, and with increased levels of Zn, the activity of CA in serum increased linearly, and the activity of CA in eggshell glands increased exponentially. In the present study, with increased Zn supplementation, the activity of CA increased. The increase in CA activity showed that organic Zn has a better effect than inorganic Zn on calcium carbonate formation. As a Zn-containing enzyme, the activity of AKP is affected by Zn deposition, which reflects the body's absorption and utilization of Zn. In the current experiment, with increased levels of Zn, AKP activity increased, and the activity of AKP was higher in the organic group than in the inorganic group. This may be due to the higher utilization of organic Zn. The liver is the center of nutrient metabolism (Feng et al., 2010). It should be noted that this study focused on the activity of CA and AKP in the liver, but eggshell glands also play an important role in eggshell formulation. To clearly elucidate the mechanism by which Zn affects eggshell quality, the activities of Zn-containing enzymes and mineral deposition in eggshell glands require further study. In conclusion, the supplementation of MHA chelates could have beneficial effects on laying hens, including improving eggshell quality and promoting Zn and Ca deposition in eggshells, as well as increasing CA activity in the liver. Moreover, the recommendation level of MHA-Zn in the diet of aged laying hens is 40 mg/kg. 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Supplementation of a corn-soybean meal diet with manganese and zinc improves eggshell quality in laying hens. Pak. J. Biol. Sci.  8: 1311– 1317. Google Scholar CrossRef Search ADS   Zhang Y. N., Zhang H. J., Wang J., Yue H. Y., Qi X. L., Wu S. G., Qi G. H.. 2017. Effect of dietary supplementation of organic or inorganic zinc on carbonic anhydrase activity in eggshell formation and quality of aged laying hens. Poult. Sci.  96: 2176– 2183. Google Scholar CrossRef Search ADS PubMed  © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effects of methionine hydroxyl analog chelated zinc on laying performance, eggshell quality, eggshell mineral deposition, and activities of Zn-containing enzymes in aged laying hens

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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10.3382/ps/pey203
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Abstract

ABSTRACT The study aimed to determine the effects of methionine hydroxyl analog chelated zinc (MHA-Zn) on laying performance, eggshell quality and mineral deposits, and the activities of Zn-containing enzymes on aged laying hens. A total of 960 layers (Hy-Line Grey, 57 wk old) were fed a basal diet (Zn: 35.08 mg/kg) without extra zinc for 2 wk. During the ensuing 14 wk, birds were randomly divided into 4 groups according to body weight and laying rate, with 8 replicates per treatment, and each group had 8 replicates of 30 hens. Four levels of Zn (ZnSO4: 80 mg/kg; MHA-Zn: 20, 40, 80 mg/kg) were added to the diet, respectively. The results shown that dietary Zn did not affect laying rate, average egg weight, average daily feed intake, or feed conversion ratio (P > 0.05); however, compared to the inorganic group, dietary supplementation with 40 or 80 mg/kg MHA-Zn decreased broken egg rate significantly in the whole period (P < 0.05), while significantly increased eggshell weight in week 62 to 72, eggshell thickness and eggshell strength in wk 66 to 72, eggshell weight percent and eggshell density in week 62 to 72 (P < 0.05). Besides, dietary supplementation with different sources and levels of Zn did not affect ash concentration of eggshell (P > 0.05), whereas dietary supplementation with 80 mg/kg MHA-Zn improved the Zn and Ca concentrations of eggshells and carbonic anhydrase (CA) activity of liver, and 40 mg/kg MHA-Zn increased Zn concentration of liver (P < 0.05). Moreover, no significant differences in alkaline phosphatase activity were observed among the treatment groups (P > 0.05). Therefore, dietary supplementation with 40 mg/kg MHA-Zn can improve eggshell quality by promoting Ca deposition and CA activity. INTRODUCTION Zinc (Zn) acts as a necessary micromineral for layer, and it is a component of more than 300 enzymes, mainly involved in the metabolism of cells, immune, and antioxides (Yan and Waldroup, 2006; Yi et al., 2007). Functions of Zn can be categorized as catalytic (metalloenzymes), structural (e.g., Zn finger domains of proteins), and regulatory (e.g., metal response elements of gene promoters). For the later period of laying, there are some changes in the layers, characterized by the decreasing of reproductive system and antioxide states, bigger and heavier eggs, and poor eggshell quality. It was reported there was 8% egg production losses resulted by the poor eggshell quality, which bring a great economic losses for poultry industry (Nys, 2001; Abdallan et al., 2009). To improve eggshell quality, researchers have taken great efforts in the genetics, environment, and nutrition, especially in the micromineral nutrition (Vallee and Falchuk, 1993; Batal et al., 2001; Berg et al., 2004; Gheisari et al., 2010). For example, zinc is a component of carbonic anhydrase (CA) enzyme, which involved in supplying carbonate ions during eggshell formation. Is it possible for microminerals such as Zn, Mn, and Cu to change the formation of CaCO3 calcite crystals and the construction of the eggshell, and finally affect the physical property of eggshell (Nys et al., 1999; Creech et al., 2004; Holm et al., 2006; Ao et al., 2009; Pierce et al., 2009; Yildiz et al., 2010). However, Zn deficiency or excess can cause metabolic disorders or imbalance and other symptoms, affecting normal production performance (Tabatabaie et al., 2007). Mabe et al. (2003) reported that dietary supplementation with 30 or 60 mg/kg Zn could improve the eggshell strength of aged layers. Zamani et al. (2005) observed that a diet containing 50, 100, or 150 mg/kg Zn could significantly improve eggshell thickness, and supplementation with 150 mg/kg Zn