F. A. Steward Micronutrients, Div. of Heritage Technologies, LLC, Indianapolis, IN 46231, USA

Transcription

1 Biol Trace Elem Res (2010) 138: DOI /s Effect of Dietary Supplementation with Copper Sulfate or Tribasic Copper Chloride on the Growth Performance, Liver Copper Concentrations of Broilers Fed in Floor Pens, and Stabilities of Vitamin E and Phytase in Feeds Lin Lu & Run L. Wang & Zheng J. Zhang & Fred A. Steward & Xugang Luo & Bin Liu Received: 11 January 2010 / Accepted: 20 January 2010 / Published online: 20 February 2010 # Springer Science+Business Media, LLC 2010 Abstract An experiment was conducted using a total of 840, 1-day-old, Arbor Acres commercial male broilers to compare copper (Cu) sulfate and tribasic Cu chloride (TBCC, Cu 2 (OH) 3 Cl) as sources of supplemental Cu for broilers fed in floor pens. Chicks were randomly allotted to one of seven treatments for six replicate pens of 20 birds each, and were fed a basal corn soybean meal diet (10.20 mg/kg Cu) supplemented with 0, 100, 150, or 200 mg/kg Cu from either Cu sulfate or TBCC for 21 days. Chicks fed 200 mg/kg Cu as TBCC had a higher (P<0.05) average daily gain (ADG) than those consuming other diets. Liver Cu contents of broilers fed diets supplemented with TBCC were numerically lower (P>0.05) than those of broilers fed diets supplemented with Cu sulfate. The vitamin E contents and the phytase activities in the feed fortified with TBCC were higher (P<0.01) and numerically higher (P>0.05) compared with those in the feeds fortified with Cu sulfate stored at room temperature, respectively. The vitamin E contents in liver and plasma of L. Lu : X. Luo (*) : B. Liu Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), No. 2 Yuanmingyuan West Road, Haidian, Beijing , People s Republic of China wlysz@263.net L. Lu : X. Luo : B. Liu State Key Laboratory of Animal Nutrition, Beijing , People s Republic of China F. A. Steward Micronutrients, Div. of Heritage Technologies, LLC, Indianapolis, IN 46231, USA Present Address: R. L. Wang Department of Animal Science, Guangdong Ocean University, Zhanjiang , People s Republic of China Present Address: Z. J. Zhang Beijing Oasis Kerey Mineral Nutrition and Technology Company, Beijing , People s Republic of China

2 182 Lu et al. broilers fed diets supplemented with TBCC were higher (P<0.05) than those of birds fed diets supplemented with Cu sulfate. This result indicates that TBCC is more effective than Cu sulfate in improving the growth of broilers fed in floor pens, and it is chemically less active than Cu sulfate in promoting the undesirable oxidation of vitamin E in feeds. Keywords Broilers. Copper. Growth performance. Liver copper concentration. Phytase activity. Vitamin E Introduction The essentiality of copper (Cu) for poultry and livestock is well documented [1]. Knowledge of the effect of supplemental Cu sources on performance of animals is critical in the selection of a source for use in poultry and livestock production. Micronutrients Tribasic Copper Chloride (TBCC, Cu 2 (OH) 3 Cl) is a new Cu source, which is a more concentrated form of Cu than Cu sulfate pentahydrate (CuSO4 5H 2 O) (57% vs. 25% Cu). It is also believed that TBCC is a less reactive and destructive form of Cu relative to Cu sulfate because it has lower hygroscopicity and solubility in water. Therefore, TBCC may be a potential Cu source with higher bioavailability and less oxidant-promoting activity than Cu sulfate. Some studies indicated that TBCC improved performance of poultry, pig, and cattle more efficiently as compared to Cu sulfate [2 6]. It was also demonstrated that TBCC could improve the stability of animal feeds by reducing oxidation reactions and reducing the loss of vitamin in feeds [2, 7 9]. In addition, Luo et al. [10] reported that TBCC is