Safety evaluation of zinc methionine in laying hens: Effects on laying performance, clinical blood parameters, organ development, and histopathology

Safety evaluation of zinc methionine in laying hens: Effects on laying performance, clinical... Abstract The study was conducted to investigate whether high-dose zinc methionine (Zn-Met) affected the safety of laying hens, including laying performance, hematological parameters, serum chemical parameters, organ index, and histopathology. A total of 540 20-week-old Hy-Line White laying hens was randomly allocated to 6 groups with 6 replicates of 15 birds each. Birds were fed diets supplemented with 0 (control), 70, 140, 350, 700, or 1,400 mg Zn/kg diet as Zn-Met. The experiment lasted for 8 wk after a 2-week acclimation period. Results showed that dietary supplementation with 70 or 140 mg Zn/kg diet as Zn-Met significantly increased average daily egg mass (ADEM), laying rate (LR), and feed conversion ratio (FCR) (P < 0.05) and lowered broken and soft-shelled egg ratio (BSER) (P < 0.05) in comparison with the control group; no significant differences were detected among hens fed with 0, 350, or 700 mg Zn/kg as Zn-Met (P > 0.05); hens administered 1,400 mg Zn/kg showed a significant increase in BSER and remarkable decreases in ADEM, LR, and FCR (P < 0.001). There were no significant differences among hens receiving 0, 70, 140, 350, or 700 mg Zn/kg as Zn-Met in serum chemical parameters (P > 0.05); supplementation with 1,400 mg Zn/kg as Zn-Met remarkably elevated the concentrations of serum total bilirubin (TBILI), glucose (GLU), uric acid (UA), and creatinine (CRE) (P < 0.001), and enhanced activities of serum glutamic oxalacetic transaminase (GOP) and alkaline phosphatase (AKP) (P < 0.001) compared with the control group. No significant histopathological changes were found in hens administered 0, 70, 140, 350, or 700 mg Zn/kg as Zn-Met, while significant histological lesions were observed in the heart, liver, lung, and kidney tissues of hens receiving 1,400 mg Zn/kg as Zn-Met. No significant differences were detected in hematological parameters or organ index (P > 0.05). In conclusion, a nominal Zn concentration of 700 mg/kg as Zn-Met is considered to be no-observed-adverse-effect level following daily administration to hens for 56 days. INTRODUCTION Zinc methionine (Zn-Met), an organic source of Zn, has become increasingly popular in poultry production because dietary supplementation with optimal levels of Zn-Met was reported to promote laying performance, improve egg quality, elevate hatchability, and enhance the capabilities to resist diseases and stresses in poultry (Pimentel et al., 1991; Kienholz et al., 1992; Moreng et al., 1992; Hamidi and Pourreza, 2009; Wang et al., 2014; Jahanian and Rasouli, 2015; Mohammadi et al., 2015). However, available data and literature concerning the safety of Zn-Met in laying hens are not known. Much attention should be attached to the potential detrimental effects of excessive exposure to Zn-Met in hens. It is well acknowledged that Zn is an essential trace element for the health and growth of animals, and is involved in diverse biological and metabolic processes, including DNA synthesis, gene expression regulation, and nutrient metabolism (Bray and Bettger, 1990; Prasad, 2009). Although zinc is essential for animals, long-term or excessive exposure to zinc has adverse or even toxic effects on various systems in the body, such as the reproductive, immune, digestive, neural, and respiratory systems (Porea et al., 2000; Walther et al., 2002; Palmiter, 2004; Tetsuchikawahara et al., 2005). Zn at relatively low to moderate concentrations plays crucial roles in cellular metabolism and cell proliferation, but Zn at extremely high concentrations can interfere with intracellular Zn ion homeostasis and trigger cytotoxicity, which is characterized by the breakdown of mitochondrial membrane potential, caspase activation, and cell apoptosis (Fosmire, 1990; Kao et al., 2012; Lemire et al., 2010; Wang et al., 2017). Hence, this study was conducted to investigate whether high-dose Zn-Met affected the safety of hens, including laying performance, hematological parameters, serum chemical parameters, organ development, and histopathology. In the present study, high-dose Zn-Met was chosen to provide significant exposure to the test material, while other dose levels were set to provide adequate and convincing data for risk evaluation. MATERIALS AND METHODS Study Design The study was designed in accordance with the guidelines for safety evaluation of feed additives in animals by the European Food Safety Authority (EFSA) and Ministry of Agriculture of China. A total of 540 20-week-old Hy-Line White laying hens with similar body weights and performances was randomly allocated to 6 groups with 6 replicates of 15 birds each (90 hens per group). All birds were acclimated to a basal diet and the environment for 2 weeks. The birds received diets supplemented with 0 (control), 70 [the maximum recommended available dose (MRAD)], 140 (2-fold of MRAD), 350 (5-fold of MRAD), 700 (10-fold of MRAD), or 1400 mg Zn/kg diet (20-fold of MRAD) as Zn-Met for 8 weeks. Clinical observations were conducted throughout the experiment, and laying performance was determined. At the end of the feeding trial, 72 birds (2 hens from each replicate, 12 hens per group) were sacrificed and subjected to full postmortem examinations. Jugular vein blood samples were collected for a clinical pathological examination, while selected visceral organs were weighed, and specified tissues were taken for subsequent histopathological examinations. Birds, Diets, and Management This study was conducted at Feed Science Institute, Zhejiang University. Zn-Met was provided by JiningHeshi Biotechnology Co., Ltd (Shandong, China) with 97% purity. Birds were kept in the ventilated room with a lighting schedule of 16-h light/8-h dark cycle. Housing temperature and relative humidity were maintained at 23 ± 5°C and 65% ± 5%, respectively. Three hens were raised in an individual battery cage (45 × 45 × 50 cm). All hens were allowed access to water all the time and fed ad libitum twice daily. The composition and nutrient levels of the basal diet are presented in Table 1. All the experimental protocols were approved by the Animal Care and Use Committee of Zhejiang University. Table 1. Ingredients and nutrient levels of basal diet (air-dry basis). Items  Ingredients  Contents (%)  Corn  59.20  Soybean meal  26.50  Soybean oil  2.00  Calcium hydrophosphate  1.20  Limestone  8.60  Salt  0.30  Methionine  0.20  Premix1  2.00  Total  100.00  Nutrient levels2    ME (MJ/kg)  11.40  Crude protein (%)  16.27  Calcium (%)  3.48  Total phosphorus (%)  0.56  Available phosphorus (%)  0.38  Lysine (%)  0.82  Met+Cys (%)  0.67  Zinc3 (mg/kg)  34.21  Items  Ingredients  Contents (%)  Corn  59.20  Soybean meal  26.50  Soybean oil  2.00  Calcium hydrophosphate  1.20  Limestone  8.60  Salt  0.30  Methionine  0.20  Premix1  2.00  Total  100.00  Nutrient levels2    ME (MJ/kg)  11.40  Crude protein (%)  16.27  Calcium (%)  3.48  Total phosphorus (%)  0.56  Available phosphorus (%)  0.38  Lysine (%)  0.82  Met+Cys (%)  0.67  Zinc3 (mg/kg)  34.21  1Premix provided the following per kilogram of diet: Mn, 65 mg; I 1 mg; Fe, 60 mg; Cu, 8 mg; Se, 0.3 mg; vitamin A, 12,500 IU; vitamin D3, 4125 IU, vitamin E, 15 IU; vitamin K, 2 mg; thiamine, 1 mg; riboflavin, 8.5 mg; calcium pantothenate, 50 mg; niacin, 32.5 mg; pyridoxine, 8 mg; folic aicd, 5 mg; vitamin B12, 5 mg; choline cloride, 500 mg. 2Calculated from tables of feed composition and nutritive values provided by Feed Database in China (2004). 