resulted in the thickest eggshells. Inorganic mineral elements are wildly used for its cheaper price. But there are existing antagonistic effect among different minerals and easily getting the formation of complex with other ingredients such as phytate, tannin in the diets in the intestinal, which result in the decreasing of the utilization (Swiatkiewicz and Koreleski, 2008). However, organic microminerals are characterized by reducing the chelating effects in the intestinal, decreasing metal ions interaction, increasing mineral steability in the lower pH value, more abosoption channel, higher abosorption and lower pollution, it is more and more used in the layer diets (Wedekind et al., 1992; Cao et al., 2000). It is generally accepted that organic trace elements (amino acid complex) have beneficial effects on eggshell quality (Henry et al., 1989; Neto et al., 2011). Cheng and Guo (2004) found that 60 mg/kg amino acid zinc (AA-Zn) decreased the broken egg rate. Abbas et al. (2010) reported that organic minerals could be utilized at a lower concentration in the diet than inorganic minerals, without having a negative impact on production performance. Similarly, Swiatkiewicz and Koreleski (2008) demonstrated that amino-acid-chelated trace elements could reduce adverse effects on the eggshell quality of aged layers. Trace elements may improve eggshell quality by promoting Ca deposition and the activities of key enzymes involved in eggshell formation (Zamani et al., 2005). Zn is found in the active center of CA and plays an important role in the conversion of carbon dioxide to bicarbonate (Bakst and Holm, 2003). Sun et al. (2012) reported that organic trace elements could improve CA activity in the liver. Alkaline phosphatase (AKP) is a Zn-containing enzyme, and its activity of serum or liver has been used as indicator of Zn status. Qin et al. (2017) showed that dietary supplementation with 60 mg/kg Zn significantly increased serum AKP activity. Previous studies have shown that organic Zn has more advantages than inorganic Zn in improving eggshell quality. However, the optimum supplementation amount has not been fully studied. The present study was designed to evaluate the effect of organic Zn on productivity, eggshell quality and mineral deposits, as well as activities of Zn-containing enzymes in late-phase laying hens. MATERIALS AND METHODS Animal Management and Diet Treatment A total of 960 layers (Hy-Line Grey, 57 wk old) were fed a basal diet (Zn = 35.08) without extra Zn to exhaust Zn that had previously accumulated. After 2 wk, the birds were randomly divided into 4 groups according to body weight and laying egg rate, with 8 replicates per treatment, and each group had 8 replicates of 30 hens. During the following 14 wk, the 4 groups were fed diets with 4 different levels of Zn added to the basal diet (ZnSO4: 80 mg/kg; MHA-Zn: 20, 40, 80 mg/kg). The experimental protocol used in the study was approved by the Animal Care and Use Committee of Northwest A & F University, Yangling, China. Research on live animals met the guidelines approved by the Institutional Animal Care and Use Committee. The basal diet was formulated to provide all other nutrients except Zn in accordance with the nutrient recommendations from NRC (1994). The dietary composition and nutrient levels are listed in Table 1. Table 1. Composition and nutrient levels of the basal diet (air-dry basis). Ingredients  %  Nutrients  %  Corn  58.50  ME (MJ/kg)  12.29  Soybean meal  23.00  Crude protein2  15.47  Ground limestone  8.74  Calcium2  3.60  CaHPO4  1.10  Total phosphorus  0.62  Wheat bran  7.50  Available phosphorus  0.32  Soybean oil  0.50  Lys  0.94  NaCl  0.24  Met  0.44  Met  0.16  Thr  0.70  Thr  0.03  Zinc2 (mg/kg)  35.08  Lys  0.05      Premix1  0.18      Total  100.00      Ingredients  %  Nutrients  %  Corn  58.50  ME (MJ/kg)  12.29  Soybean meal  23.00  Crude protein2  15.47  Ground limestone  8.74  Calcium2  3.60  CaHPO4  1.10  Total phosphorus  0.62  Wheat bran  7.50  Available phosphorus  0.32  Soybean oil  0.50  Lys  0.94  NaCl  0.24  Met  0.44  Met  0.16  Thr  0.70  Thr  0.03  Zinc2 (mg/kg)  35.08  Lys  0.05      Premix1  0.18      Total  100.00      1Supplied the following per kilogram of diet: Vitamin A 10,000 IU, VD3 31, 800 IU, VE 10 IU, VK 10 mg, VB125 μg, thiamine l mg, riboflavin 4.5 mg, calcium pantothenate 50 mg, niacin 24.5 mg, pyridoxine 5 mg, biotin 1 mg, folic acid 1 mg, choline 500 mg, Mn (as manganese sulfate) 60 mg, I (as calcium iodate) 0.4 mg, Fe (as ferrous sulfate) 60 mg, Cu (as copper sulfate) 8 mg, Se (sodium selenite) 0.30 mg. 2Analyzed values, each value based on triplicate determinations. View Large Table 1. Composition and nutrient levels of the basal diet (air-dry basis). Ingredients  %  Nutrients  %  Corn  58.50  ME (MJ/kg)  12.29  Soybean meal  23.00  Crude protein2  15.47  Ground limestone  8.74  Calcium2  3.60  CaHPO4  1.10  Total phosphorus  0.62  Wheat bran  7.50  Available phosphorus  0.32  Soybean oil  0.50  Lys  0.94  NaCl  0.24  Met  0.44  Met  0.16  Thr  0.70  Thr  0.03  Zinc2 (mg/kg)  35.08  Lys  0.05      Premix1  0.18      Total  100.00      Ingredients  %  Nutrients  %  Corn  58.50  ME (MJ/kg)  12.29  Soybean meal  23.00  Crude protein2  15.47  Ground limestone  8.74  Calcium2  3.60  CaHPO4  1.10  Total phosphorus  0.62  Wheat bran  7.50  Available phosphorus  0.32  Soybean oil  0.50  Lys  0.94  NaCl  0.24  Met  0.44  Met  0.16  Thr  0.70  Thr  0.03  Zinc2 (mg/kg)  35.08  Lys  0.05      Premix1  0.18      Total  100.00      1Supplied the following per kilogram of diet: Vitamin A 10,000 IU, VD3 31, 800 IU, VE 10 IU, VK 10 mg, VB125 μg, thiamine l mg, riboflavin 4.5 mg, calcium pantothenate 50 mg, niacin 24.5 mg, pyridoxine 5 mg, biotin 1 mg, folic acid 1 mg, choline 500 mg, Mn (as manganese sulfate) 60 mg, I (as calcium iodate) 0.4 mg, Fe (as ferrous sulfate) 60 mg, Cu (as copper sulfate) 8 mg, Se (sodium selenite) 0.30 mg. 2Analyzed values, each value based on triplicate determinations. View Large The inorganic trace elements in the diet were ZnSO4 (Zn ≥ 34.5%), CuSO4 (Cu ≥ 25.0%), FeSO4 (Fe ≥ 30.0%), MnSO4 (Mn ≥ 32.0%), Na2SeO3 (S ≥ 1.0%), and Ca (IO3)2 (I ≥ 1.0%). The organic Zn was MHA-Zn (MINTREX), purchased from Novus International Trading (Shanghai) Company. The actual concentrations of the total Zn analyzed in the 4 treatment diets are shown in Table 2. Table 2. Concentrations of Zn in experimental treatment diets (mg/kg).1 Dietary zinc supplementation  Calculated  Analyzed1  80 mg/kg ZnSO4  115.08  115.53  20 mg/kg MHA-Zn2  55.08  55.38  40 mg/kg MHA-Zn  75.08  75.97  80 mg/kg MHA-Zn  115.08  114.18  Dietary zinc supplementation  Calculated  Analyzed1  80 mg/kg ZnSO4  115.08  115.53  20 mg/kg MHA-Zn2  55.08  55.38  40 mg/kg MHA-Zn  75.08  75.97  80 mg/kg MHA-Zn  115.08  114.18  1Value based on triplicate determinations. 