Bioavailability of organic and inorganic zinc sources in male broilers

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1 Bioavailability of organic and inorganic zinc sources in male broilers L. Star,* 1 J. D. van der Klis,* C. Rapp, and T. L. Ward * Schothorst Feed Research, PO Box 533, 8200 AM Lelystad, the Netherlands; Zinpro Animal Nutrition, Akkerdistel 2E, 5831 PJ Boxmeer, the Netherlands; and Zinpro Corporation, Viking Drive Suite 240, Eden Prairie, MN ABSTRACT The objective of the current study was to determine the bioavailability of an organic zinc source (Availa-Zn) compared with zinc sulfate in a Europeantype broiler diet. A total of 480 one-day-old male Ross 308 broilers were housed in 48 digestibility cages (10 birds per cage), being randomly divided over 9 treatments. At d 3, the number of birds was standardized to 8. Birds were fed a basal wheat-maize-soya diet (containing 33.5 mg of Zn/kg) with different supplementation levels of zinc (reference zinc source: inorganic zinc sulfate: 0, 5, 10, 15, 20, and 40 mg of Zn/kg of feed; test zinc source: Availa-Zn: 0, 5, 10, 15 mg of Zn/kg of INTRODUCTION Zinc is an essential trace element for birds as a cofactor in many enzymes and involved in protein synthesis, carbohydrate and energy metabolism, and other biochemical reactions. Among other processes, it plays a role in DNA and RNA synthesis, tissue growth and repair, bone mineralization, and blood clotting (as reviewed by Salim et al., 2008). Due to its central role, a zinc deficiency will cause numerous physical and pathological changes including skin lesions, decreased growth, general disability of bones and joints, poor feathering, and reduced immunity to infection of several diseases. Zinc requirement of chicks was first defined to be 30 mg/kg of feed (Roberson and Schaible, 1958). Since then, research provided new evidence for the NRC (1994) to set the requirement of broilers at 40 mg/ kg of feed. As summarized by Salim et al. (2008), zinc supplementation up to a level of about 30 to 50 mg of Zn/kg of feed mainly increased feed intake and weight gain and improved feed efficiency, whereas effects of higher levels of Zn in the diet were less consistent. feed). Production performance and tibia zinc content were measured. There were no differences in production performance between the different zinc sources when fed to broilers until 21 d of age. Tibia zinc content was increased linearly with the dietary zinc content up to 20 mg/kg zinc sulfate. The relative biological value of organic zinc was 1.64 compared with zinc sulfate as a reference zinc source (1.00), as indicated by the slope ratio of the linear response curves for both zinc sources, using tibia zinc content as a response parameter. In a practical European broiler diet, the organic Availa-Zn had a higher bioavailability than inorganic zinc sulfate. Key words: Availa-Zn, bioavailability, performance, tibia ash, zinc sulfate 2012 Poultry Science 91 : Poultry Science Association Inc. Received March 18, Accepted September 1, Corresponding author: lstar@schothorst.nl Zinc is added to broiler diets usually as inorganic feed grade zinc sulfate, zinc chloride, zinc oxide, or one of the organic forms complexed to amino acids, proteins, or carbohydrates. The nutritional value of mineral sources depends on the concentration in the feed; potential interactions with, for example, calcium; the amount of the element that is absorbed, the bioavailability of the element to the bird (Salim et al., 2008), or a combination of these. In recent years, organic zinc sources have been used increasingly due to their potentially higher zinc bioavailability (Kidd et al., 1996; Salim et al., 2010). However, some studies indicated only small or no differences in bioavailability of Zn between organic and inorganic sources (Pimentel et al., 1991; Ammerman et al., 1995). The composition of the diet can also influence bioavailability. A major antagonist known to decrease zinc availability is phytate that is prevalent in whole grains (O Dell and Savage, 1960). Addition of phytase, an enzyme that degrades phytate, to broiler diets is a common practice in Europe. Accurate values for the bioavailability of different organic and inorganic zinc sources are important to ensure adequate zinc supplementation of broiler diets. This study was carried out to determine the bioavailability of an organic zinc source (Availa-Zn) compared 3115