3Analyzed by inductively coupled plasma atomic emission spectrometry. View Large Sample Collection and Analytical Determination Clinical Observations. Close cage-side observations were performed 3 times daily throughout the feeding trial period. Mortality and abnormal behaviors in clinical circumstances, including dullness, anorexia, ruffled feather, molt, lethargy, depression, recumbence, shrunken eyes, and mucous mixed droppings, were recorded. Laying Performance. Egg number, total egg weight, and number of broken and soft-shelled eggs of each replicate were monitored daily, and feed consumption on a replicate basis was recorded at weekly intervals. Egg weight (EW), average daily egg mass (ADEM), average daily feed intake (ADFI), laying rate (LR), and feed conversion ratio (FCR) were then calculated based on the collected data. The LR was expressed as average hen-day production, calculated from the total number of eggs divided by the total number of hens. The FCR was calculated as grams of egg produced per gram of feed consumed. Clinical Blood Parameters. At the end of the feeding trial, 2 hens from each replicate (12 hens per group) were chosen randomly. Each hen was weighed and sacrificed by exsanguination via the jugular vein. Jugular vein blood samples for hematological and clinical chemical analysis were collected. 2 mL of blood sample were collected in EDTA-containing tubes for hematology, whereas 5 mL of blood were taken for clinical chemical analysis. Hematological parameters of the collected samples involving white blood cells (WBC), red blood cells (RBC), platelet count (PLT), hematocrit (HCT), hemoglobin (HGB), and red cell distribution width (RCDW) were measured by A Sysmex XE2100 automated hematology analyzer (Sysmex Co., Kobe, Japan). Serum samples were prepared by centrifuging at 3500 × g for 10 min at 4 °C. Chemical parameters, including the activities of glutamic pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) and the concentrations of total bilirubin (TBILI), alkaline phosphatase (AKP), total protein (TP), albumin (ALB), glucose (GLU), uric acid (UA), and creatinine (CRE), were measured in serum samples using a RX Daytona Chemical Analyzer (Randox, London, UK). These parameters were determined in order to cover a range of potential toxicities, including the disturbances to material metabolism and the damage to the major organs. Gross Pathology. After exsanguination via the jugular vein, the hens were subjected to full postmortem examinations for gross lesions in the heart, liver, lung, and kidney, including swelling, bleeding, and necrosis of organs, as well as the chalky white deposits of urate on the surface of the visceral organs. Organ Index. After postmortem examinations, the heart, liver, spleen, lung, and kidney were collected and weighed. The organ index was calculated via the formula: organ index (%) = organ weight/body weight × 100. Histopathology. Portions of the heart, liver, lung, and kidney tissues were fixed in 10% buffered formalin (Chemical Laboratory Center of Zhejiang University, Hangzhou, China). For histological analysis, the formalin-fixed samples were processed into wax sections of 5 μm thickness and stained with hematoxylin-eosin (H&E) in accordance with the method reported by Jadhav et al. (2007). Light microscopic examination for histopathological alterations was conducted under a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). The corresponding images were captured by DigiLab II digital microscope mutual systems (Motic Groups, Xiamen, China). Statistical Analysis All data were analyzed using a one-way analysis of variance (ANOVA, SPSS 20.0 for Windows, SPSS Inc., Chicago, IL). When significant differences were detected, a 5% variance average was used, with a Duncan's multiple comparison test for comparing among the groups. A P-value of less than 0.05 was considered significant. RESULTS Clinical Observations No mortality or significant molt occurred throughout the feeding trial. All hens appeared normal without any significant clinical signs of intoxication during the entire experimental period. Laying Performance Effects of different levels of dietary Zn-Met supplementation on laying performance of hens are presented in Table 2. ADFI and EW were unaffected by dietary Zn-Met supplementation (P > 0.05). Compared with the control group, dietary supplementation with 70 or 140 mg Zn/kg diet as Zn-Met significantly increased ADEM, LR, and FCR (P < 0.05) and considerably lowered BSER (P < 0.001). Hens fed with 1,400 mg Zn/kg diet as Zn-Met showed a significant increase in BSER (P < 0.001) and remarkable decreases in ADEM, LR, and FCR (P < 0.001). There were no statistically significant differences regarding the measured parameters of laying performance among the hens supplemented with 0, 350, or 700 mg Zn/kg diet as Zn-Met (P > 0.05). Table 2. Laying performance of hens during a study to evaluate zinc methionine (Zn-Met) safety.1 Added Zn levels  Laying performance  mg/kg  EW, g  ADEM, g/hen/d  ADFI, g/hen/d  LR, %  FCR, %  BSER, %  0  53.62  47.58b  97.88  88.73b  48.54b  0.61b  70  53.90  48.87a  97.69  90.68a  50.25a  0.21c  140  53.56  48.45a  98.03  90.47a  49.75a  0.26c  350  53.34  47.51b  97.80  89.07a,b  48.78b  0.57b  700  53.61  47.25b  98.59  88.13b  48.31b  0.65b  1400  53.11  42.52b  94.97  80.07c  44.84c  1.98a  SEM  0.615  0.604  0.881  0.919  0.574  0.081  P-value              Linear  0.489  0.000  0.067  0.000  0.000  0.000  Quadratic  0.127  0.000  0.781  0.000  0.000  0.002  Added Zn levels  Laying performance  mg/kg  EW, g  ADEM, g/hen/d  ADFI, g/hen/d  LR, %  FCR, %  BSER, %  0  53.62  47.58b  97.88  88.73b  48.54b  0.61b  70  53.90  48.87a  97.69  90.68a  50.25a  0.21c  140  53.56  48.45a  98.03  90.47a  49.75a  0.26c  350  53.34  47.51b  97.80  89.07a,b  48.78b  0.57b  700  53.61  47.25b  98.59  88.13b  48.31b  0.65b  1400  53.11  42.52b  94.97  80.07c  44.84c  1.98a  SEM  0.615  0.604  0.881  0.919  0.574  0.081  P-value              Linear  0.489  0.000  0.067  0.000  0.000  0.000  Quadratic  0.127  0.000  0.781  0.000  0.000  0.002  1Results are the means of 6 replicates of 15 hens each. EW = egg weight; ADEM = average daily egg mass; ADFI = average daily feed intake; LR = laying rate; FCR = feed conversion ratio; BSER = broken and soft-shelled egg ratio. a-cValues within a column with no common superscripts differ significantly (P < 0.05). View Large Clinical Blood Parameters Hematological parameters of hens administered different levels of Zn-Met are displayed in Table 3. There were no statistically significant differences in any of the determined hematological parameters (P > 0.05). Table 3. Hematological parameters of hens during a study to evaluate zinc