2MHA-Zn: methionine hydroxy analog chelate zinc, methionine yielded 80%. View Large Table 2. Concentrations of Zn in experimental treatment diets (mg/kg).1 Dietary zinc supplementation  Calculated  Analyzed1  80 mg/kg ZnSO4  115.08  115.53  20 mg/kg MHA-Zn2  55.08  55.38  40 mg/kg MHA-Zn  75.08  75.97  80 mg/kg MHA-Zn  115.08  114.18  Dietary zinc supplementation  Calculated  Analyzed1  80 mg/kg ZnSO4  115.08  115.53  20 mg/kg MHA-Zn2  55.08  55.38  40 mg/kg MHA-Zn  75.08  75.97  80 mg/kg MHA-Zn  115.08  114.18  1Value based on triplicate determinations. 2MHA-Zn: methionine hydroxy analog chelate zinc, methionine yielded 80%. View Large Chemical Analysis To analyze the dietary Zn concentration, a sample from each treatment was prepared according to Mabe et al. (2003). The Zn concentrations in diets were measured using flame atomic absorption spectrophotometry (No. 968.08). DM (No. 934.01), CP (No. 976.05), calcium (927.02), and phosphorus (No. 965.17) were determined using standard procedures from Association of Official Analytical Chemists (AOAC). Laying Performance and Eggshell Quality The number of eggs produced and the number of broken eggs were recorded every day. The amount of feed provided and residual feed were measured weekly. The laying rate, average egg weight, average daily feed intake(DFI), feed conversion rate (FCR), and the broken egg rate were calculated based on the records every 7 wk. At the end of 60, 62, 64, 66, 68, 70, and 72 wk, 24 eggs from each treatment group (3 eggs/replicate, 8 replicates) were obtained for the measurement of egg weight, eggshell weight, eggshell strength, and eggshell thickness. Egg weight and eggshell weight were measured using electronic balance (accuracy, 0.01 g). Eggshell strength and eggshell thickness were measured using eggshell strength tester (EFG-0503, Robotmation, Japan) and eggshell thickness tester (ETG-1601A, Robotmation, Japan), respectively. Eggshell weight percent and eggshell density were calculated using the following formulas: The eggshell weight percent was calculated as eggshell weight/egg weight × 100; eggshell density (g/100 cm2) was calculated as I = (C/S) × 100, where C is eggshell weight (g) and S (cm2) is shell surface (S = 4.68 × P2/3, where P is egg weight [g]) (Gheisar et al., 2010). Eggshell Mineral Deposit At the end of week 62, 66, 70, and 72, 3 eggs from each replicate with 8 replicates per treatment were taken. Washing eggshell membrane, dry at 80°C overnight, weighed the eggshell. The Zn concentrations in eggshell were measured using flame atomic absorption spectrophotometry (No. 968.08). Calcium (927.02) and phosphorus (No. 965.17) were determined using standard procedures from AOAC. Zn-containing Enzyme Activity At the end of the trial (72 wk), 1 bird from each replicate was chosen according to body weight. Those birds were sacrificed. The liver was taken and frozen in liquid nitrogen and then stored at –80°C. Frozen livers were homogenized and then centrifuged at 4000 r/min for 10 min at 4°C, and the supernatant was stored at –80°C until analysis. All samples were measured in triplicate, at the appropriate dilutions, to obtain enzyme activities in the linear range of standard curves constructed with pure enzymes. The protein concentration of supernatants was determined using a Coomassie Brilliant Blue kit assay with BSA as the standard. Meanwhile, CA and AKP activities were measured using CA assay kits and AKP assay kits. These 3 kits were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. The CA and AKP activities were assayed spectrophotometrically using colorimetric methods (Bio-Tek). Statistical Analysis Data were analyzed by one-way ANOVA using SPSS 21.0 (SPSS software for Windows, release 22.0, SPSS Inc., Chicago, IL). Duncan's test was used for multiple comparisons, and a P-value of 0.05 or less was declared significant. RESULTS Production Performance Production performance data are listed in Table 3. During the entire trial, neither dietary Zn levels nor sources affected the laying rate, average egg weight, average DFI, and FCR (P > 0.05). In addition, layers fed 40 mg/kg MHA-Zn exhibited the lowest FCR, but no significant differences were observed between the treatments. Moreover, the results indicated that the broken egg rate decreased with the increasing of MHA-Zn supplementation. In addition, at the same Zn level, the broken egg rate was significantly lower in the organic group than in the inorganic group (P < 0.05). Table 3. Effects of Zn on production performance of aged laying hens at the end of 66 and 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Laying rate (%)  Week 59 to 66  85.88 ± 0.82  86.10 ± 0.85  86.15 ± 0.78  86.13 ± 1.28  0.934  Week 67 to 72  82.31 ± 1.18  82.65 ± 1.47  82.97 ± 1.43  82.64 ± 1.24  0.804  Average egg weight (g)  Week 59 to 66  64.94 ± 0.36  64.89 ± 0.38  65.01 ± 0.58  64.91 ± 0.43  0.954  Week 67 to 72  66.19 ± 0.49  66.10 ± 0.52  66.17 ± 0.53  66.26 ± 0.59  0.941  Average daily feed intake (g)  Week 59 to 66  121.18 ± 0.48  121.34 ± 0.43  121.22 ± 0.50  121.25 ± 0.48  0.918  Week 67 to 72  121.15 ± 1.21  120.71 ± 1.28  120.53 ± 0.83  121.03 ± 1.34  0.716  Feed conversion rate  Week 59 to 66  2.17 ± 0.02  2.17 ± 0.03  2.16 ± 0.03  2.17 ± 0.04  0.942  Week 67 to 72  2.22 ± 0.04  