2 3116 Star et al. Table 1. Experimental design with feed grade zinc sulfate as a reference zinc source in a practical European broiler diet. MATERIALS AND METHODS Birds and Housing A total of 480 one-day-old male Ross 308 broiler chickens were used in a dose-response study evaluating the bioavailability of 2 different zinc sources: 1) feed grade zinc sulfate (ZnSO 4 7H 2 O) used as a reference zinc source and 2) Availa-Zn (Zinpro Corporation, Eden Prairie, MN), an organic zinc source based on single amino acids from hydrolyzed soy protein. The study was performed with 9 dietary treatments consisting of a diet without added zinc, zinc sulfate providing 5, 10, 15, 20, and 40 mg/kg of zinc and organic zinc providing 5, 10, and 15 mg/kg of zinc (Table 1). Each treatment consisted of 5 (zinc sulfate) or 6 replicates (organic zinc). At d 0, broilers arrived at the poultry facility of Schothorst Feed Research (Lelystad, the Netherlands) and were housed in digestibility cages (made of stainless steel). Based on BW, 10 birds were allotted to each replicate cage resulting in a similar mean BW per cage. Birds were housed in these cages until the end of the experiment at d 21. At d 3, the number of birds was standardized to 8 per cage. Standardization (based on BW) was done first by removing birds with visual aberrations and second at random to decrease the number to 8. Ambient temperature was gradually decreased by approximately 0.5 C per day from 32 C at the start of the experiment to 24 C at the end of the experiment. Feed and water were supplied for ad libitum intake throughout the entire experiment. The Institutional Animal Care and Use Committee approved each protocol. Experimental Diets Supplemented dose (mg/kg of feed) Number of replicates 1 Control Zinc sulfate Zinc sulfate Zinc sulfate Zinc sulfate Zinc sulfate Organic zinc Organic zinc Organic zinc Availa-Zn, Zinpro Corporation, Eden Prairie, MN. Table 2. Composition of the basal diet without added zinc sources Item The broilers were supplied a pelleted basal wheatmaize-soya diet without supplemental zinc from day of arrival to 3 d of age. A basal diet (Table 2) was formulated, which contained 33.5 mg of Zn/kg. Experimental diets were manufactured by adding the respective zinc source to the basal diet. All diets met or exceeded nutrient requirements of broilers according to Dutch standards (CVB, 2007), except for dietary zinc. From d 3 onward, the birds were fed their respective pelleted experimental diets until the end of the experiment (d 21). Feed Analysis Basal diet Ingredient (%) Maize (Zn) Wheat (Zn) Soybean meal Hipro (Zn) Maize gluten meal >60% CP 6.00 Soybean oil 3.00 Monocalcium phosphate (Zn) 0.38 Limestone 1.12 Salt 0.42 Premix Lysine-HCl (l, 79%) 0.38 Methionine (dl, 99%) 0.15 Threonine (l, 98%) 0.02 Tryptophan (l, 98%) 0.02 Nutrient (g/kg) Moisture 124 Ash 43 CP 201 Crude fat 56 Crude fiber 23 Starch 417 AME (kcal/kg) 3,018 Phosphorus 4.4 Calcium 5.6 Zinc (mg/kg) 27.7 Manganese (mg/kg) 19.0 Copper (mg/kg) 5.2 Digestible lysine From the feedstuffs with (Zn), the zinc level was analyzed before experimental diets were formulated. 2 Supplies per kilogram of diet: vitamin A, 12,000 IE; vitamin D 3, 2,400 IU; vitamin E, 30 mg; vitamin K 3, 1.5 mg; vitamin B 1, 2.0 mg; vitamin B 2, 7.5 mg; pantothenic acid, 10 mg; niacin, 35 mg; biotin, 200 µg; vitamin B 12, 20 µg; folic acid, 1.0 mg; vitamin B 6, 3.5 mg; choline chloride, 460 mg; Fe, 80 mg (as FeSO 4 H 2 O); Cu, 12 mg (as CuSO 4 5H 2 O); Mn, 85 mg (as MnO); I, 0.8 mg (as KI); Se, 0.1 mg (as Na 2 SeO 3 ); Co, 0.4 mg (as CoSO 4 7H 2 O); antioxidant, 125 mg Oxytrap PXN; phytase (Natuphos 5000G, BASF AG, Ludwigshafen, Germany), 0.1 g; glucanase/xylanase (Rovabio Excel AP, Adisseo SAS, Antony, France), 0.05 g; anticoccidial (Clinacox 0.5%, Huvepharma, Antwerp, Belgium), 0.2 g. Prior to feed manufacturing, zinc levels were analyzed in critical feedstuffs (e.g., maize, wheat, soybean meal, and monocalcium phosphate) and supplemental zinc sources. Based on the analyses the experimental diets were optimized according to the experimental setup (Table 1). Thereafter, the zinc level was analyzed in each of the experimental diets. Furthermore, the basal diet was analyzed for moisture (ISO 6496), ash (NEN 3329), CP (ISO/CD 15670), crude fat (ISO DIS 6492), crude fiber (ISO 6865:2001), calcium (ISO 6869:2001), and phosphorus (ISO 6491:1999). All analysis in feedstuffs and experimental diets were done by Schothorst Feed Research. Analysis in the supplemental zinc sources (zinc sulfate and organic zinc) was done by Zinpro Corporation before the start of the experiment.