methionine (Zn-Met) safety.1 Added Zn levels  Hematological parameters  mg/kg  WBC, 109/L  RBC, 1012/L  PLT, 109/L  HCT, %  HGB, g/L  RCDW, %  0  266.29  2.86  1.04  32.61  86.42  39.54  70  272.23  2.90  1.25  32.82  85.80  38.21  140  266.84  2.89  1.33  32.56  84.67  38.47  350  268.57  2.88  1.29  32.66  85.33  40.05  700  268.97  2.87  1.17  31.63  83.42  39.96  1400  288.13  2.89  1.17  33.28  84.75  40.32  SEM  4.456  0.029  0.087  0.298  0.722  0.063  P-value              Linear  0.205  0.199  0.541  0.083  0.093  0.478  Quadratic  0.186  0.436  0.209  0.571  0.176  0.192  Added Zn levels  Hematological parameters  mg/kg  WBC, 109/L  RBC, 1012/L  PLT, 109/L  HCT, %  HGB, g/L  RCDW, %  0  266.29  2.86  1.04  32.61  86.42  39.54  70  272.23  2.90  1.25  32.82  85.80  38.21  140  266.84  2.89  1.33  32.56  84.67  38.47  350  268.57  2.88  1.29  32.66  85.33  40.05  700  268.97  2.87  1.17  31.63  83.42  39.96  1400  288.13  2.89  1.17  33.28  84.75  40.32  SEM  4.456  0.029  0.087  0.298  0.722  0.063  P-value              Linear  0.205  0.199  0.541  0.083  0.093  0.478  Quadratic  0.186  0.436  0.209  0.571  0.176  0.192  1Results are the means of 6 replicates of 2 hens each. WBC = white blood cells; RBC = red blood cells; PLT = platelet count; HCT = hematocrit; HGB = hemoglobin; RCDW = red cell distribution width. View Large Effects of dietary supplementation with different levels of Zn-Met on blood chemical parameters of hens are shown in Table 4. No statistically significant differences were found among all the treatments in serum GPT, TP, or ALB (P > 0.05). Dietary supplementation with 1,400 mg Zn/kg diet as Zn-Met significantly elevated the concentrations of serum, TBILI, GLU, and CA, as well as CRE (P < 0.001), and enhanced the activities of serum GOP and AKP in comparison with the control group (P < 0.001). No significant differences were observed in any of the blood chemical parameters among the hens fed 0, 70, 140, 350, or 700 mg Zn/kg diet as Zn-Met (P > 0.05). Table 4. Blood chemical parameters of hens during a study to evaluate zinc methionine (Zn-Met) safety.1   Added Zn-Met, mg/kg    P-value  Items  0  70  140  350  700  1,400  SEM  Linear  Quadratic  GPT, U/L  3.17  3.07  3.22  3.35  3.36  3.51  0.021  0.079  0.618  GOT, U/L  11.83b  10.89b  11.03b  11.19b  11.61b  16.38a  0.149  0.000  0.242  TBILI, μmol/L  4.38b  4.30b  4.67b  4.73b  4.80b  7.54a  0.041  0.000  0.326  AKP, U/L  212.57b  202.41b  205.91b  208.84b  210.42b  247.71a  2.491  0.000  0.605  TP, g/L  78.39  79.76  80.38  80.35  79.49  78.28  0.987  0.097  0.479  ALB, g/L  35.54  36.64  36.29  35.73  35.57  35.01  0.573  0.095  0.205  GLU, mmol/L  10.31b  10.11b  10.15b  10.19b  10.23b  14.35a  0.192  0.000  0.357  UA, mg/L  149.23b  144.26b  143.97b  145.40b  146.18b  172.59a  1.554  0.000  0.452  CRE, μmol/L  41.66b  39.28b  40.42b  40.77b  41.28b  51.84a  0.739  0.000  0.145    Added Zn-Met, mg/kg    P-value  Items  0  70  140  350  700  1,400  SEM  Linear  Quadratic  GPT, U/L  3.17  3.07  3.22  3.35  3.36  3.51  0.021  0.079  0.618  GOT, U/L  11.83b  10.89b  11.03b  11.19b  11.61b  16.38a  0.149  0.000  0.242  TBILI, μmol/L  4.38b  4.30b  4.67b  4.73b  4.80b  7.54a  0.041  0.000  0.326  AKP, U/L  212.57b  202.41b  205.91b  208.84b  210.42b  247.71a  2.491  0.000  0.605  TP, g/L  78.39  79.76  80.38  80.35  79.49  78.28  0.987  0.097  0.479  ALB, g/L  35.54  36.64  36.29  35.73  35.57  35.01  0.573  0.095  0.205  GLU, mmol/L  10.31b  10.11b  10.15b  10.19b  10.23b  14.35a  0.192  0.000  0.357  UA, mg/L  149.23b  144.26b  143.97b  145.40b  146.18b  172.59a  1.554  0.000  0.452  CRE, μmol/L  41.66b  39.28b  40.42b  40.77b  41.28b  51.84a  0.739  0.000  0.145  1Results are the means of 6 replicates of 2 hens each. GPT = glutamic pyruvic transaminase; GOT = glutamic oxalacetic transaminase; TBILI = total bilirubin; AKP = alkaline phosphatase; TP = total protein; ALB = albumin; GLU = glucose; UA = uric acid; CRE = creatinine. a,bValues within a row with no common superscripts differ significantly (P < 0.05). View Large Gross Pathology No hens showed obvious macroscopic lesions in the major organs, including the heart, liver, lung, and kidney at necropsy. Organ Index Effects of different levels of dietary Zn-Met supplementation on body weight and organ index of hens are shown in Table 5. No significant difference was detected in the body weight of hens (P > 0.05). Dietary Zn-Met supplementation did not remarkably affect cardiac index, hepatic index, splenic index, pulmonary index, or renal index of all the hens (P > 0.05). Table 5. Organ development of hens during a study to evaluate zinc-methionine (Zn-Met) safety.1   Added Zn-Met, mg/kg    P-value  Items  0  70  140  350  700  1400  SEM  Linear  Quadratic  Body weight, g  1598.38  1614.5  1623.26  1605.54  1590.96  1611.05  37.28  0.153  0.231  Cardiac index, %  0.386  0.395  0.403  0.389  0.374  0.382  0.009  0.097  0.254  Hepatic index, %  1.835  1.909  1.895  1.887  1.768  1.821  0.051  0.258  0.136  Splenic index, %  0.112  0.117  0.115  0.116  0.117  0.114  0.003  0.314  0.106  Pulmonary index, %  0.432  0.445  0.449  0.437  0.449  0.458  0.013  0.264  0.328  Renal index, %  0.589  0.605  0.594  0.603  0.615  0.612  0.015  0.349  0.753    Added Zn-Met, mg/kg    P-value  Items  0  70  140  350  700  1400  SEM  Linear  Quadratic  Body weight, g  1598.38  1614.5  1623.26  1605.54  1590.96  1611.05  37.28  0.153  0.231  Cardiac index, %  0.386  0.395  0.403  0.389  0.374  0.382  0.009  0.097  0.254  Hepatic index, %  1.835  1.909  1.895  1.887  1.768  1.821  0.051  0.258  0.136  Splenic index, %  0.112  0.117  0.115  0.116  0.117  0.114  0.003  0.314  0.106  Pulmonary index, %  0.432  0.445  0.449  0.437  0.449  0.458  0.013  0.264  0.328  Renal index, %  0.589  0.605  0.594  0.603  0.615  0.612  0.015  0.349  0.753  1Results are the means of 6 replicates of 2 hens each. View Large Histopathology No significant histopathological lesions were found in the heart, liver, lung, and kidney of hens in the control group (Figure 1). Microscopic results of the heart, liver, lung, and kidney in hens fed with 70, 140, 350, or 700 mg Zn/kg as Zn-Met are basically consistent with those in hens from the control group. However, extensive pathological changes in the heart, liver, lung, and kidney were found in hens receiving 1,400 mg Zn/kg as Zn-Met (Figure 1). In contrast to the control group, hens supplemented with 1,400 mg Zn/kg diet as Zn-Met developed severe cardiac damage, characterized by obvious fracture and disarray of myocardial fibers, and infiltration of inflammatory cells (Figure A1 and A2). Hepatic pathological alternations also were observed in hens receiving the highest dose (1,400 mg Zn/kg diet), evidenced by marked leukocyte aggregation, slight fatty degeneration, and necrosis of local tissue, as well as mild congestion (Figure B1 and B2). Microscopic examination of lung section revealed that dietary supplementation with 1,400 mg/kg diet as Zn-Met resulted in discernible leukocyte accumulation, pulmonary interstitial hyperplasia, as well as degeneration and necrosis of local tissue (Figure C1 and C2). When compared to the control group, hens treated with 1,400 mg Zn/kg