2.21 ± 0.04  2.20 ± 0.05  2.21 ± 0.04  0.604  Broken egg rate (%)  Week 59 to 66  2.77 ± 0.18a  2.83 ± 0.18a  2.17 ± 0.27b  2.14 ± 0.30b  <0.001  Week 67 to 72  3.75 ± 0.40a  3.87 ± 0.38a  2.88 ± 0.40b  2.86 ± 0.23b  <0.001  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Laying rate (%)  Week 59 to 66  85.88 ± 0.82  86.10 ± 0.85  86.15 ± 0.78  86.13 ± 1.28  0.934  Week 67 to 72  82.31 ± 1.18  82.65 ± 1.47  82.97 ± 1.43  82.64 ± 1.24  0.804  Average egg weight (g)  Week 59 to 66  64.94 ± 0.36  64.89 ± 0.38  65.01 ± 0.58  64.91 ± 0.43  0.954  Week 67 to 72  66.19 ± 0.49  66.10 ± 0.52  66.17 ± 0.53  66.26 ± 0.59  0.941  Average daily feed intake (g)  Week 59 to 66  121.18 ± 0.48  121.34 ± 0.43  121.22 ± 0.50  121.25 ± 0.48  0.918  Week 67 to 72  121.15 ± 1.21  120.71 ± 1.28  120.53 ± 0.83  121.03 ± 1.34  0.716  Feed conversion rate  Week 59 to 66  2.17 ± 0.02  2.17 ± 0.03  2.16 ± 0.03  2.17 ± 0.04  0.942  Week 67 to 72  2.22 ± 0.04  2.21 ± 0.04  2.20 ± 0.05  2.21 ± 0.04  0.604  Broken egg rate (%)  Week 59 to 66  2.77 ± 0.18a  2.83 ± 0.18a  2.17 ± 0.27b  2.14 ± 0.30b  <0.001  Week 67 to 72  3.75 ± 0.40a  3.87 ± 0.38a  2.88 ± 0.40b  2.86 ± 0.23b  <0.001  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a,bMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc. View Large Table 3. Effects of Zn on production performance of aged laying hens at the end of 66 and 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Laying rate (%)  Week 59 to 66  85.88 ± 0.82  86.10 ± 0.85  86.15 ± 0.78  86.13 ± 1.28  0.934  Week 67 to 72  82.31 ± 1.18  82.65 ± 1.47  82.97 ± 1.43  82.64 ± 1.24  0.804  Average egg weight (g)  Week 59 to 66  64.94 ± 0.36  64.89 ± 0.38  65.01 ± 0.58  64.91 ± 0.43  0.954  Week 67 to 72  66.19 ± 0.49  66.10 ± 0.52  66.17 ± 0.53  66.26 ± 0.59  0.941  Average daily feed intake (g)  Week 59 to 66  121.18 ± 0.48  121.34 ± 0.43  121.22 ± 0.50  121.25 ± 0.48  0.918  Week 67 to 72  121.15 ± 1.21  120.71 ± 1.28  120.53 ± 0.83  121.03 ± 1.34  0.716  Feed conversion rate  Week 59 to 66  2.17 ± 0.02  2.17 ± 0.03  2.16 ± 0.03  2.17 ± 0.04  0.942  Week 67 to 72  2.22 ± 0.04  2.21 ± 0.04  2.20 ± 0.05  2.21 ± 0.04  0.604  Broken egg rate (%)  Week 59 to 66  2.77 ± 0.18a  2.83 ± 0.18a  2.17 ± 0.27b  2.14 ± 0.30b  <0.001  Week 67 to 72  3.75 ± 0.40a  3.87 ± 0.38a  2.88 ± 0.40b  2.86 ± 0.23b  <0.001  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Laying rate (%)  Week 59 to 66  85.88 ± 0.82  86.10 ± 0.85  86.15 ± 0.78  86.13 ± 1.28  0.934  Week 67 to 72  82.31 ± 1.18  82.65 ± 1.47  82.97 ± 1.43  82.64 ± 1.24  0.804  Average egg weight (g)  Week 59 to 66  64.94 ± 0.36  64.89 ± 0.38  65.01 ± 0.58  64.91 ± 0.43  0.954  Week 67 to 72  66.19 ± 0.49  66.10 ± 0.52  66.17 ± 0.53  66.26 ± 0.59  0.941  Average daily feed intake (g)  Week 59 to 66  121.18 ± 0.48  121.34 ± 0.43  121.22 ± 0.50  121.25 ± 0.48  0.918  Week 67 to 72  121.15 ± 1.21  120.71 ± 1.28  120.53 ± 0.83  121.03 ± 1.34  0.716  Feed conversion rate  Week 59 to 66  2.17 ± 0.02  2.17 ± 0.03  2.16 ± 0.03  2.17 ± 0.04  0.942  Week 67 to 72  2.22 ± 0.04  2.21 ± 0.04  2.20 ± 0.05  2.21 ± 0.04  0.604  Broken egg rate (%)  Week 59 to 66  2.77 ± 0.18a  2.83 ± 0.18a  2.17 ± 0.27b  2.14 ± 0.30b  <0.001  Week 67 to 72  3.75 ± 0.40a  3.87 ± 0.38a  2.88 ± 0.40b  2.86 ± 0.23b  <0.001  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a,bMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc. View Large Eggshell Quality Eggshell quality was shown in Table 4. Compared to inorganic group, groups with organic Zn did not influenced eggshell weight at the end of 60 wk; however, dietary supplementation with 40 or 80 mg/kg MHA-Zn significantly improved eggshell weight in week 62 to 72. Besides, the results showed that eggshell strength and eggshell thickness were not significantly affected by different levels and sources in week 60 to 64, whereas dietary supplementation with 40 or 80 mg/kg MHA-Zn exhibited higher eggshell strength in week 66 to 72. Dietary supplementation with 20 mg/kg MHA-Zn got the lowest eggshell thickness and eggshell strength, but the difference was not remarkable. Moreover, dietary supplementation with 40 or 80 mg/kg MHA-Zn significantly improved the eggshell weight percent and eggshell density (P < 0.05) compared with the control group in week 62 to 72. However, dietary supplementation with 20 mg/kg MHA-Zn did not exhibit a substantial difference. Table 4. Effects of Zn on the eggshell quality of aged laying hens at the end of 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Eggshell weight (g)  Week 60  7.82 ± 0.27a,b  7.75 ± 0.21b  8.04 ± 0.38a,b  8.13 ± 0.45a  0.081  Week 62  7.64 ± 0.38b  7.66 ± 0.29b  8.15 ± 0.25a  8.07 ± 0.32a  0.003  Week 64  7.41 ± 0.50b  7.40 ± 0.30b  7.91 ± 0.26a  7.92 ± 0.25a  0.003  Week 66  7.36 ± 0.40b  7.29 ± 0.23b  7.84 ± 0.40a  7.79 ± 0.40a  0.007  Week 68  7.28 ± 0.30b  7.29 ± 0.45b  7.78 ± 0.38a  7.81 ± 0.39a  0.009  Week 70  7.12 ± 0.35b  7.11 ± 0.40b  7.70 ± 0.48a  7.71 ± 0.64a  0.017  Week 72  6.89 ± 0.29b  6.90 ± 0.27b  7.58 ± 0.38a  7.54 ± 0.63a  0.001  Eggshell thickness (mm)  Week 60  0.402 ± 0.019  0.392 ± 0.005  0.401 ± 0.016  0.402 ± 0.018  0.484  Week 62  0.377 ± 0.016  0.373 ± 0.010  0.381 ± 0.010  0.385 ± 0.011  0.249  Week 64  0.362 ± 0.010  0.360 ± 0.013  0.372 ± 0.014  0.374 ± 0.012  0.080  Week 66  0.353 ± 0.014b  0.351 ± 0.014b  0.370 ± 0.021a  0.373 ± 0.019a  0.025  Week 68  0.336 ± 0.014b  0.335 ± 0.013b  0.362 ± 0.008a  0.364 ± 0.017a  <0.001  Week 70  0.324 ± 0.008b  0.321 ± 0.016b  0.365 ± 0.012a  0.363 ± 0.016a  <0.001  Week 72  0.308 ± 0.012b  0.306 ± 0.011b  0.352 ± 0.012a  0.354 ± 0.011a  <0.001  Eggshell strength (N)  Week 60  4.25 ± 0.19  4.21 ± 0.26  4.25 ± 0.22  4.24 ± 0.10  0.979  Week 62  3.96 ± 0.20  3.91 ± 0.13  4.02 ± 0.28  4.05 ± 0.31  0.634  Week 64  3.65 ± 0.24a,b  3.63 ± 0.30b  3.99 ± 0.30a  3.98 ± 0.18a,b  0.010  Week 66  3.50 ± 0.23b  3.50 ± 0.13b  3.83 ± 0.23a  3.80 ± 0.27a  0.003  Week 68  3.43 ± 0.22b  3.44 ± 0.30b  3.79 ± 0.21a,b  3.83 ± 0.31a  0.005  Week 70  3.32 ± 0.27b  3.26 ± 0.24b  3.66 ± 0.16a  3.63 ± 0.20a  0.001  Week 