safer and more effective than Cu sulfate, and it is chemically less active than Cu sulfate in promoting the oxidation of vitamin E in feed. However, the additional levels (150, 300, and 450 mg/kg) of Cu as TBCC used in their study were much higher than levels normally used in the commercial poultry industry. It was documented that Cu as Cu sulfate at low additional levels ( 150 mg/kg) in diets is of anti-microbial effect for broilers [11, 12]. Broilers fed in floor pens are easily susceptible to bacterial diseases. It is speculated that Cu as TBCC supplemented to diets at low levels might be more effective in inhibiting the bacterial diseases and promoting the growth performance of the broilers fed in floor pens than Cu as Cu sulfate. In addition, TBCC may improve the stability of organic compounds like phytase in broilers feeds more effectively than Cu sulfate. However, no experiment on the effect of TBCC on the performance of broilers fed in floor pens and phytase stability in their feeds has been done so far. The objectives of the following study were to compare the efficacy of Cu as feed grade TBCC with that in the sulfate form in improving growth of broilers fed in floor pens and to determine if Cu source influenced the stability of dietary vitamin E and phytase in stored feed. Materials and Methods Experimental Design and Treatments A completely randomized design involving a 2 4 factorial arrangement of treatments was used in this study. Two supplemental Cu sources were TBCC and feed-grade Cu sulfate (CuSO 4 5H 2 O), respectively. The Cu sources were added to the basal diet at 0, 100, 150, or 200 mg/kg Cu. The diets were fed for 21 days. Because two Cu sources shared the same basal diet supplemented with Cu at the requirement level, there were a total of seven treatments in this experiment.

3 Effect of Copper in Feeds 183 Birds and Diets A total of 840, 1-day-old, Arbor Acres commercial male chicks were used in the 21-day experiment. Broilers were randomly allotted by body weight to one of seven treatments for six replicate floor pens of 20 birds each. Broilers were housed on the mixed litter (2/3 used litter at the bottom plus 1/3 new litter on the top). Birds were maintained on a 24-h constant-light schedule. Feed and tap water were available ad libitum. The corn soybean meal basal diet (Table 1) was in mash form, and formulated to meet the requirements of starting chicks [13]. The Cu products were added to the basal diet according to the experimental treatments. Dietary actually analyzed Cu concentrations are shown in Table 2. The analyzed content of vitamin E in the premix was 64 IU/kg, and the content of supplemental vitamin E in feeds was 16 IU/kg. In addition, 30 kg of the basal diet were removed at the time of mixing and supplemented with phytase (BASF, Mount Olive, NJ) at 1,000 PU/kg. This mixture was then divided into three equal parts. One part was used as the control treatment, and the other two were supplemented with 200 mg/kg Cu as either TBCC or Cu sulfate in order to investigate the effect of Cu from two sources on phytase stability in feeds. Therefore, there were a total of three treatments in the phytase stability test. Sample Collections and Preparations Feed samples were taken from all the treatments of the animal feeding trial and submitted for Cu analysis prior to the initiation of the trial to confirm Cu contents in the diets. Ten Table 1 Composition of the Basal Diet for Broilers (As-fed Basis) Ingredient Composition (g/kg) Composition (g/kg) Amount Corn Metabolizable energy (MJ/kg) Soybean meal Crude protein b Fish meal 43.0 Lysine 12.7 Soybean oil 36.0 Methionine 5.9 Calcium hydrogen phosphate 12.5 Methionine + cystine 9.3 Limestone 10.5 Calcium b 10.0 Salt 3.0 Nonphytate phosphorus 5.2 Methionine 2.1 Copper