diet as Zn-Met exhibited slight swelling of renal tubular epithelial cells, visible aggregation of inflammatory cells, and mild congestion in kidney tissue (Figure D1 and D2). Figure 1. View largeDownload slide Photomicrographs of histological sections (H&E staining). A1, B1, C1, and D1 represent heart, liver, lung, and kidney of hens receiving 0 mg Zn/kg (control) as Zn-Met, respectively. A2, B2, C2, and D2 represent heart, liver, lung, and kidney of hens administered 1,400 mg Zn/kg as Zn-Met, respectively. Figure 1. View largeDownload slide Photomicrographs of histological sections (H&E staining). A1, B1, C1, and D1 represent heart, liver, lung, and kidney of hens receiving 0 mg Zn/kg (control) as Zn-Met, respectively. A2, B2, C2, and D2 represent heart, liver, lung, and kidney of hens administered 1,400 mg Zn/kg as Zn-Met, respectively. DISCUSSION Zn is an essential trace element for animals and humans, but long-term or excessive exposure to Zn could cause detrimental impacts on health and performance (Dewar et al., 1983; Marrs et al., 1988; Richards et al., 1989; Park et al., 2004; Reis et al., 2010). Our present study found that dietary supplementation with no more than 700 mg Zn/kg as Zn-Met had no significantly negative effects on laying performance; however, supplementation with as high as 1,400 mg Zn/kg as Zn-Met for 8 wk exerted some adverse impacts on performance of layers. In accordance with the previous studies (Hermayer et al., 1977; Palafox and Ho-A, 1980), our current research showed a significant decrease in LR of hens supplemented with 1,400 mg Zn/kg as Zn-Met compared with all other groups. Additionally, in hens treated with 1,400 mg Zn/kg as Zn-Met, a significant increase in BSER and considerable decreases in ADEM and FCR were observed, implying that long-term exposure to excessive (1,400 mg Zn/kg) Zn-Met could have adverse effects on laying performance of hens. An excessive amount of Zn supplementation could interfere with the utilization of other nutrients, particularly its antagonistic trace elements (Fosmire, 1990; Romaña et al., 2011), which may be responsible for the negative effects of excessive Zn-Met on performance of layers. It is well known that UA is the end product of liver protein catabolism, and CRE is the end product of muscle creatine metabolism. It was reported that blood UA and CRE concentrations are critical clinical indices for monitoring the renal functions (Edwards et al., 1989; Andreasen et al., 1996; Valchev et al., 2013). The present results demonstrated that serum UA and CRE concentrations were significantly elevated in hens fed with 1,400 mg Zn/kg as Zn-Met in comparison with all other groups, which indicated that long-term exposure to high-dose Zn-Met could trigger the renal function impairment of hens. Under normal physiological condition, GPT and GOT mainly exist in cytoplasm and mitochondria, and once the liver is badly impaired, higher numbers of intracellular GPT and GOT are released into the serum (Madsen et al., 1958; Shimokawa et al., 1977). Activities of GPT, GOT and AKP, and TBILI level in serum are usually used to evaluate the hepatic and hepatobiliary functions in clinical examination (Iheukwumere et al., 2007; Itoh et al., 2008; Rekha et al., 2011). In the current study, it was showed that supplementation with no more than 700 mg Zn/kg as Zn-Met did not affect serum chemical parameters of layers. However, we found that dietary supplementation with 1,400 mg Zn/kg as Zn-Met markedly enhanced serum GOT and AKP activities, and improved serum TBILI level compared with all other groups, which implied that longer-term exposure to excessive (1,400 mg Zn/kg) Zn-Met may be detrimental to the functions of hepatic and hepatobiliary systems in hens. Interestingly, it was found that serum GLU concentration in hens receiving 1,400 mg Zn/kg as Zn-Met was notably elevated, which suggested that excessive (1,400 mg Zn/kg) Zn-Met exposure may disturb carbohydrate metabolism of laying hens (Li, 2014). Organ indices, which reflect the status of organ development in animals, are important parameters in the safety evaluation study (Dunnington and Siegel, 1995; Barri et al., 2008; Kabeir et al., 2008; Maatjens et al., 2016). Our results revealed that dietary Zn-Met supplementation did not affect the organ indices, which indicated that supplementation with 1,400 mg Zn/kg as Zn-Met could have no observed adverse effect on organ development. It is acknowledged that histopathology is a crucial technology support in the safety evaluation study. It was reported that excessive zinc supplementation may produce toxic effects and cause damage in the organs of animals (Smith and Embling, 1993). In the present study, no significant histological lesions were observed in organs of hens supplemented with no more than 700 mg Zn/kg as Zn-Met, whereas supplementation with as high as 1,400 mg Zn/kg as Zn-Met caused obvious injuries in some organs of layers. Gasaway and Buss (1972) demonstrated that significant lesions in liver were observed in domestic mallard ducks receiving 3,000 mg Zn/kg as zinc carbonate. The study by Dewar et al. (1983) showed that histological injuries in organs occurred in chicks fed 2,000 mg Zn/kg as zinc oxide. The finding by Abdel-Warith et al. (2011) presented that long-term exposure to 6 mg/L of zinc chloride caused hepatic damage in Nile tilapia. Consistent with the above reports, our present study found histological changes in the liver of hens administrated 1,400 mg Zn/kg as Zn-Met. In addition, major organs, including the heart, lung, and kidney, were damaged to some extent in 1,400 mg Zn/kg group, which indicated that long-term excessive (1,400 mg Zn/kg) Zn-Met exposure could give rise to histological lesions of major organs in laying hens. The detrimental effects of excess Zn-Met on histology of layers may be attributed to the fact that a considerable number of intracellular Zn retention triggers cytotoxicity and causes mitochondrial dysfunction as well as cell apoptosis (Lemire et al., 2010; Kao et al., 2012; Li et al., 2012). Even though no significant macroscopic alterations were observed in hens receiving 1,400 mg Zn/kg as Zn-Met, a continuously prolonged exposure to high-level Zn-Met may trigger gross pathological changes. In summary, our results reveal that dietary supplementation with up to 700 mg Zn/kg diet as Zn-Met has no negative effects on laying performance, clinical blood parameters, organ development, or histological architectures, while long-term exposure to 1,400 mg Zn/kg as Zn-Met has an adverse effect on laying performance, disturbs the hepatic and renal functions, and also causes histological lesions of visceral organs in laying hens. Hence, a nominal Zn concentration of 700 mg/kg as Zn-Met is considered non-adversarial following daily administration to hens for 56 days. Acknowledgements The study was funded by National Key Technology Research and Development Program (2013BAD20B02) and Feed Quality and Safety Supervision Project of Ministry of Agriculture of China (2014). 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Safety evaluation of zinc methionine in laying hens: Effects on laying performance, clinical blood parameters, organ development, and histopathology