72  3.27 ± 0.12b  3.29 ± 0.29b  3.57 ± 0.17a  3.57 ± 0.15a  0.001  Eggshell weight percent (%)  Week 60  12.11 ± 0.41a,b  12.01 ± 0.36b  12.45 ± 0.59a,b  12.64 ± 0.60a  0.071  Week 62  11.78 ± 0.52b  11.83 ± 0.41b  12.53 ± 0.31a  12.41 ± 0.48a  0.002  Week 64  11.37 ± 0.80b  11.35 ± 0.49b  12.14 ± 0.48a  12.12 ± 0.46a  0.008  Week 66  11.17 ± 0.60b  11.16 ± 0.50b  11.91 ± 0.51a  11.87 ± 0.39a  0.004  Week 68  10.98 ± 0.42b  10.99 ± 0.66b  11.71 ± 0.62a  11.78 ± 0.73a  0.016  Week 70  10.67 ± 0.56b  10.67 ± 0.73b  11.55 ± 0.56a  11.59 ± 0.98a  0.016  Week 72  10.35 ± 0.33b  10.37 ± 0.52b  11.39 ± 0.64a  11.26 ± 0.58a  <0.001  Eggshell density (%)  Week 60  10.38 ± 0.35a,b  10.29 ± 0.30b  10.67 ± 0.50a,b  10.82 ± 0.52a  0.076  Week 62  10.11 ± 0.50b  10.15 ± 0.36b  10.77 ± 0.28a  10.66 ± 0.40a  0.003  Week 64  9.77 ± 0.68b  9.76 ± 0.40b  10.44 ± 0.38a  10.43 ± 0.35a  0.005  Week 66  9.64 ± 0.50b  9.60 ± 0.37b  10.28 ± 0.45a  10.23 ± 0.38a  0.003  Week 68  9.49 ± 0.37b  9.51 ± 0.57b  10.13 ± 0.52a  10.19 ± 0.58a  0.012  Week 70  9.24 ± 0.45b  9.24 ± 0.59b  10.01 ± 0.52a  10.03 ± 0.83a  0.014  Week 72  8.96 ± 0.31b  8.98 ± 0.41b  9.86 ± 0.52a  9.76 ± 0.61a  <0.001  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Eggshell weight (g)  Week 60  7.82 ± 0.27a,b  7.75 ± 0.21b  8.04 ± 0.38a,b  8.13 ± 0.45a  0.081  Week 62  7.64 ± 0.38b  7.66 ± 0.29b  8.15 ± 0.25a  8.07 ± 0.32a  0.003  Week 64  7.41 ± 0.50b  7.40 ± 0.30b  7.91 ± 0.26a  7.92 ± 0.25a  0.003  Week 66  7.36 ± 0.40b  7.29 ± 0.23b  7.84 ± 0.40a  7.79 ± 0.40a  0.007  Week 68  7.28 ± 0.30b  7.29 ± 0.45b  7.78 ± 0.38a  7.81 ± 0.39a  0.009  Week 70  7.12 ± 0.35b  7.11 ± 0.40b  7.70 ± 0.48a  7.71 ± 0.64a  0.017  Week 72  6.89 ± 0.29b  6.90 ± 0.27b  7.58 ± 0.38a  7.54 ± 0.63a  0.001  Eggshell thickness (mm)  Week 60  0.402 ± 0.019  0.392 ± 0.005  0.401 ± 0.016  0.402 ± 0.018  0.484  Week 62  0.377 ± 0.016  0.373 ± 0.010  0.381 ± 0.010  0.385 ± 0.011  0.249  Week 64  0.362 ± 0.010  0.360 ± 0.013  0.372 ± 0.014  0.374 ± 0.012  0.080  Week 66  0.353 ± 0.014b  0.351 ± 0.014b  0.370 ± 0.021a  0.373 ± 0.019a  0.025  Week 68  0.336 ± 0.014b  0.335 ± 0.013b  0.362 ± 0.008a  0.364 ± 0.017a  <0.001  Week 70  0.324 ± 0.008b  0.321 ± 0.016b  0.365 ± 0.012a  0.363 ± 0.016a  <0.001  Week 72  0.308 ± 0.012b  0.306 ± 0.011b  0.352 ± 0.012a  0.354 ± 0.011a  <0.001  Eggshell strength (N)  Week 60  4.25 ± 0.19  4.21 ± 0.26  4.25 ± 0.22  4.24 ± 0.10  0.979  Week 62  3.96 ± 0.20  3.91 ± 0.13  4.02 ± 0.28  4.05 ± 0.31  0.634  Week 64  3.65 ± 0.24a,b  3.63 ± 0.30b  3.99 ± 0.30a  3.98 ± 0.18a,b  0.010  Week 66  3.50 ± 0.23b  3.50 ± 0.13b  3.83 ± 0.23a  3.80 ± 0.27a  0.003  Week 68  3.43 ± 0.22b  3.44 ± 0.30b  3.79 ± 0.21a,b  3.83 ± 0.31a  0.005  Week 70  3.32 ± 0.27b  3.26 ± 0.24b  3.66 ± 0.16a  3.63 ± 0.20a  0.001  Week 72  3.27 ± 0.12b  3.29 ± 0.29b  3.57 ± 0.17a  3.57 ± 0.15a  0.001  Eggshell weight percent (%)  Week 60  12.11 ± 0.41a,b  12.01 ± 0.36b  12.45 ± 0.59a,b  12.64 ± 0.60a  0.071  Week 62  11.78 ± 0.52b  11.83 ± 0.41b  12.53 ± 0.31a  12.41 ± 0.48a  0.002  Week 64  11.37 ± 0.80b  11.35 ± 0.49b  12.14 ± 0.48a  12.12 ± 0.46a  0.008  Week 66  11.17 ± 0.60b  11.16 ± 0.50b  11.91 ± 0.51a  11.87 ± 0.39a  0.004  Week 68  10.98 ± 0.42b  10.99 ± 0.66b  11.71 ± 0.62a  11.78 ± 0.73a  0.016  Week 70  10.67 ± 0.56b  10.67 ± 0.73b  11.55 ± 0.56a  11.59 ± 0.98a  0.016  Week 72  10.35 ± 0.33b  10.37 ± 0.52b  11.39 ± 0.64a  11.26 ± 0.58a  <0.001  Eggshell density (%)  Week 60  10.38 ± 0.35a,b  10.29 ± 0.30b  10.67 ± 0.50a,b  10.82 ± 0.52a  0.076  Week 62  10.11 ± 0.50b  10.15 ± 0.36b  10.77 ± 0.28a  10.66 ± 0.40a  0.003  Week 64  9.77 ± 0.68b  9.76 ± 0.40b  10.44 ± 0.38a  10.43 ± 0.35a  0.005  Week 66  9.64 ± 0.50b  9.60 ± 0.37b  10.28 ± 0.45a  10.23 ± 0.38a  0.003  Week 68  9.49 ± 0.37b  9.51 ± 0.57b  10.13 ± 0.52a  10.19 ± 0.58a  0.012  Week 70  9.24 ± 0.45b  9.24 ± 0.59b  10.01 ± 0.52a  10.03 ± 0.83a  0.014  Week 72  8.96 ± 0.31b  8.98 ± 0.41b  9.86 ± 0.52a  9.76 ± 0.61a  <0.001  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a,bMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc. View Large Table 4. Effects of Zn on the eggshell quality of aged laying hens at the end of 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Eggshell weight (g)  Week 60  7.82 ± 0.27a,b  7.75 ± 0.21b  8.04 ± 0.38a,b  8.13 ± 0.45a  0.081  Week 62  7.64 ± 0.38b  7.66 ± 0.29b  8.15 ± 0.25a  8.07 ± 0.32a  0.003  Week 64  7.41 ± 0.50b  7.40 ± 0.30b  7.91 ± 0.26a  7.92 ± 0.25a  0.003  Week 66  7.36 ± 0.40b  7.29 ± 0.23b  7.84 ± 0.40a  7.79 ± 0.40a  0.007  Week 68  7.28 ± 0.30b  7.29 ± 0.45b  7.78 ± 0.38a  7.81 ± 0.39a  0.009  Week 70  7.12 ± 0.35b  7.11 ± 0.40b  7.70 ± 0.48a  7.71 ± 0.64a  0.017  Week 72  6.89 ± 0.29b  6.90 ± 0.27b  7.58 ± 0.38a  7.54 ± 0.63a  0.001  Eggshell thickness (mm)  Week 60  0.402 ± 0.019  0.392 ± 0.005  0.401 ± 0.016  0.402 ± 0.018  0.484  Week 62  0.377 ± 0.016  0.373 ± 0.010  0.381 ± 0.010  0.385 ± 0.011  0.249  Week 64  0.362 ± 0.010  0.360 ± 0.013  0.372 ± 0.014  0.374 ± 0.012  0.080  Week 66  0.353 ± 0.014b  0.351 ± 0.014b  0.370 ± 0.021a  0.373 ± 0.019a  0.025  Week 68  0.336 ± 0.014b  0.335 ± 0.013b  0.362 ± 0.008a  0.364 ± 0.017a  <0.001  Week 70  0.324 ± 0.008b  0.321 ± 0.016b  0.365 ± 0.012a  0.363 ± 0.016a  <0.001  Week 72  0.308 ± 0.012b  0.306 ± 0.011b  0.352 ± 0.012a  0.354 ± 0.011a  <0.001  Eggshell strength (N)  Week 60  4.25 ± 0.19  4.21 ± 0.26  4.25 ± 0.22  4.24 ± 0.10  0.979  Week 62  3.96 ± 0.20  3.91 ± 0.13  4.02 ± 0.28  4.05 ± 0.31  0.634  Week 64  3.65 ± 0.24a,b  3.63 ± 0.30b  3.99 ± 0.30a  3.98 ± 0.18a,b  0.010  Week 66  3.50 ± 0.23b  3.50 ± 0.13b  3.83 ± 0.23a  3.80 ± 0.27a  0.003  Week 68  3.43 ± 0.22b  3.44 ± 0.30b  3.79 ± 0.21a,b  3.83 ± 0.31a  0.005  Week 70  3.32 ± 0.27b  3.26 ± 0.24b  3.66 ± 0.16a  3.63 ± 0.20a  0.001  Week 72  3.27 ± 0.12b  3.29 ± 0.29b  3.57 ± 0.17a  3.57 ± 0.15a  0.001  Eggshell weight percent (%)  Week 60  12.11 ± 0.41a,b  12.01 ± 0.36b  12.45 ± 0.59a,b  12.64 ± 0.60a  0.071  Week 62  11.78 ± 0.52b  11.83 ± 0.41b  12.53 ± 0.31a  12.41 ± 0.48a  0.002  Week 64  11.37 ± 0.80b  11.35 ± 0.49b  12.14 ± 