b, mg/kg Vitamin-mineral premix a 3.1 Magnesium b 0.17 Manganese b (mg/kg) Iron b (mg/kg) Copper b (mg/kg) Zinc b (mg/kg) a Ingredients supplied per kilogram of diet: vitamin A (as all-trans retinol acetate), 14,400 IU; cholecalciferol, 4,896 IU; vitamin E (as all-rac-α-tocopherol acetate), 16 IU; vitamin K (as menadione sodium bisulfate), 2.3 mg; thiamin (as thiamin mononitrate), 1.2 mg; riboflavin, 9.8 mg; vitamin B 6, 2.3 mg; vitamin B 12, mg; pantothenic acid calcium, 11.5 mg; niacin, 37.4 mg; folic acid, 1.2 mg; biotin, mg; choline (as choline chloride), 700 mg; Cu(CuSO 4 5H 2 O), 8 mg; Zn (ZnSO 4 7H 2 O), 80 mg; Fe (FeSO 4 7H 2 O), 80 mg; Mn (MnSO 4 H 2 O ), 100 mg, I (KI), 0.35 mg; Se (Na 2 SeO 3 ), 0.2 mg; The vitamin-mineral premix did not contain an antioxidant b Determined by analysis

4 184 Lu et al. Table 2 Added and Analyzed Copper Concentrations (mg/kg) of Diets for Broilers a Values based on chemical analysis of triplicate samples of diets, and reported on an as-fed basis Mn sources Add Cu Dietary Cu (day 1 to 21) a Control CuSO 4 5H 2 O TBCC replicate feed samples were taken from the diets of the treatments supplemented with 200 mg/kg Cu from two sources, respectively, for Cu analysis to compare the distribution of Cu in feed mixing between the two sources. In addition, ten replicate feed samples were taken from the control and the treatments with 200 mg/kg added Cu from two sources in the animal feeding trial for vitamin E analysis, and another ten replicate samples were also taken from the diet of the control treatment and the treatments with 200 mg/kg added Cu from two sources in the phytase stability test for phytase activity analysis. On days 10, 21, 31, and 41 of feed storage at room temperature (18±5 C), ten replicate feed samples were taken from the control treatment and the treatments with 200 mg/kg added Cu from two sources in the animal feeding trial, respectively, for vitamin E analysis, and ten replicate samples were taken from the diets of all three treatments in the phytase stability test, respectively, for phytase activity analysis. At the end of the animal feeding trial, chicks were weighed by each pen following a 12-h fast, and three chicks were chosen from every pen according to the pen average body weight. Blood samples were taken from each of the three birds via cardiac puncture, which were then centrifuged to harvest plasma samples for plasma vitamin E analysis. After the three chicks were killed by cervical dislocation, liver samples were collected for vitamin E and Cu analyses. All plasma and liver samples were stored at 50 C until analyses. Three samples of plasma or livers of the birds from each pen were pooled in equal ratios into one sample before analysis. Sample Analysis Copper concentrations in feeds, livers, and two sources were measured by inductively coupled plasma emission spectroscopy (IRIS Intrepid II, TE, Madison, WI, USA) according to a method described by Luo et al. [10]. Copper contents in feed samples supplemented with 200 mg/kg Cu from two sources were analyzed, and variation coefficients of analyzed values were calculated to compare the distribution of Cu in feed mixing between the two sources. Validations of the mineral analysis were conducted using bovine liver powder (National Institute of Standards and Technology, Beijing, China) as a standard reference. The contents of vitamin E in feed (ten replicate assays) and plasma (triplicate assays) were determined as described by Luo et al. [10]. Liver vitamin E (triplicate assays) was determined as described for plasma vitamin E by homogenizing 0.4 g of liver tissue in 3.6 ml (wt/vol) of cold saline (0.9%, wt/vol) and then extracting it with heptane. The activity of phytase in feeds was determined by the method of Engelen et al. [14].