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Oxford University Press
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0032-5791
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Abstract

Abstract The study was conducted to investigate whether high-dose zinc methionine (Zn-Met) affected the safety of laying hens, including laying performance, hematological parameters, serum chemical parameters, organ index, and histopathology. A total of 540 20-week-old Hy-Line White laying hens was randomly allocated to 6 groups with 6 replicates of 15 birds each. Birds were fed diets supplemented with 0 (control), 70, 140, 350, 700, or 1,400 mg Zn/kg diet as Zn-Met. The experiment lasted for 8 wk after a 2-week acclimation period. Results showed that dietary supplementation with 70 or 140 mg Zn/kg diet as Zn-Met significantly increased average daily egg mass (ADEM), laying rate (LR), and feed conversion ratio (FCR) (P < 0.05) and lowered broken and soft-shelled egg ratio (BSER) (P < 0.05) in comparison with the control group; no significant differences were detected among hens fed with 0, 350, or 700 mg Zn/kg as Zn-Met (P > 0.05); hens administered 1,400 mg Zn/kg showed a significant increase in BSER and remarkable decreases in ADEM, LR, and FCR (P < 0.001). There were no significant differences among hens receiving 0, 70, 140, 350, or 700 mg Zn/kg as Zn-Met in serum chemical parameters (P > 0.05); supplementation with 1,400 mg Zn/kg as Zn-Met remarkably elevated the concentrations of serum total bilirubin (TBILI), glucose (GLU), uric acid (UA), and creatinine (CRE) (P < 0.001), and enhanced activities of serum glutamic oxalacetic transaminase (GOP) and alkaline phosphatase (AKP) (P < 0.001) compared with the control group. No significant histopathological changes were found in hens administered 0, 70, 140, 350, or 700 mg Zn/kg as Zn-Met, while significant histological lesions were observed in the heart, liver, lung, and kidney tissues of hens receiving 1,400 mg Zn/kg as Zn-Met. No significant differences were detected in hematological parameters or organ index (P > 0.05). In conclusion, a nominal Zn concentration of 700 mg/kg as Zn-Met is considered to be no-observed-adverse-effect level following daily administration to hens for 56 days. INTRODUCTION Zinc methionine (Zn-Met), an organic source of Zn, has become increasingly popular in poultry production because dietary supplementation with optimal levels of Zn-Met was reported to promote laying performance, improve egg quality, elevate hatchability, and enhance the capabilities to resist diseases and stresses in poultry (Pimentel et al., 1991; Kienholz et al., 1992; Moreng et al., 1992; Hamidi and Pourreza, 2009; Wang et al., 2014; Jahanian and Rasouli, 2015; Mohammadi et al., 2015). However, available data and literature concerning the safety of Zn-Met in laying hens are not known. Much attention should be attached to the potential detrimental effects of excessive exposure to Zn-Met in hens. It is well acknowledged that Zn is an essential trace element for the health and growth of animals, and is involved in diverse biological and metabolic processes, including DNA synthesis, gene expression regulation, and nutrient metabolism (Bray and Bettger, 1990; Prasad, 2009). Although zinc is essential for animals, long-term or excessive exposure to zinc has adverse or even toxic effects on various systems in the body, such as the reproductive, immune, digestive, neural, and respiratory systems (Porea et al., 2000; Walther et al., 2002; Palmiter, 2004; Tetsuchikawahara et al., 2005). Zn at relatively low to moderate concentrations plays crucial roles in cellular metabolism and cell proliferation, but Zn at extremely high concentrations can interfere with intracellular Zn ion homeostasis and trigger cytotoxicity, which is characterized by the breakdown of mitochondrial membrane potential, caspase activation, and cell apoptosis (Fosmire, 1990; Kao et al., 2012; Lemire et al., 2010; Wang et al., 2017). Hence, this study was conducted to investigate whether high-dose Zn-Met affected the safety of hens, including laying performance, hematological parameters, serum chemical parameters, organ development, and histopathology. In the present study, high-dose Zn-Met was chosen to provide significant exposure to the test material, while other dose levels were set to provide adequate and convincing data for risk evaluation. MATERIALS AND METHODS Study Design The study was designed in accordance with the guidelines for safety evaluation of feed additives in animals by the European Food Safety Authority (EFSA) and Ministry of Agriculture of China. A total of 540 20-week-old Hy-Line White laying hens with similar body weights and performances was randomly allocated to 6 groups with 6 replicates of 15 birds each (90 hens per group). All birds were acclimated to a basal diet and the environment for 2 weeks. The birds received diets supplemented with 0 (control), 70 [the maximum recommended available dose (MRAD)], 140 (2-fold of MRAD), 350 (5-fold of MRAD), 700 (10-fold of MRAD), or 1400 mg Zn/kg diet (20-fold of MRAD) as Zn-Met for 8 weeks. Clinical observations were conducted throughout the experiment, and laying performance was determined. At the end of the feeding trial, 72 birds (2 hens from each replicate, 12 hens per group) were sacrificed and subjected to full postmortem examinations. Jugular vein blood samples were collected for a clinical pathological examination, while selected visceral organs were weighed, and specified tissues were taken for subsequent histopathological examinations. Birds, Diets, and Management This study was conducted at Feed Science Institute, Zhejiang University. Zn-Met was provided by JiningHeshi Biotechnology Co., Ltd (Shandong, China) with 97% purity. Birds were kept in the ventilated room with a lighting schedule of 16-h light/8-h dark cycle. Housing temperature and relative humidity were maintained at 23 ± 5°C and 65% ± 5%, respectively. Three hens were raised in an individual battery cage (45 × 45 × 50 cm). All hens were allowed access to water all the time and fed ad libitum twice daily. The composition and nutrient levels of the basal diet are presented in Table 1. All the experimental protocols were approved by the Animal Care and Use Committee of Zhejiang University. Table 1. Ingredients and nutrient levels of basal diet (air-dry basis). Items  Ingredients  Contents (%)  Corn  59.20  Soybean meal  26.50  Soybean oil  2.00  Calcium hydrophosphate  1.20  Limestone  8.60  Salt  0.30  Methionine  0.20  Premix1  2.00  Total  100.00  Nutrient levels2    ME (MJ/kg)  11.40  Crude protein (%)  16.27  Calcium (%)  3.48  Total phosphorus (%)  0.56  Available phosphorus (%)  0.38  Lysine (%)  0.82  Met+Cys (%)  0.67  Zinc3 (mg/kg)  34.21  Items  Ingredients  Contents (%)  Corn  59.20  Soybean meal  26.50  Soybean oil  2.00  Calcium hydrophosphate  1.20  Limestone  8.60  Salt  0.30  Methionine  0.20  Premix1  2.00  Total  100.00  Nutrient levels2    ME (MJ/kg)  11.40  Crude protein (%)  16.27  Calcium (%)  3.48  Total phosphorus (%)  0.56  Available phosphorus (%)  0.38  Lysine (%)  0.82  Met+Cys (%)  0.67  Zinc3 (mg/kg)  34.21  1Premix provided the following per kilogram of diet: Mn, 65 mg; I 1 mg; Fe, 60 mg; Cu, 8 mg; Se, 0.3 mg; vitamin A, 12,500 IU; vitamin D3, 4125 IU, vitamin E, 15 IU; vitamin K, 2 mg; thiamine, 1 mg; riboflavin, 8.5 mg; calcium pantothenate, 50 mg; niacin, 32.5 mg; pyridoxine, 8 mg; folic aicd, 5 mg; vitamin B12, 5 mg; choline cloride, 500 mg. 2Calculated from tables of feed composition and nutritive values provided by Feed Database in China (2004). 