3 Tibia Samples At the termination of the experiment (d 21), 4 birds per cage were euthanized by intracardiac injection with T61 (0.1 ml/kg of BW; Intervet Nederland BV, Boxmeer, the Netherlands), and the left and right tibia were removed and pooled per replicate. A pooled sample of 4 right tibiae per cage was autoclaved for 20 min and cleaned of soft tissue. The pooled sample of 4 right tibiae was dried for 24 h at 103 C and then ashed at 550 C in a muffle furnace. This was followed by wet ashing of bone ash with HNO 3 and H 2 O 2. Left tibiae and remaining right tibiae were stored at 20 C. Tibia Zn was determined in the tibia ash fraction (ISO 6869:2001). Production Performance Body weight of birds per cage was measured at d 0, 3 (to standardize mean BW per cage), and 21 of the experiment. Feed intake per cage was measured from d 3 to 21. Feed conversion ratio (FCR) was calculated from d 3 to 21. Mortality was registered from d 0 until d 21. Statistical Analysis Observations were marked as outliers and excluded from the data set before statistical analyses if an observation exceeding the parameter mean by more than 2.5 times the SD. The experimental data were analyzed by ANOVA (GLM procedure) using GenStat statistical software (14th edition, VSN International Ltd., Hemel Hempstead, UK). Data were analyzed according to the following model: Y ij = μ + Block i + j + e ij, where Y is the response parameter, μ is the overall mean, Block is the effect of replicated block (i = 1 to 5 or 6), is the effect of zinc source and dose level (j = 1 to 9), and e is the random error term. means were compared by least significant Table 3. Analyzed zinc levels in each of the experimental diets BIOAVAILABILITY OF A ZINC AMINO ACID COMPLEX Analyzed zinc level in the diet (mg/kg) differences. A P-value < 0.05 was considered to be statistically significant, whereas 0.05 P < 0.10 was considered to be a near-significant trend. The dose-response relationship between zinc level on tibia zinc content was tested for linearity per zinc source. Subsequently, the slope ratios of both zinc sources were calculated. The slope ratio of zinc sulfate was used as a reference to estimate the relative bioavailability of the organic zinc source. Dietary Zinc Levels RESULTS In each of the experimental diets, the zinc level was determined (Table 3). The control diet was not supplemented with zinc, so zinc levels in the diet originated from feedstuffs only. The analyzed zinc level in control diet A was 33.5 mg/kg. This was higher than intended, but lower than recommended by the NRC (1994). The levels of added zinc calculated as the difference between analyzed zinc content of a given diet minus zinc content of the control diet were in line with the expected dose levels. Zinc analyses in experimental diets, corrected for added zinc from the reference and test zinc sources, indicated that the zinc content in the basal diet most likely was overestimated and should be 31.7 instead of 33.5 mg/kg. Results described below will refer to the expected supplementation levels. Tibia Zinc Content Results on tibia ash content and tibia zinc content are given in Table 4. Tibia ash content varied between 456 and 464 g/kg of fat-free DM, which is considered to be normal. These values were not affected by dietary treatment (P > 0.05). However, zinc content in tibia ash of birds fed a diet supplemented with at least 15 mg/kg of zinc as zinc sulfate or at least 10 mg/kg of zinc as organic zinc was significantly increased (P < 0.05) compared with the unsupplemented control. These values were not significantly different from the highest total zinc content observed for zinc sulfate at a Expected zinc supplementation (mg/kg) Realized zinc supplementation 1 (mg/kg) Control Zinc sulfate Zinc sulfate Zinc sulfate Zinc sulfate Zinc sulfate Organic zinc Organic zinc Organic zinc Calculated as the analyzed zinc content of a given diet minus zinc content of the control diet. 2 Calculated zinc level in control diet was 27.7 mg/kg (Table 2). 3 Availa-Zn, Zinpro Corporation, Eden Prairie, MN.