5 Effect of Copper in Feeds 185 Statistical Analysis Data were analyzed by least squares analysis of variance using the General Linear Models (GLM) procedure of the SAS Institute [15]. The replicate pen served as the experimental unit. For the data of vitamin E contents and phytase activities in feeds, the model included the main effect of treatment and time, and their interaction. For other data, the model included the main effect of Cu source and supplemental Cu level, and their interaction. Liver Cu concentrations exhibited variance heterogeneity, and were subjected to log10 transformation prior to analysis. Multiple linear regression equation was calculated by least squares using the GLM procedure of SAS. In this study, the supplemental Cu level intervals were so small that the log10 transformed liver Cu concentrations had no linear relations with Cu intake; thus, relative bioavailability value of Cu as TBCC was not able to be determined by slope ratio comparison from multiple linear regressions. Linear regression equations of vitamin E contents or phytase activities in feed against time were calculated by least squares using the GLM procedure of SAS. In all cases, P<0.05 was considered to be statistically significant. Results Copper Contents of Sources and Uniformity of Copper Mixing in Feeds Analyzed Cu concentrations were 25.4% and 56.7% for CuSO 4 5H 2 O and TBCC, respectively. When the added Cu level was 200 mg/kg, the variation coefficients of the Cu concentrations in the feeds supplemented with either Cu sulfate or TBCC were 2.94 and 2.05, respectively. Growth Performance of Broilers Copper source, added Cu level, or an interaction between Cu source and level did not affect (P>0.05) average daily feed intake (ADFI) and feed per gain (F/G), but affected (P<0.05) average daily gain (ADG) (Table 3).Chicksfed200mg/kgCuasTBCChadahigher (P<0.05) ADG than those consuming other diets, and no differences in ADG were detected (P>0.05) among birds fed other diets. Copper Concentrations in Liver Liver Cu was not affected (P>0.05) by Cu source, added Cu level, and their interaction (Table 4). However, chicks fed diets supplemented with 200 mg/kg Cu tended to have a higher (P=0.07) liver Cu concentrations than those fed the control diet. Chicks fed TBCC diets tended to have a lower (P=0.07) liver Cu concentration than those fed Cu sulfate diets. Oxidation Stability of Vitamin E During storage, vitamin E contents in feeds decreased linearly (P<0.05) with time regardless of Cu treatment (Table 5). However, vitamin E contents in the feed fortified with 200 mg/kg Cu as TBCC were always higher (P<0.01) than those in the feed added with 200 mg/kg Cu as Cu sulfate (Table 6). When the feeds were stored at room temperature for 10, 21, 31, and 41 days, vitamin E contents in the control feed decreased by 12.8%, 19.8%, 26.4%, and 29.1%, and vitamin E contents in the feed supplemented with Cu sulfate decreased by 35.5%, 58.0%, 76.9%, and 78.2%, whereas vitamin E contents in the feed fortified with TBCC

6 186 Lu et al. Table 3 Effects of Dietary Copper Source and Level on Growth Performance of Broilers Fed 21 d (n=6) Added Cu source Added Cu level (mg/kg) ADFI (g) ADG (g) F/G (g/g) Control B 1.41 CuSO 4 5H 2 O B B B 1.41 TBCC B B A 1.38 Pooled SE Added Cu source CuSO 4 5H 2 O TBCC Pooled SE Added Cu level Pooled SE P-value Source Level Source Level Means with different superscripts within a column differ (P<0.05) reduced by 11.5%, 27.1%, 26.6%, and 26.4%, respectively, in comparison with the value at the beginning of this experiment. Therefore, the loss percentages of vitamin E contents in the feed fortified with TBCC at days 10, 21, 31, and 41 of feed storage were 24.0%, 30.9%, 50.3%, and 51.8% less than those in the feed fortified with CuSO 4,respectively. Liver vitamin E contents were affected (P<0.05) by Cu source or an interaction between Cu source and level, but not affected (P>0.05) by added Cu level (Table 4). Liver vitamin E contents of broilers fed 100 and 150 mg/kg Cu as TBCC were 17.9% (P<0.01) and 0.7% (P>0.05) higher than those of the chicks consuming the same levels of Cu as Cu sulfate, respectively. Copper source and added Cu level affected (P<0.05) plasma vitamin E contents (Table 4). Plasma vitamin E contents of broilers in the treatments of 100, 150, and 200 mg/kg Cu as TBCC were 15.0% (P<0.01), 7.7% (P>0.05), and 8.8% (P>0.05) higher than those of the chicks consuming the same level of Cu as Cu sulfate, respectively. The results of vitamin E contents in plasma and liver further confirmed that prooxidant activity of TBCC was significantly lower than that of Cu sulfate. Phytase Activities in Feeds During feed storage, phytase activities in feeds decreased linearly with time regardless of Cu treatments (Table 5). Phytase activities in feeds at day 41 decreased by 41.4% in comparison with the value at the beginning of this experiment. Though the statistical analysis was not significant (P>0.05), phytase activities in the feed fortified with 200 mg/kg Cu as TBCC were always numerically higher than those in the feed fortified with 200 mg/kg Cu as Cu sulfate (Table 6).