3Analyzed by inductively coupled plasma atomic emission spectrometry. View Large Sample Collection and Analytical Determination Clinical Observations. Close cage-side observations were performed 3 times daily throughout the feeding trial period. Mortality and abnormal behaviors in clinical circumstances, including dullness, anorexia, ruffled feather, molt, lethargy, depression, recumbence, shrunken eyes, and mucous mixed droppings, were recorded. Laying Performance. Egg number, total egg weight, and number of broken and soft-shelled eggs of each replicate were monitored daily, and feed consumption on a replicate basis was recorded at weekly intervals. Egg weight (EW), average daily egg mass (ADEM), average daily feed intake (ADFI), laying rate (LR), and feed conversion ratio (FCR) were then calculated based on the collected data. The LR was expressed as average hen-day production, calculated from the total number of eggs divided by the total number of hens. The FCR was calculated as grams of egg produced per gram of feed consumed. Clinical Blood Parameters. At the end of the feeding trial, 2 hens from each replicate (12 hens per group) were chosen randomly. Each hen was weighed and sacrificed by exsanguination via the jugular vein. Jugular vein blood samples for hematological and clinical chemical analysis were collected. 2 mL of blood sample were collected in EDTA-containing tubes for hematology, whereas 5 mL of blood were taken for clinical chemical analysis. Hematological parameters of the collected samples involving white blood cells (WBC), red blood cells (RBC), platelet count (PLT), hematocrit (HCT), hemoglobin (HGB), and red cell distribution width (RCDW) were measured by A Sysmex XE2100 automated hematology analyzer (Sysmex Co., Kobe, Japan). Serum samples were prepared by centrifuging at 3500 × g for 10 min at 4 °C. Chemical parameters, including the activities of glutamic pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) and the concentrations of total bilirubin (TBILI), alkaline phosphatase (AKP), total protein (TP), albumin (ALB), glucose (GLU), uric acid (UA), and creatinine (CRE), were measured in serum samples using a RX Daytona Chemical Analyzer (Randox, London, UK). These parameters were determined in order to cover a range of potential toxicities, including the disturbances to material metabolism and the damage to the major organs. Gross Pathology. After exsanguination via the jugular vein, the hens were subjected to full postmortem examinations for gross lesions in the heart, liver, lung, and kidney, including swelling, bleeding, and necrosis of organs, as well as the chalky white deposits of urate on the surface of the visceral organs. Organ Index. After postmortem examinations, the heart, liver, spleen, lung, and kidney were collected and weighed. The organ index was calculated via the formula: organ index (%) = organ weight/body weight × 100. Histopathology. Portions of the heart, liver, lung, and kidney tissues were fixed in 10% buffered formalin (Chemical Laboratory Center of Zhejiang University, Hangzhou, China). For histological analysis, the formalin-fixed samples were processed into wax sections of 5 μm thickness and stained with hematoxylin-eosin (H&E) in accordance with the method reported by Jadhav et al. (2007). Light microscopic examination for histopathological alterations was conducted under a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). The corresponding images were captured by DigiLab II digital microscope mutual systems (Motic Groups, Xiamen, China). Statistical Analysis All data were analyzed using a one-way analysis of variance (ANOVA, SPSS 20.0 for Windows, SPSS Inc., Chicago, IL). When significant differences were detected, a 5% variance average was used, with a Duncan's multiple comparison test for comparing among the groups. A P-value of less than 0.05 was considered significant. RESULTS Clinical Observations No mortality or significant molt occurred throughout the feeding trial. All hens appeared normal without any significant clinical signs of intoxication during the entire experimental period. Laying Performance Effects of different levels of dietary Zn-Met supplementation on laying performance of hens are presented in Table 2. ADFI and EW were unaffected by dietary Zn-Met supplementation (P > 0.05). Compared with the control group, dietary supplementation with 70 or 140 mg Zn/kg diet as Zn-Met significantly increased ADEM, LR, and FCR (P < 0.05) and considerably lowered BSER (P < 0.001). Hens fed with 1,400 mg Zn/kg diet as Zn-Met showed a significant increase in BSER (P < 0.001) and remarkable decreases in ADEM, LR, and FCR (P < 0.001). There were no statistically significant differences regarding the measured parameters of laying performance among the hens supplemented with 0, 350, or 700 mg Zn/kg diet as Zn-Met (P > 0.05). Table 2. Laying performance of hens during a study to evaluate zinc methionine (Zn-Met) safety.1 Added Zn levels  Laying performance  mg/kg  EW, g  ADEM, g/hen/d  ADFI, g/hen/d  LR, %  FCR, %  BSER, %  0  53.62  47.58b  97.88  88.73b  48.54b  0.61b  70  53.90  48.87a  97.69  90.68a  50.25a  0.21c  140  53.56  48.45a  98.03  90.47a  49.75a  0.26c  350  53.34  47.51b  97.80  89.07a,b  48.78b  0.57b  700  53.61  47.25b  98.59  88.13b  48.31b  0.65b  1400  53.11  42.52b  94.97  80.07c  44.84c  1.98a  SEM  0.615  0.604  0.881  0.919  0.574  0.081  P-value              Linear  0.489  0.000  0.067  0.000  0.000  0.000  Quadratic  0.127  0.000  0.781  0.000  0.000  0.002  Added Zn levels  Laying performance  mg/kg  EW, g  ADEM, g/hen/d  ADFI, g/hen/d  LR, %  FCR, %  BSER, %  0  53.62  47.58b  97.88  88.73b  48.54b  0.61b  70  53.90  48.87a  97.69  90.68a  50.25a  0.21c  140  53.56  48.45a  98.03  90.47a  49.75a  0.26c  350  53.34  47.51b  97.80  89.07a,b  48.78b  0.57b  700  53.61  47.25b  98.59  88.13b  48.31b  0.65b  1400  53.11  42.52b  94.97  80.07c  44.84c  1.98a  SEM  0.615  0.604  0.881  0.919  0.574  0.081  P-value              Linear  0.489  0.000  0.067  0.000  0.000  0.000  Quadratic  0.127  0.000  0.781  0.000  0.000  0.002  1Results are the means of 6 replicates of 15 hens each. EW = egg weight; ADEM = average daily egg mass; ADFI = average daily feed intake; LR = laying rate; FCR = feed conversion ratio; BSER = broken and soft-shelled egg ratio. a-cValues within a column with no common superscripts differ significantly (P < 0.05). View Large Clinical Blood Parameters Hematological parameters of hens administered different levels of Zn-Met are displayed in Table 3. There were no statistically significant differences in any of the determined hematological parameters (P > 0.05). Table 3. Hematological parameters of hens during a study to evaluate zinc