4 3118 Star et al. Table 4. Effect of different zinc sources and supplementation levels on tibia ash content and tibia zinc content of broilers at 21 d of age supplementation level of 40 mg/kg of feed. For the organic zinc source, an increase of tibia zinc content was observed with increasing dose levels. Bioavailability of Zinc Sources The zinc contents in tibia ash were used to calculate the relative bioavailability of the organic zinc source compared with zinc sulfate (reference). Therefore, linear and quadratic responses were tested for each zinc source first. Both zinc sources gave a linear response (P < 0.05) up to the highest level of supplementation. However, the response on the highest dose level for zinc sulfate showed high leverage, i.e., showing a lower response than expected based on the dose level. This probably indicates that the plateau level was about to be reached at a near adequate dietary zinc concentration. As the 40 mg/kg of zinc sulfate treatment would therefore reduce the slope for the reference, this highest dose level was omitted from the slope analyses. The linear slope ratio for zinc sulfate and organic zinc are given in Figure 1. The following dose-response relationship was found: Zn in tibia ash (mg/kg) = (SE 4.18) (SE 0.468) Availa-Zn (SE 0.385) ZnSO 4 ; P-value of the dose: <0.001; P-value of the Zn source: Supplemented dose (mg of Zn/kg of feed) Tibia ash (g/kg of fat free DM) Production Performance Tibia zinc (mg/kg of ash) 1 Control d 2 Zinc sulfate bcd 3 Zinc sulfate cd 4 Zinc sulfate abc 5 Zinc sulfate abc 6 Zinc sulfate a 7 Organic zinc bcd 8 Organic zinc abc 9 Organic zinc ab P-value a d Means with different superscript within a column differ significantly (P < 0.05). 1 Availa-Zn, Zinpro Corporation, Eden Prairie, MN. Results on feed intake, BW gain (BWG), and FCR are given in Table 5. Feed conversion ratio in birds supplemented with 5 mg/kg of organic zinc was significantly higher (P < 0.05) compared with all other dietary treatments and was caused by a numerical increase in feed intake. Feed intake and BWG were not significantly affected (P > 0.05) by dietary zinc source and dose level. Feed intake from d 3 to 21 varied between 1,215 to 1,281 g/bird and BWG varied between 847 and 878 among dietary treatments. The unsupplemented control group performed relatively well. Only The slopes of the different linear dose-response relationships indicate the bioavailability: a higher slope indicates a higher bioavailability because the zinc content in tibia ash is highly responsive to each unit of zinc supplementation until the requirement is met, whereas a lower slope indicates lower bioavailability (a smaller response per unit of zinc supplementation). The slopes of zinc sulfate and organic zinc are 1.21 and 1.99, respectively, indicating a relative bioavailability of 164% for the organic zinc source based on using zinc sulfate as reference. Figure 1. Linear dose response relationship between supplemental zinc sources [zinc sulfate or organic zinc (Availa-Zn, Zinpro Corporation, Eden Prairie, MN)] and tibia zinc content (mg/kg of ash). Zinc supplementation was based on the expected dosed values.