0.48a  12.12 ± 0.46a  0.008  Week 66  11.17 ± 0.60b  11.16 ± 0.50b  11.91 ± 0.51a  11.87 ± 0.39a  0.004  Week 68  10.98 ± 0.42b  10.99 ± 0.66b  11.71 ± 0.62a  11.78 ± 0.73a  0.016  Week 70  10.67 ± 0.56b  10.67 ± 0.73b  11.55 ± 0.56a  11.59 ± 0.98a  0.016  Week 72  10.35 ± 0.33b  10.37 ± 0.52b  11.39 ± 0.64a  11.26 ± 0.58a  <0.001  Eggshell density (%)  Week 60  10.38 ± 0.35a,b  10.29 ± 0.30b  10.67 ± 0.50a,b  10.82 ± 0.52a  0.076  Week 62  10.11 ± 0.50b  10.15 ± 0.36b  10.77 ± 0.28a  10.66 ± 0.40a  0.003  Week 64  9.77 ± 0.68b  9.76 ± 0.40b  10.44 ± 0.38a  10.43 ± 0.35a  0.005  Week 66  9.64 ± 0.50b  9.60 ± 0.37b  10.28 ± 0.45a  10.23 ± 0.38a  0.003  Week 68  9.49 ± 0.37b  9.51 ± 0.57b  10.13 ± 0.52a  10.19 ± 0.58a  0.012  Week 70  9.24 ± 0.45b  9.24 ± 0.59b  10.01 ± 0.52a  10.03 ± 0.83a  0.014  Week 72  8.96 ± 0.31b  8.98 ± 0.41b  9.86 ± 0.52a  9.76 ± 0.61a  <0.001  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Eggshell weight (g)  Week 60  7.82 ± 0.27a,b  7.75 ± 0.21b  8.04 ± 0.38a,b  8.13 ± 0.45a  0.081  Week 62  7.64 ± 0.38b  7.66 ± 0.29b  8.15 ± 0.25a  8.07 ± 0.32a  0.003  Week 64  7.41 ± 0.50b  7.40 ± 0.30b  7.91 ± 0.26a  7.92 ± 0.25a  0.003  Week 66  7.36 ± 0.40b  7.29 ± 0.23b  7.84 ± 0.40a  7.79 ± 0.40a  0.007  Week 68  7.28 ± 0.30b  7.29 ± 0.45b  7.78 ± 0.38a  7.81 ± 0.39a  0.009  Week 70  7.12 ± 0.35b  7.11 ± 0.40b  7.70 ± 0.48a  7.71 ± 0.64a  0.017  Week 72  6.89 ± 0.29b  6.90 ± 0.27b  7.58 ± 0.38a  7.54 ± 0.63a  0.001  Eggshell thickness (mm)  Week 60  0.402 ± 0.019  0.392 ± 0.005  0.401 ± 0.016  0.402 ± 0.018  0.484  Week 62  0.377 ± 0.016  0.373 ± 0.010  0.381 ± 0.010  0.385 ± 0.011  0.249  Week 64  0.362 ± 0.010  0.360 ± 0.013  0.372 ± 0.014  0.374 ± 0.012  0.080  Week 66  0.353 ± 0.014b  0.351 ± 0.014b  0.370 ± 0.021a  0.373 ± 0.019a  0.025  Week 68  0.336 ± 0.014b  0.335 ± 0.013b  0.362 ± 0.008a  0.364 ± 0.017a  <0.001  Week 70  0.324 ± 0.008b  0.321 ± 0.016b  0.365 ± 0.012a  0.363 ± 0.016a  <0.001  Week 72  0.308 ± 0.012b  0.306 ± 0.011b  0.352 ± 0.012a  0.354 ± 0.011a  <0.001  Eggshell strength (N)  Week 60  4.25 ± 0.19  4.21 ± 0.26  4.25 ± 0.22  4.24 ± 0.10  0.979  Week 62  3.96 ± 0.20  3.91 ± 0.13  4.02 ± 0.28  4.05 ± 0.31  0.634  Week 64  3.65 ± 0.24a,b  3.63 ± 0.30b  3.99 ± 0.30a  3.98 ± 0.18a,b  0.010  Week 66  3.50 ± 0.23b  3.50 ± 0.13b  3.83 ± 0.23a  3.80 ± 0.27a  0.003  Week 68  3.43 ± 0.22b  3.44 ± 0.30b  3.79 ± 0.21a,b  3.83 ± 0.31a  0.005  Week 70  3.32 ± 0.27b  3.26 ± 0.24b  3.66 ± 0.16a  3.63 ± 0.20a  0.001  Week 72  3.27 ± 0.12b  3.29 ± 0.29b  3.57 ± 0.17a  3.57 ± 0.15a  0.001  Eggshell weight percent (%)  Week 60  12.11 ± 0.41a,b  12.01 ± 0.36b  12.45 ± 0.59a,b  12.64 ± 0.60a  0.071  Week 62  11.78 ± 0.52b  11.83 ± 0.41b  12.53 ± 0.31a  12.41 ± 0.48a  0.002  Week 64  11.37 ± 0.80b  11.35 ± 0.49b  12.14 ± 0.48a  12.12 ± 0.46a  0.008  Week 66  11.17 ± 0.60b  11.16 ± 0.50b  11.91 ± 0.51a  11.87 ± 0.39a  0.004  Week 68  10.98 ± 0.42b  10.99 ± 0.66b  11.71 ± 0.62a  11.78 ± 0.73a  0.016  Week 70  10.67 ± 0.56b  10.67 ± 0.73b  11.55 ± 0.56a  11.59 ± 0.98a  0.016  Week 72  10.35 ± 0.33b  10.37 ± 0.52b  11.39 ± 0.64a  11.26 ± 0.58a  <0.001  Eggshell density (%)  Week 60  10.38 ± 0.35a,b  10.29 ± 0.30b  10.67 ± 0.50a,b  10.82 ± 0.52a  0.076  Week 62  10.11 ± 0.50b  10.15 ± 0.36b  10.77 ± 0.28a  10.66 ± 0.40a  0.003  Week 64  9.77 ± 0.68b  9.76 ± 0.40b  10.44 ± 0.38a  10.43 ± 0.35a  0.005  Week 66  9.64 ± 0.50b  9.60 ± 0.37b  10.28 ± 0.45a  10.23 ± 0.38a  0.003  Week 68  9.49 ± 0.37b  9.51 ± 0.57b  10.13 ± 0.52a  10.19 ± 0.58a  0.012  Week 70  9.24 ± 0.45b  9.24 ± 0.59b  10.01 ± 0.52a  10.03 ± 0.83a  0.014  Week 72  8.96 ± 0.31b  8.98 ± 0.41b  9.86 ± 0.52a  9.76 ± 0.61a  <0.001  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a,bMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc. View Large Eggshell Mineral Deposit The results of eggshell mineral deposit were shown in Table 5. The concentration of ash in eggshells was not affected by dietary supplementation or the source of Zn in the whole period. Compared with inorganic group, dietary supplementation with 80 mg/kg MHA-Zn improved the Ca concentration in eggshells at week 62, 70, and 72, and Zn concentration in eggshell at week 66, 70, and 72. On the other hand, dietary supplementation with 40 mg/kg MHA-Zn increased Zn and Ca concentration in liver, whereas group with 20 mg/kg MHA-Zn decreased Zn and Ca concentration but the difference was not significant. Moreover, P concentration in liver was not influenced by Zn level and source during the entire trial. Table 5. Effects of Zn on the mineral content in eggshell of aged laying hens.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Ash content of eggshell (%)  Week 62  94.39 ± 1.28  94.16 ± 1.55  93.04 ± 3.88  92.21 ± 2.86  0.325  Week 66  93.78 ± 1.14  92.65 ± 2.43  93.85 ± 0.95  93.26 ± 0.61  0.340  Week 70  93.21 ± 1.26  93.33 ± 0.99  93.05 ± 1.66  93.14 ± 2.05  0.986  Week 72  93.31 ± 0.84  93.04 ± 0.96  92.99 ± 0.99  93.64 ± 0.79  0.473  Ca content of eggshell (%)  Week 62  37.28 ± 1.70b  37.42 ± 2.36b  38.86 ± 0.95a,b  39.21 ± 0.96a  0.046  Week 66  37.14 ± 2.18  36.68 ± 2.50  38.32 ± 1.98  39.02 ± 0.89  0.098  Week 70  37.42 ± 1.06a,b  36.70 ± 1.65b  37.97 ± 1.07a,b  38.74 ± 1.47a  0.039  Week 72  37.17 ± 1.58b  36.71 ± 1.03b  37.18 ± 1.58a  38.70 ± 1.11a  0.048  P content of eggshell (%)  Week 62  1.06 ± 0.07  1.03 ± 0.09  1.05 ± 0.03  1.04 ± 0.09  0.817  Week 66  1.01 ± 0.05  0.96 ± 0.08  1.01 ± 0.03  1.02 ± 0.06  0.214  Week 70  0.99 ± 0.03  0.99 ± 0.03  1.00 ± 0.04  0.99 ± 0.05  0.988  Week 72  0.99 ± 0.04a  0.95 ± 0.02b  0.98 ± 0.01a,b  1.00 ± 0.05a  0.011  Zn content of eggshell (mg/kg)  Week 62  81.29 ± 7.22  80.00 ± 7.58  81.71 ± 6.35  82.23 ± 5.29  0.933  Week 66  70.26 ± 7.04b,c  68.98 ± 8.92c  76.95 ± 3.47a,b  80.91 ± 5.56a  0.006  Week 70  69.90 ± 8.88b,c  68.07 ± 7.81c  76.33 ± 3.70a,b  77.94 ± 3.66a  0.030  Week 72  65.56 ± 7.49b  64.50 ± 6.43b  75.23 ± 3.33a  73.25 ± 6.15a  0.005  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Ash content of eggshell (%)  Week 62  94.39 ± 1.28  94.16 ± 1.55  93.04 ± 3.88  