7 Effect of Copper in Feeds 187 Table 4 Effects of Dietary Copper Source and Level on Vitamin E Contents in Plasma and Liver, and Liver Copper Concentrations of 21-day-old Broilers (n=6; GLM of Log 10 Transformed Liver Cu Concentration) Added Cu source Added Cu level(mg/kg) Liver Cu a, b (µg/g) Liver vitamin E b, c (µg/g) Plasma vitamin E b, c (µg/ml) Control A 13.1 CuSO 4 5H 2 O C AB BC 10.2 TBCC A AB BC 11.1 Pooled SE Added Cu source CuSO 4 5H 2 O B TBCC A Pooled SE Added Cu level A B BC C Pooled SE P-value Source Level < Source Level Means with different superscript capital letters within a column differ (P<0.05) a GLM of log 10 transformed liver Cu concentration b Fresh basis, by analysis c α-tocopherol Discussion The results from Cu contents of sources and uniformity of Cu mixing in feeds indicate that TBCC was advantageous for better mixing quality in feeds. This was attributable to the smaller particles and better flow characteristic of TBCC than those of Cu sulfate. Table 5 Regressions of Vitamin E Contents or Phytase Activities in Feed (Y) on Time of Storage (X) Dependent variable (Y) Treatment Regression equation r 2 P-value Vitamin E contents in the feed from the treatments Control Y=-0.412X CuSO4 5H 2 O Y=-1.164X TBCC Y=-0.397X Phytase activities in the feed from the treatments Control Y= X+1, CuSO4 5H 2 O Y= X+1, TBCC Y= X+1,

8 188 Lu et al. Table 6 Vitamin E Contents and Phytase Activities in Feeds Supplemented with 200 mg/kg Copper as Either TBCC or Cu Sulfate During Different Time of Storage Item Cu source Time of storage (d) Vitamin E a,b,c Control 59.2±6.65 A 51.6±2.74 A 47.5±2.29 A 43.6±2.91 A 42.0±1.89 A CuSO 4 5H 2 O 60.5±4.79 A 39.0±2.73 B 25.4±5.61 C 14.0±2.29 B 13.2±2.59 B TBCC 59.8±4.86 A 52.9±4.06 A 43.6±4.06 B 43.9±3.27 A 44.0±2.99 A Phytase activities a,b Control 1,296±124 1,014±152 1,008± ± ±99 CuSO4 5H 2 O 1,240± ± ±95 757± ±117 TBCC 1,293±187 1,001± ± ± ±206 Means with different superscripts within a column differ (P<0.05) a Reported on an as-fed basis, by analysis b Each value represents the mean ± SD of 10 replicate samples c α-tocopherol After Braude [16] first reported that the addition of a high level (ten times the requirement level) of Cu improved the growth performance of fattening piglets, high levels of Cu have been widely used as a growth promoter in animal production. Spears et al. [3] and Cromwell et al. [5] found that TBCC was a new supplemental Cu source to replace Cu sulfate in livestock production. The results from the current study indicate that supplemental TBCC improved ADG without increasing ADFI, and the addition of 200 mg/kg Cu was optimal for improving ADG of broilers, which was in agreement with the above findings. In this study, the supplemental Cu level intervals were so small that the log 10 transformed liver Cu concentrations had no linear relations with Cu intake; thus, relative bioavailability value of Cu as TBCC was not able to be determined by slope ratio comparison from multiple linear regressions. The results from this study showed that chicks fed TBCC had a lower Cu residue in liver than those fed Cu sulfate, indicating that TBCC might be a safer product than Cu sulfate. Compared with CuSO 4, the prooxidant activity and water solubility of TBCC was lower [2, 17]. Hooge et al. [17] and Luo et al. [10] have shown a reduction in vitamin degradation in mixed feed when TBCC rather than Cu sulfate was included as the Cu supplement. In the current study, TBCC was less active than Cu sulfate in promoting the oxidation of vitamin E in feeds, which is consistent with the results of previous research [10, 17]. Luo et al. [10] also reported that both serum and liver vitamin E of chicks mirrored the trends in the feed sample. The results of vitamin E contents in plasma and liver in this study further confirmed that prooxidant activity of TBCC was significantly lower than that of Cu sulfate. The results from this study indicate that TBCC tended to maintain a higher phytase activity than CuSO 4 in broiler feeds, but no significant differences were observed. This might be due to the following reasons: (1) the study was conducted in September and October when the environmental temperature was relatively low, (2) the phytase used in this study was well coated, and (3) Cu sources were added to the complete diets where Cu had fewer chances to get access to the phytase. Therefore, a further study will be needed to address the effect of Cu source on the stability of the uncoated phytase in premixes under a high temperature in order to demonstrate TBCC s significant advantage over Cu sulfate in maintaining the phytase stability in the feed.