5 BIOAVAILABILITY OF A ZINC AMINO ACID COMPLEX 3119 Table 5. Effect of different zinc sources and supplementation levels on feed intake, BW gain (BWG), and feed conversion ratio (FCR) of broilers from 3 to 21 d of age addition of 15 or 20 mg/kg of zinc sulfate resulted in a numerically higher BWG, and 20 mg/kg of zinc sulfate in a numerically lower FCR than the unsupplemented control. Mortality ranged from 0 to 8.3% and was not different (P > 0.05) between dietary treatments (data not shown). DISCUSSION Supplemented dose (mg of Zn/kg of feed) In this study, the effect of zinc sulfate (inorganic source) was compared with an organic zinc source (Availa-Zn) on tibia Zn in broilers up to 21 d of age. The basal diet without supplemental zinc contained 33.5 mg of Zn/kg. This was most probably overestimated because the average content of all experimental diets minus the supplementation was 31.7 mg/kg. The analyzed dose levels of both zinc sources were in line with the calculated dose levels. The organic zinc source had a significant higher bioavailability than zinc sulfate. The bioavailability was approximately 64% higher for the organic zinc source compared with zinc sulfate as indicated by the slope ratio for the organic zinc source on the one hand and zinc sulfate as a reference on the other hand. The higher bioavailability of organic zinc sources as measured by bone ash zinc content was also found by others. The bioavailability of the current study was similar to the zinc bioavailability found by Wedekind et al. (1992) who found a bioavailability ranging from 177 to 206% for organic zinc compared with zinc sulfate (100%) by using a basal diet with 45 mg of Zn/kg of diet. Cao et al. (2000) found smaller differences in bioavailability between zinc sulfate and organic zinc sources by using a basal diet with 58 or 100 mg of Zn/kg of diet. The higher bioavailability indicates that more zinc was absorbed and deposited in bone tissue from organic zinc sources compared with inorganic sources. Irrespective of zinc source, production performance (except for FCR) was not affected by zinc supplementation. This is in accordance with other studies (Jondreville et al., 2007; Schlegel et al., 2010; Salim et al., 2012). Birds fed the control diet performed relatively well. The zinc level in the unsupplemented control diet Feed intake (g) BWG (g) FCR (g/g) 1 Control 0 1, a 2 Zinc sulfate 5 1, a 3 Zinc sulfate 10 1, a 4 Zinc sulfate 15 1, ab 5 Zinc sulfate 20 1, a 6 Zinc sulfate 40 1, ab 7 Organic zinc 1 5 1, b 8 Organic zinc 10 1, ab 9 Organic zinc 15 1, a P-value a,b Means with different superscript within a column differ significantly (P < 0.05). 1 Availa-Zn, Zinpro Corporation, Eden Prairie, MN. most likely was not strongly limiting for BWG in broilers housed in digestibility cages because there were no differences in production performance among the different zinc sources, except for the increased FCR at the 5 mg/kg Availa-Zn supplementation. Swiatkiewicz et al. (2001) used diets with added phytase and supplemented with up to 40 mg/kg of zinc fed over a 24-d period. Irrespective of phytase level, they found a higher BWG and improved FCR when birds were fed diets with supplemental zinc. The shorter duration of the current study might explain the lack of response to dietary zinc supplementation if the experimental period was not long enough to cause a depletion of zinc in the birds fed the unsupplemented diet. Although production performance was not improved by organic zinc supplementation, a clear dose-response relationship could be shown between dietary zinc content and zinc content in tibia ash. This proves that the zinc level in the control was below the animal s requirement for maximum zinc deposition. Zinc is involved in many biochemical reactions, and in further research it might be of interest to test the effect of a higher bioavailability on skin (foot pad dermatitis, wound healing) or immunity (for instance, the antibody reaction to a vaccine or a common disease in broilers such as necrotic enteritis). It was already shown by Salim et al. (2010, 2012) that dietary organic zinc was more effective in increasing the zinc concentration in skin and improving skin quality. However, this was not directly related to practical problems such as foot pad dermatitis. In conclusion, a clear dose-response relationship was shown between dietary zinc and tibia zinc content. The elevated tibia zinc content with increasing dietary zinc level proves that the zinc level in the unsupplemented diet was below the requirement of the birds for maximum zinc deposition. In a practical European diet, the organic zinc source Availa-Zn had a higher bioavailability than zinc sulfate, being approximately 64% higher. REFERENCES Ammerman, C. B., D. H. Baker, and A. J. Lewis Bioavailability of Nutrients for Animals: Amino Acids, Minerals and Vitamins. Academic Press, San Diego, CA.

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