methionine (Zn-Met) safety.1 Added Zn levels  Hematological parameters  mg/kg  WBC, 109/L  RBC, 1012/L  PLT, 109/L  HCT, %  HGB, g/L  RCDW, %  0  266.29  2.86  1.04  32.61  86.42  39.54  70  272.23  2.90  1.25  32.82  85.80  38.21  140  266.84  2.89  1.33  32.56  84.67  38.47  350  268.57  2.88  1.29  32.66  85.33  40.05  700  268.97  2.87  1.17  31.63  83.42  39.96  1400  288.13  2.89  1.17  33.28  84.75  40.32  SEM  4.456  0.029  0.087  0.298  0.722  0.063  P-value              Linear  0.205  0.199  0.541  0.083  0.093  0.478  Quadratic  0.186  0.436  0.209  0.571  0.176  0.192  Added Zn levels  Hematological parameters  mg/kg  WBC, 109/L  RBC, 1012/L  PLT, 109/L  HCT, %  HGB, g/L  RCDW, %  0  266.29  2.86  1.04  32.61  86.42  39.54  70  272.23  2.90  1.25  32.82  85.80  38.21  140  266.84  2.89  1.33  32.56  84.67  38.47  350  268.57  2.88  1.29  32.66  85.33  40.05  700  268.97  2.87  1.17  31.63  83.42  39.96  1400  288.13  2.89  1.17  33.28  84.75  40.32  SEM  4.456  0.029  0.087  0.298  0.722  0.063  P-value              Linear  0.205  0.199  0.541  0.083  0.093  0.478  Quadratic  0.186  0.436  0.209  0.571  0.176  0.192  1Results are the means of 6 replicates of 2 hens each. WBC = white blood cells; RBC = red blood cells; PLT = platelet count; HCT = hematocrit; HGB = hemoglobin; RCDW = red cell distribution width. View Large Effects of dietary supplementation with different levels of Zn-Met on blood chemical parameters of hens are shown in Table 4. No statistically significant differences were found among all the treatments in serum GPT, TP, or ALB (P > 0.05). Dietary supplementation with 1,400 mg Zn/kg diet as Zn-Met significantly elevated the concentrations of serum, TBILI, GLU, and CA, as well as CRE (P < 0.001), and enhanced the activities of serum GOP and AKP in comparison with the control group (P < 0.001). No significant differences were observed in any of the blood chemical parameters among the hens fed 0, 70, 140, 350, or 700 mg Zn/kg diet as Zn-Met (P > 0.05). Table 4. Blood chemical parameters of hens during a study to evaluate zinc methionine (Zn-Met) safety.1   Added Zn-Met, mg/kg    P-value  Items  0  70  140  350  700  1,400  SEM  Linear  Quadratic  GPT, U/L  3.17  3.07  3.22  3.35  3.36  3.51  0.021  0.079  0.618  GOT, U/L  11.83b  10.89b  11.03b  11.19b  11.61b  16.38a  0.149  0.000  0.242  TBILI, μmol/L  4.38b  4.30b  4.67b  4.73b  4.80b  7.54a  0.041  0.000  0.326  AKP, U/L  212.57b  202.41b  205.91b  208.84b  210.42b  247.71a  2.491  0.000  0.605  TP, g/L  78.39  79.76  80.38  80.35  79.49  78.28  0.987  0.097  0.479  ALB, g/L  35.54  36.64  36.29  35.73  35.57  35.01  0.573  0.095  0.205  GLU, mmol/L  10.31b  10.11b  10.15b  10.19b  10.23b  14.35a  0.192  0.000  0.357  UA, mg/L  149.23b  144.26b  143.97b  145.40b  146.18b  172.59a  1.554  0.000  0.452  CRE, μmol/L  41.66b  39.28b  40.42b  40.77b  41.28b  51.84a  0.739  0.000  0.145    Added Zn-Met, mg/kg    P-value  Items  0  70  140  350  700  1,400  SEM  Linear  Quadratic  GPT, U/L  3.17  3.07  3.22  3.35  3.36  3.51  0.021  0.079  0.618  GOT, U/L  11.83b  10.89b  11.03b  11.19b  11.61b  16.38a  0.149  0.000  0.242  TBILI, μmol/L  4.38b  4.30b  4.67b  4.73b  4.80b  7.54a  0.041  0.000  0.326  AKP, U/L  212.57b  202.41b  205.91b  208.84b  210.42b  247.71a  2.491  0.000  0.605  TP, g/L  78.39  79.76  80.38  80.35  79.49  78.28  0.987  0.097  0.479  ALB, g/L  35.54  36.64  36.29  35.73  35.57  35.01  0.573  0.095  0.205  GLU, mmol/L  10.31b  10.11b  10.15b  10.19b  10.23b  14.35a  0.192  0.000  0.357  UA, mg/L  149.23b  144.26b  143.97b  145.40b  146.18b  172.59a  1.554  0.000  0.452  CRE, μmol/L  41.66b  39.28b  40.42b  40.77b  41.28b  51.84a  0.739  0.000  0.145  1Results are the means of 6 replicates of 2 hens each. GPT = glutamic pyruvic transaminase; GOT = glutamic oxalacetic transaminase; TBILI = total bilirubin; AKP = alkaline phosphatase; TP = total protein; ALB = albumin; GLU = glucose; UA = uric acid; CRE = creatinine. a,bValues within a row with no common superscripts differ significantly (P < 0.05). View Large Gross Pathology No hens showed obvious macroscopic lesions in the major organs, including the heart, liver, lung, and kidney at necropsy. Organ Index Effects of different levels of dietary Zn-Met supplementation on body weight and organ index of hens are shown in Table 5. No significant difference was detected in the body weight of hens (P > 0.05). Dietary Zn-Met supplementation did not remarkably affect cardiac index, hepatic index, splenic index, pulmonary index, or renal index of all the hens (P > 0.05). Table 5. Organ development of hens during a study to evaluate zinc-methionine (Zn-Met) safety.1   Added Zn-Met, mg/kg    P-value  Items  0  70  140  350  700  1400  SEM  Linear  Quadratic  Body weight, g  1598.38  1614.5  1623.26  1605.54  1590.96  1611.05  37.28  0.153  0.231  Cardiac index, %  0.386  0.395  0.403  0.389  0.374  0.382  0.009  0.097  0.254  Hepatic index, %  1.835  1.909  1.895  1.887  1.768  1.821  0.051  0.258  0.136  Splenic index, %  0.112  0.117  0.115  0.116  0.117  0.114  0.003  0.314  0.106  Pulmonary index, %  0.432  0.445  0.449  0.437  0.449  0.458  0.013  0.264  0.328  Renal index, %  0.589  0.605  0.594  0.603  0.615  0.612  0.015  0.349  0.753    Added Zn-Met, mg/kg    P-value  Items  0  70  140  350  700  1400  SEM  Linear  Quadratic  Body weight, g  1598.38  1614.5  1623.26  1605.54  1590.96  1611.05  37.28  0.153  0.231  Cardiac index, %  0.386  0.395  0.403  0.389  0.374  0.382  0.009  0.097  0.254  Hepatic index, %  1.835  1.909  1.895  1.887  1.768  1.821  0.051  0.258  0.136  Splenic index, %  0.112  0.117  0.115  0.116  0.117  0.114  0.003  0.314  0.106  Pulmonary index, %  0.432  0.445  0.449  0.437  0.449  0.458  0.013  0.264  0.328  Renal index, %  0.589  0.605  0.594  0.603  0.615  0.612  0.015  0.349  0.753  1Results are the means of 6 replicates of 2 hens each. View Large Histopathology No significant histopathological lesions were found in the heart, liver, lung, and kidney of hens in the control group (Figure 1). Microscopic results of the heart, liver, lung, and kidney in hens fed with 70, 140, 350, or 700 mg Zn/kg as Zn-Met are basically consistent with those in hens from the control group. However, extensive pathological changes in the heart, liver, lung, and kidney were found in hens receiving 1,400 mg Zn/kg as Zn-Met (Figure 1). In contrast to the control group, hens supplemented with 1,400 mg Zn/kg diet as Zn-Met developed severe cardiac damage, characterized by obvious fracture and disarray of myocardial fibers, and infiltration of inflammatory cells (Figure A1 and A2). Hepatic pathological alternations also were observed in hens receiving the highest dose (1,400 mg Zn/kg diet), evidenced by marked leukocyte aggregation, slight fatty degeneration, and necrosis of local tissue, as well as mild congestion (Figure B1 and B2). Microscopic examination of lung section revealed that dietary supplementation with 1,400 mg/kg diet as Zn-Met resulted in discernible leukocyte accumulation, pulmonary interstitial hyperplasia, as well as degeneration and necrosis of local tissue (Figure C1 and C2). When compared to the control group, hens treated with 1,400 mg Zn/kg diet