92.21 ± 2.86  0.325  Week 66  93.78 ± 1.14  92.65 ± 2.43  93.85 ± 0.95  93.26 ± 0.61  0.340  Week 70  93.21 ± 1.26  93.33 ± 0.99  93.05 ± 1.66  93.14 ± 2.05  0.986  Week 72  93.31 ± 0.84  93.04 ± 0.96  92.99 ± 0.99  93.64 ± 0.79  0.473  Ca content of eggshell (%)  Week 62  37.28 ± 1.70b  37.42 ± 2.36b  38.86 ± 0.95a,b  39.21 ± 0.96a  0.046  Week 66  37.14 ± 2.18  36.68 ± 2.50  38.32 ± 1.98  39.02 ± 0.89  0.098  Week 70  37.42 ± 1.06a,b  36.70 ± 1.65b  37.97 ± 1.07a,b  38.74 ± 1.47a  0.039  Week 72  37.17 ± 1.58b  36.71 ± 1.03b  37.18 ± 1.58a  38.70 ± 1.11a  0.048  P content of eggshell (%)  Week 62  1.06 ± 0.07  1.03 ± 0.09  1.05 ± 0.03  1.04 ± 0.09  0.817  Week 66  1.01 ± 0.05  0.96 ± 0.08  1.01 ± 0.03  1.02 ± 0.06  0.214  Week 70  0.99 ± 0.03  0.99 ± 0.03  1.00 ± 0.04  0.99 ± 0.05  0.988  Week 72  0.99 ± 0.04a  0.95 ± 0.02b  0.98 ± 0.01a,b  1.00 ± 0.05a  0.011  Zn content of eggshell (mg/kg)  Week 62  81.29 ± 7.22  80.00 ± 7.58  81.71 ± 6.35  82.23 ± 5.29  0.933  Week 66  70.26 ± 7.04b,c  68.98 ± 8.92c  76.95 ± 3.47a,b  80.91 ± 5.56a  0.006  Week 70  69.90 ± 8.88b,c  68.07 ± 7.81c  76.33 ± 3.70a,b  77.94 ± 3.66a  0.030  Week 72  65.56 ± 7.49b  64.50 ± 6.43b  75.23 ± 3.33a  73.25 ± 6.15a  0.005  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a–cMeans with different superscripts within the same row are significantly different (P < 0.05). View Large Table 5. Effects of Zn on the mineral content in eggshell of aged laying hens.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Ash content of eggshell (%)  Week 62  94.39 ± 1.28  94.16 ± 1.55  93.04 ± 3.88  92.21 ± 2.86  0.325  Week 66  93.78 ± 1.14  92.65 ± 2.43  93.85 ± 0.95  93.26 ± 0.61  0.340  Week 70  93.21 ± 1.26  93.33 ± 0.99  93.05 ± 1.66  93.14 ± 2.05  0.986  Week 72  93.31 ± 0.84  93.04 ± 0.96  92.99 ± 0.99  93.64 ± 0.79  0.473  Ca content of eggshell (%)  Week 62  37.28 ± 1.70b  37.42 ± 2.36b  38.86 ± 0.95a,b  39.21 ± 0.96a  0.046  Week 66  37.14 ± 2.18  36.68 ± 2.50  38.32 ± 1.98  39.02 ± 0.89  0.098  Week 70  37.42 ± 1.06a,b  36.70 ± 1.65b  37.97 ± 1.07a,b  38.74 ± 1.47a  0.039  Week 72  37.17 ± 1.58b  36.71 ± 1.03b  37.18 ± 1.58a  38.70 ± 1.11a  0.048  P content of eggshell (%)  Week 62  1.06 ± 0.07  1.03 ± 0.09  1.05 ± 0.03  1.04 ± 0.09  0.817  Week 66  1.01 ± 0.05  0.96 ± 0.08  1.01 ± 0.03  1.02 ± 0.06  0.214  Week 70  0.99 ± 0.03  0.99 ± 0.03  1.00 ± 0.04  0.99 ± 0.05  0.988  Week 72  0.99 ± 0.04a  0.95 ± 0.02b  0.98 ± 0.01a,b  1.00 ± 0.05a  0.011  Zn content of eggshell (mg/kg)  Week 62  81.29 ± 7.22  80.00 ± 7.58  81.71 ± 6.35  82.23 ± 5.29  0.933  Week 66  70.26 ± 7.04b,c  68.98 ± 8.92c  76.95 ± 3.47a,b  80.91 ± 5.56a  0.006  Week 70  69.90 ± 8.88b,c  68.07 ± 7.81c  76.33 ± 3.70a,b  77.94 ± 3.66a  0.030  Week 72  65.56 ± 7.49b  64.50 ± 6.43b  75.23 ± 3.33a  73.25 ± 6.15a  0.005  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    Ash content of eggshell (%)  Week 62  94.39 ± 1.28  94.16 ± 1.55  93.04 ± 3.88  92.21 ± 2.86  0.325  Week 66  93.78 ± 1.14  92.65 ± 2.43  93.85 ± 0.95  93.26 ± 0.61  0.340  Week 70  93.21 ± 1.26  93.33 ± 0.99  93.05 ± 1.66  93.14 ± 2.05  0.986  Week 72  93.31 ± 0.84  93.04 ± 0.96  92.99 ± 0.99  93.64 ± 0.79  0.473  Ca content of eggshell (%)  Week 62  37.28 ± 1.70b  37.42 ± 2.36b  38.86 ± 0.95a,b  39.21 ± 0.96a  0.046  Week 66  37.14 ± 2.18  36.68 ± 2.50  38.32 ± 1.98  39.02 ± 0.89  0.098  Week 70  37.42 ± 1.06a,b  36.70 ± 1.65b  37.97 ± 1.07a,b  38.74 ± 1.47a  0.039  Week 72  37.17 ± 1.58b  36.71 ± 1.03b  37.18 ± 1.58a  38.70 ± 1.11a  0.048  P content of eggshell (%)  Week 62  1.06 ± 0.07  1.03 ± 0.09  1.05 ± 0.03  1.04 ± 0.09  0.817  Week 66  1.01 ± 0.05  0.96 ± 0.08  1.01 ± 0.03  1.02 ± 0.06  0.214  Week 70  0.99 ± 0.03  0.99 ± 0.03  1.00 ± 0.04  0.99 ± 0.05  0.988  Week 72  0.99 ± 0.04a  0.95 ± 0.02b  0.98 ± 0.01a,b  1.00 ± 0.05a  0.011  Zn content of eggshell (mg/kg)  Week 62  81.29 ± 7.22  80.00 ± 7.58  81.71 ± 6.35  82.23 ± 5.29  0.933  Week 66  70.26 ± 7.04b,c  68.98 ± 8.92c  76.95 ± 3.47a,b  80.91 ± 5.56a  0.006  Week 70  69.90 ± 8.88b,c  68.07 ± 7.81c  76.33 ± 3.70a,b  77.94 ± 3.66a  0.030  Week 72  65.56 ± 7.49b  64.50 ± 6.43b  75.23 ± 3.33a  73.25 ± 6.15a  0.005  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a–cMeans with different superscripts within the same row are significantly different (P < 0.05). View Large Activities of Zn-containing Enzymes As shown in Table 6, both the dietary supplementation levels and sources of Zn significantly influenced CA activity (P < 0.05). With the increasing of MHA-Zn supplementation, the activity of CA increased accordingly. At equal levels of Zn supplementation, the group with organic Zn showed significantly improved CA activity. In addition, 40 mg/kg MHA-Zn also greatly increased CA activity. Moreover, we found that AKP activity increased with the increasing of Zn supplementation and dietary supplementation with 40 or 80 mg/kg MHA-Zn, the activity was higher than inorganic group. Table 6. Effects of Zn on CA and AKP activity in the livers of aged hens at the end of 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    CA (ng/mL)2  76.4 ± 1.35c  71.44 ± 3.10b  79.52 ± 1.20b  82.54 ± 2.47a  <0.001  AKP(U/gprot)2  43.70 ± 2.98a,b  40.89 ± 4.23b  43.94 ± 2.30a,b  46.22 ± 2.40a  0.064  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    CA (ng/mL)2  76.4 ± 1.35c  71.44 ± 3.10b  79.52 ± 1.20b  82.54 ± 2.47a  <0.001  AKP(U/gprot)2  43.70 ± 2.98a,b  40.89 ± 4.23b  43.94 ± 2.30a,b  46.22 ± 2.40a  0.064  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a–cMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc; CA: carbonic anhydrase; AKP: alkaline phosphatase. View Large Table 6. Effects of Zn on CA and AKP activity in the livers of aged hens at the end of 72 wk.1 Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    CA (ng/mL)2  76.4 ± 1.35c  71.44 ± 3.10b  79.52 ± 1.20b  82.54 ± 2.47a  <0.001  AKP(U/gprot)2  43.70 ± 2.98a,b  40.89 ± 4.23b  43.94 ± 2.30a,b  46.22 ± 2.40a  0.064  Items  Experimental treatment  P-Value    80 mg/kg ZnSO4  20 mg/kg MHA-Zn2  40 mg/kg MHA-Zn  80 mg/kg MHA-Zn    CA (ng/mL)2  76.4 ± 1.35c  71.44 ± 3.10b  79.52 ± 1.20b  82.54 ± 2.47a  <0.001  AKP(U/gprot)2  43.70 ± 2.98a,b  40.89 ± 4.23b  43.94 ± 2.30a,b  46.22 ± 2.40a  0.064  1Data represent the means from 8 replicates per treatment. Values are the means ± SD. a–cMeans with different superscripts within the same row are significantly different (P < 0.05). 