9 Effect of Copper in Feeds 189 Acknowledgements This study was supported by the earmarked fund for Modern Agro-industry Technology Research System (No. nycytx-42-g2-04), the special research fund for public benefit industries from Ministry of Agriculture of the People s Republic of China (No ), and Micronutrients, Div. of Heritage Technologies, LLC, Indianapolis, IN. References 1. Davis GK, Mertz W (1987) Copper. In: Mertz W (ed) Trace elements in human and animal nutrition, Vol. I. Academic, New York, pp Miles RD, O Keefe SF, Henry PR, Ammerman CB, Luo XG (1998) The effect of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability and dietary prooxidant activity. Poult Sci 77: Spears JW, Kegley EB, Mullis LA, Wise TA (1997) Bioavailability of copper chloride in cattle. J Anim Sci 75(Suppl 1):265, Abstr 4. Liu Z, Bryant MM, Roland DA Sr (2005) Layer performance and phytase retention as influenced by copper sulfate pentahydrate and tribasic copper chloride. J Appl Poult Res 14: Cromwell GL, Lindemann MD, Monegue HJ, Hall DD, Orr DE Jr (1998) Tribasic copper chloride and copper sulfate as copper sources for weanling pigs. J Anim Sci 76: PARC Institute (1997) Evaluation of TBCC and copper sulfate in diets upon the performance of commercial chicken broilers. PARC Institute, Easton 7. PARC Institute (1999) Study of effect of copper source on vitamin stability. PARC Institute, Easton 8. PARC Institute (1997) Evaluation of TBCC impact on vitamin loss during pelleting. PARC Institute, Easton 9. Micronutrients (2003) TBCC Notes: technical information for users of micronutrients TBCC: vitamin stability. Accessed 29 June Luo XG, Ji F, Lin YX, Steward F, Lu L, Liu B, Yu SX (2005) Effects of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability and oxidation stability of vitamin E in feed. Poult Sci 84: PARC Institute (1999) Study of effects of copper source on crop mycosis and performance with coccidia and candida albican challenge. PARC Institute, Easton 12. PARC Institute (1998) In vitro antimicrobial screening vs. E. Coli, Salmonella and Camplobacter: TBCC appeared significantly more discouraging to microbial growth than copper sulfate. PARC Institute, Easton 13. National Research Council (1994) Nutrient requirements of poultry, 9th edn. National Academic Press, Washington DC 14. Engelen AJ, Van der Heeft FC, Randsdorp PHG, Smit ELC (1994) Simple and rapid determination of phytase activity. J AOAC Int 77: SAS (2002) User s guide: statistics. Version, 9th edn. SAS Inst., Cary 16. Braude R (1945) Some observations on the need for copper in the diet of fattening pigs. J Agric Sci 35: Hooge DM, Steward FA, McNaughton JL (2000) Improved stabilities of vitamin A, D 3, E and riboflavin with tribasic copper chloride (TBCC) compared to copper sulfate pentahydrate in crumbled broiler starter feed. Poult Sci 79(suppl 1):43 44