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Effect of copper- and zinc-methionine supplementation on bioavailability, mineral status and tissue concentrations of copper and zinc in ewes
ARTICLE IN PRESS Journal of Trace Elements in Medicine and Biology 24 (2010) 89–94
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Nutrition
Effect of copper- and zinc-methionine supplementation on bioavailability, mineral status and tissue concentrations of copper and zinc in ewes D.T. Pal n, N.K.S. Gowda, C.S. Prasad 1, R. Amarnath, U. Bharadwaj, G. Suresh Babu, K.T. Sampath National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore 560 030, India
a r t i c l e in fo
abstract
Article history: Received 9 January 2008 Accepted 30 November 2009
The effect of feeding Cu- and Zn-methionine to ewes was studied in a 240d feeding trial. The plasma and tissue Cu and Zn concentrations and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity were employed to assess the relative bioavailability from Cu- and Zn-methionine. The macro and micronutrient intake, utilization, plasma mineral status, tissue accumulation of Cu and Zn as well as wool concentration of Cu and Zn were studied in ewes (n =12) fed a corn-soybean meal based basal diet with 50% more Cu and Zn supplementation over the basal diet either from Cu- and Zn-sulfate (Cu-Sulf+ Zn-Sulf group) or Cu- and Zn-methionine (Cu-Meth +Zn-Meth group). The average daily feed intake and body weight gain of ewes did not differ due to dietary supplementation of Cu- and Zn-methionine. However, dry matter intake was comparatively lower and thus resulted in better feed: gain in Cu- and Zn-methionine group as compared to ewes fed Cu- and Zn-sulfate. Supplementation of Cu and Zn over the basal diet either from methionine-chelated or sulfate sources resulted in increased plasma Cu and Zn as well as Cu/Zn-SOD activity on d-30, which indicated a positive correlation between plasma Cu and Zn and Cu/Zn-SOD activity. The gut absorption, liver concentrations of Cu and Zn, and liver Cu/Zn-SOD activity were significantly (P o0.01) higher in ewes supplemented with Cu- and Zn-methionine compared to Cu- and Zn-sulfate. Periodical analysis of wool samples indicated no significant difference in Cu and Zn content between Cu-and Zn-methionine and Cu- and Zn-sulfate groups. Feeding of Cu and Zn from methionine-chelated source resulted in reduced (P o 0.01) excretion of Cu and Zn in feces indicating their better utilization, and this will have positive implication on environment. The gut absorption values, plasma and liver tissue concentrations of Cu and Zn supported the hypothesis that Cu- and Zn-methionine supplements have better bioavailability compared to Cu- and Zn-sulfate and Cu- and Zn-dependent enzyme (Cu/Zn-SOD) could be used to determine the bioavailability of Cu and Zn. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Bioavailability Copper-methionine Superoxide dismutase Tissue minerals Zinc-methionine
Introduction Copper (Cu) and zinc (Zn) are critical trace minerals for production and reproduction in farm animals. The inorganic form of trace minerals are not sufficiently absorbed and retained. However, the organic form of these minerals are absorbed efficiently and retained in the tissue to enhance performance, improve immunity, health and reproduction compared to inorganic forms [1]. Limited research has been done concerning the biological availability of organic mineral sources in sheep. In studies with cattle [2–4], pigs [5], rats [6] and ewes [7,8] tissue concentrations of Cu and Zn were found higher for organic than inorganic sources. Lambs supplemented with Zn in the diet from Zn-lysine had higher Zn concentration in different tissues than lambs received Zn-sulfate or Zn-oxide [7,9,10].
Availability of trace minerals from common feeds is variable from poor to levels that can interfere with absorption or utilization of other minerals. Cu and Zn are the most limiting trace minerals under farm conditions [11]. The aim of the present study was to evaluate the effect of supplementing Cu- and Zn-methionine sources on gut absorption, plasma Cu and Zn status, tissue concentrations of Cu and Zn, and relative bioavailability as compared to inorganic source of Cu and Zn (Cu- and Zn-sulfate) in ewes and to ascertain the utility of Cu/Zn-superoxide dismutase (Cu/Zn SOD), as a biochemical marker to determine the bioavailability of Cu and Zn from different sources.
Materials and methods Animals and experimental design
n
Corresponding author. Tel.: + 91 80 25711304; fax: + 91 80 25711420. E-mail address: [email protected] (D.T. Pal). 1 ICAR, Krishi Bhavan, New Delhi 110 001, India.