as Zn-Met exhibited slight swelling of renal tubular epithelial cells, visible aggregation of inflammatory cells, and mild congestion in kidney tissue (Figure D1 and D2). Figure 1. View largeDownload slide Photomicrographs of histological sections (H&E staining). A1, B1, C1, and D1 represent heart, liver, lung, and kidney of hens receiving 0 mg Zn/kg (control) as Zn-Met, respectively. A2, B2, C2, and D2 represent heart, liver, lung, and kidney of hens administered 1,400 mg Zn/kg as Zn-Met, respectively. Figure 1. View largeDownload slide Photomicrographs of histological sections (H&E staining). A1, B1, C1, and D1 represent heart, liver, lung, and kidney of hens receiving 0 mg Zn/kg (control) as Zn-Met, respectively. A2, B2, C2, and D2 represent heart, liver, lung, and kidney of hens administered 1,400 mg Zn/kg as Zn-Met, respectively. DISCUSSION Zn is an essential trace element for animals and humans, but long-term or excessive exposure to Zn could cause detrimental impacts on health and performance (Dewar et al., 1983; Marrs et al., 1988; Richards et al., 1989; Park et al., 2004; Reis et al., 2010). Our present study found that dietary supplementation with no more than 700 mg Zn/kg as Zn-Met had no significantly negative effects on laying performance; however, supplementation with as high as 1,400 mg Zn/kg as Zn-Met for 8 wk exerted some adverse impacts on performance of layers. In accordance with the previous studies (Hermayer et al., 1977; Palafox and Ho-A, 1980), our current research showed a significant decrease in LR of hens supplemented with 1,400 mg Zn/kg as Zn-Met compared with all other groups. Additionally, in hens treated with 1,400 mg Zn/kg as Zn-Met, a significant increase in BSER and considerable decreases in ADEM and FCR were observed, implying that long-term exposure to excessive (1,400 mg Zn/kg) Zn-Met could have adverse effects on laying performance of hens. An excessive amount of Zn supplementation could interfere with the utilization of other nutrients, particularly its antagonistic trace elements (Fosmire, 1990; Romaña et al., 2011), which may be responsible for the negative effects of excessive Zn-Met on performance of layers. It is well known that UA is the end product of liver protein catabolism, and CRE is the end product of muscle creatine metabolism. It was reported that blood UA and CRE concentrations are critical clinical indices for monitoring the renal functions (Edwards et al., 1989; Andreasen et al., 1996; Valchev et al., 2013). The present results demonstrated that serum UA and CRE concentrations were significantly elevated in hens fed with 1,400 mg Zn/kg as Zn-Met in comparison with all other groups, which indicated that long-term exposure to high-dose Zn-Met could trigger the renal function impairment of hens. Under normal physiological condition, GPT and GOT mainly exist in cytoplasm and mitochondria, and once the liver is badly impaired, higher numbers of intracellular GPT and GOT are released into the serum (Madsen et al., 1958; Shimokawa et al., 1977). Activities of GPT, GOT and AKP, and TBILI level in serum are usually used to evaluate the hepatic and hepatobiliary functions in clinical examination (Iheukwumere et al., 2007; Itoh et al., 2008; Rekha et al., 2011). In the current study, it was showed that supplementation with no more than 700 mg Zn/kg as Zn-Met did not affect serum chemical parameters of layers. However, we found that dietary supplementation with 1,400 mg Zn/kg as Zn-Met markedly enhanced serum GOT and AKP activities, and improved serum TBILI level compared with all other groups, which implied that longer-term exposure to excessive (1,400 mg Zn/kg) Zn-Met may be detrimental to the functions of hepatic and hepatobiliary systems in hens. Interestingly, it was found that serum GLU concentration in hens receiving 1,400 mg Zn/kg as Zn-Met was notably elevated, which suggested that excessive (1,400 mg Zn/kg) Zn-Met exposure may disturb carbohydrate metabolism of laying hens (Li, 2014). Organ indices, which reflect the status of organ development in animals, are important parameters in the safety evaluation study (Dunnington and Siegel, 1995; Barri et al., 2008; Kabeir et al., 2008; Maatjens et al., 2016). Our results revealed that dietary Zn-Met supplementation did not affect the organ indices, which indicated that supplementation with 1,400 mg Zn/kg as Zn-Met could have no observed adverse effect on organ development. It is acknowledged that histopathology is a crucial technology support in the safety evaluation study. It was reported that excessive zinc supplementation may produce toxic effects and cause damage in the organs of animals (Smith and Embling, 1993). In the present study, no significant histological lesions were observed in organs of hens supplemented with no more than 700 mg Zn/kg as Zn-Met, whereas supplementation with as high as 1,400 mg Zn/kg as Zn-Met caused obvious injuries in some organs of layers. Gasaway and Buss (1972) demonstrated that significant lesions in liver were observed in domestic mallard ducks receiving 3,000 mg Zn/kg as zinc carbonate. The study by Dewar et al. (1983) showed that histological injuries in organs occurred in chicks fed 2,000 mg Zn/kg as zinc oxide. The finding by Abdel-Warith et al. (2011) presented that long-term exposure to 6 mg/L of zinc chloride caused hepatic damage in Nile tilapia. Consistent with the above reports, our present study found histological changes in the liver of hens administrated 1,400 mg Zn/kg as Zn-Met. In addition, major organs, including the heart, lung, and kidney, were damaged to some extent in 1,400 mg Zn/kg group, which indicated that long-term excessive (1,400 mg Zn/kg) Zn-Met exposure could give rise to histological lesions of major organs in laying hens. The detrimental effects of excess Zn-Met on histology of layers may be attributed to the fact that a considerable number of intracellular Zn retention triggers cytotoxicity and causes mitochondrial dysfunction as well as cell apoptosis (Lemire et al., 2010; Kao et al., 2012; Li et al., 2012). Even though no significant macroscopic alterations were observed in hens receiving 1,400 mg Zn/kg as Zn-Met, a continuously prolonged exposure to high-level Zn-Met may trigger gross pathological changes. In summary, our results reveal that dietary supplementation with up to 700 mg Zn/kg diet as Zn-Met has no negative effects on laying performance, clinical blood parameters, organ development, or histological architectures, while long-term exposure to 1,400 mg Zn/kg as Zn-Met has an adverse effect on laying performance, disturbs the hepatic and renal functions, and also causes histological lesions of visceral organs in laying hens. Hence, a nominal Zn concentration of 700 mg/kg as Zn-Met is considered non-adversarial following daily administration to hens for 56 days. Acknowledgements The study was funded by National Key Technology Research and Development Program (2013BAD20B02) and Feed Quality and Safety Supervision Project of Ministry of Agriculture of China (2014). 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Poultry ScienceOxford University Press

Published: Apr 1, 2018

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