2MHA-Zn: methionine hydroxy analog chelate zinc; CA: carbonic anhydrase; AKP: alkaline phosphatase. View Large DISCUSSION Production Performance The present study showed that 40 mg/kg MHA-Zn can be used in diets for aged laying hens with no negative effects on production performance and can reduce the broken egg rate compared with dietary supplementation with 80 mg/kg ZnSO4. Similarly, Niyomdecha et al. (2008) found that the source of Zn did not affect laying rate, egg weight, DFI, and FCR. In addition, when the level of MHA-Zn was increased, the production performance did not improve. It seems that laying performance was not a sensitive criterion for assessing the dietary Zn requirement of laying hens fed a normal diet. We inferred that the main reason for this is that the Zn supplementation in the diet had met the layers’ requirement, so the production performance did not improve with increased Zn. However, some studies shown that organic Zn has more advantages than inorganic Zn in improving the production performance of hens (Skrivan et al., 2006; Bao et al., 2007; Jahanian et al., 2008; Sunder et al., 2008). The above inconsistencies might be due to differences of Zn level, experimental phase, or type of hens. Eggshell Quality Eggshell quality is an important indicator of the production efficiency of laying hens (Lien et al., 2004; Yousaf et al., 2009). The current study found that supplementation with MHA-Zn improved eggshell quality during the late phase (66 to 72 wk), including eggshell weight, eggshell thickness, eggshell strength, eggshell weight percent, and eggshell density. These results indicate that the effects of MHA-Zn on eggshell quality developed over time. Previous studies have reported that organic Zn has beneficial effects on eggshell quality (Klecker et al. 2002; Sahin et al., 2002; Xiao et al., 2014). Zhang et al. (2017) indicated that eggshell thickness was higher in organic treatment group than inorganic treatment group, which is consistent with our findings. This may be due to the higher efficiency of mineral deposition and calcium utilization of organic Zn compared with inorganic Zn. However, previous studies indicated that Zn sources did not affect eggshell quality (Lim and Paik, 2003; Mabe et al., 2003; Gheisari et al., 2011). It seems that some of these contradictory results arose from hen age, dietary composition, the concentration of examined trace elements in the basal diets, and the source of organic minerals (amino acid complex, proteinate, etc.). Eggshell Mineral Deposit The concentration of Ca in eggshells is closely related to eggshell quality, and Ca deposition is related to Ca level in the diet and Ca metabolism as well as the metabolism of trace elements such as Zn. Our study found that the concentration of Ca in eggshells increased with the increase in zinc supplementation, and at the same Zn level, the effect of organic Zn was better than that of inorganic Zn. This may be due to the higher bioavailability of the organic form. On one hand, the higher bioavailability of MHA-Zn may be linked to the shielding of the positive charge of the mineral during chelation. This allows the mineral avoid binding to the negatively charged mucin layer and results in lower competition between minerals of similar charge in their resorption from the gut and transfer to enterocytes (Power 2006). On the other hand, the use of inorganic salts can result in poor bioavailability of the mineral, primarily because of the numerous nutrient and ingredient antagonisms that impair absorption (Abdallah et al., 2009). Activities of Zn-containing Enzymes To date, few studies have focused on how Zn affects eggshell quality (Zhang et al., 2017). Zn is found in the active site of CA, which can promote the hydrolysis of H2CO3, increase the concentration of HCO3−, and promote the deposition of calcium carbonate. Supplementation of Zn in the diet can improve CA activity, reduce the broken egg rate and increase eggshell strength (Balnave and Zhang, 1993). Zhang et al. (2017) found that dietary supplementation with Zn could increase the CA activity in serum and eggshell glands, and with increased levels of Zn, the activity of CA in serum increased linearly, and the activity of CA in eggshell glands increased exponentially. In the present study, with increased Zn supplementation, the activity of CA increased. The increase in CA activity showed that organic Zn has a better effect than inorganic Zn on calcium carbonate formation. As a Zn-containing enzyme, the activity of AKP is affected by Zn deposition, which reflects the body's absorption and utilization of Zn. In the current experiment, with increased levels of Zn, AKP activity increased, and the activity of AKP was higher in the organic group than in the inorganic group. This may be due to the higher utilization of organic Zn. The liver is the center of nutrient metabolism (Feng et al., 2010). It should be noted that this study focused on the activity of CA and AKP in the liver, but eggshell glands also play an important role in eggshell formulation. To clearly elucidate the mechanism by which Zn affects eggshell quality, the activities of Zn-containing enzymes and mineral deposition in eggshell glands require further study. In conclusion, the supplementation of MHA chelates could have beneficial effects on laying hens, including improving eggshell quality and promoting Zn and Ca deposition in eggshells, as well as increasing CA activity in the liver. Moreover, the recommendation level of MHA-Zn in the diet of aged laying hens is 40 mg/kg. 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Poultry ScienceOxford University Press

Published: Jun 1, 2018

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