0946-672X/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2009.11.007
Animal experimental protocol was approved by the Institute Ethical Committee, NIANP, Bangalore, India. Twelve ewes based
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Table 1 Dietary composition of basal diet (%). Ingredients
Concentrate feed
Para grass
Crushed corn Soybean meal Calcium carbonate Sodium chloride Mineral mixturea Vitamin A, D and Eb
55.00 43.50 1.00 0.50 0.2 0.02
– – – – – –
Chemical composition (% DM basis) OM CP Ash AIA Ca P Mg Cu (ppm) Zn (ppm)
94.55 16.82 5.45 0.59 0.56 0.69 0.34 14.66 94.17
89.26 6.05 10.74 4.20 0.57 0.34 0.40 8.67 76.40
a Mineral mixture consists of 25 mg of Fe as FeSO4, 20 mg of Mn as MnSO4 and 0.1 mg of Co as CoCl2 without Cu and Zn salts per kg of diet. b Provided 5000 IU of vitamin A, 500 IU of vitamin D3 and 187 mg of vitamin E per kg of diet.
on their body weight randomly assigned to two groups of six animals each. One group was supplemented with 50% more of Cu and Zn over the basal diet from feed grade inorganic source (copper sulfate and zinc sulfate; Cu-Sulf+ Zn-Sulf group) and the other group was supplemented with 50% more of Cu and Zn over the basal diet from commercially available feed grade organic source (copper-methionine and zinc-methionine; Cu-Meth+Zn-Meth group). The ewes were fed a corn-soybean meal and green para grass based basal diet (9.8 mg of Cu and 39.33 mg of Zn per kg of DM). The diet was formulated to be adequate in protein, energy, vitamins and minerals for this category of sheep [12]. Ingredient composition of concentrate feed is provided in Table 1. All ewes were maintained under uniform managerial conditions by housing them in a wellventilated shed with facilities for individual feeding. The ewes were dewormed with anthelmintic and were vaccinated for common diseases before the experiment. Ewes were individually fed the dry matter (DM) at 3% of their body weight to meet the nutrient requirement as per ICAR [12]. Fresh drinking water was made available at all the time. Water samples were collected at the beginning, middle and end of the study, composited and analyzed for Cu and Zn. Body weight of ewes was recorded at monthly interval till end of the study. A digestibility trial of six days duration was conducted to study the nutrient utilization and gut absorption of minerals in ewes fed organic and inorganic sources of Cu and Zn in the diet.
Sample collection The concentrate feed was provided to ewes at 08.30 h daily, chaffed green para grass (Brachiaria mutica) was offered in the afternoon. Residue if any was weighed next morning to calculate the feed intake by the ewes. Representative samples of green para grass and concentrate feed were collected daily and dried in hot air oven at 105 1C to determine the DM content and calculate the daily DM intake by ewes. Blood samples were collected on d 0 of the experiment and then subsequently at d 30, 60, 90, 120, 150, 180, 210 and 240 of the experimental period via jugular venipuncture in heparinizedtrace mineral free tubes. The whole blood was centrifuged at 800g for 10 min, plasma was aspirated into multiple polyethylene
tubes and frozen at 20 1C until analyzed for minerals and enzyme. Wool samples were collected by clipping 4 4 cm2 area from rib region at d 0, 30, 120 and 240. The dirt and grease of wool samples were removed by thoroughly rinsing the wool first with detergent followed by ethanol. The wool was finally washed several times in bidistilled water, oven dried at 60 1C for 24 h and kept in sealed plastic bags for minerals analysis. At the middle of the experiment (d 160), a digestibility trial was conducted to determine the nutrient utilization in ewes. The feed and fecal samples of the trial were collected and processed daily for DM estimation in hot air oven and then ground in a willy grinding machine to pass through 1 mm mesh and stored in plastic containers for further analysis. All the ewes were slaughtered at the end of the study and tissue samples of liver, kidney, heart, lung and muscle (Biceps femoris) from femoral region and skin from lower front leg were collected and frozen at 20 1C for further mineral and enzyme analysis. Wool was shaved from each skin sample, and visible connective tissue was separated from the skin and preserved at 20 1C for further analysis. Chemical analysis Total ash and crude protein estimation Total ash and acid insoluble ash of the experimental diets and fecal samples were estimated as per the method of A. O. A. C. [13], whereas the protein content in feed and feces was estimated in semi-automatic Kjel Plus systems (Pelican Equipments, Chennai, India). Reference material used was purified casein (Titan Biotech Indian Pvt. Ltd., New Delhi, India) for standardization of protein content in samples and crude protein content in standard casein was 87.30% on dry weight basis. Mineral analysis Samples were analyzed for minerals in atomic absorption spectrophotometer (Perkin Elmer AA Analyst 300, USA). Samples of feeds, fodders and feces were wet digested with 36 N nitric acid and 70% perchloric acid (3:1) in a microwave digestion system (Multiwave 3000, Anton Paar, USA) operated using a software at 15 bar pressure and 190 1C temperature with 10 min ramp and 30 min holding time. Wool and tissue samples (0.2 g) were also wet digested with nitric acid and perchloric acid (3:1) at 15 bar pressure and 190 1C temperature with 8 min ramp and 19 min holding times in the microwave digestion system. After completion of digestion, the clear extract obtained was transferred to volumetric flask and volume made up to 100 mL with deionized water for analysis of macro and micro minerals. Calcium (Ca), magnesium (Mg), copper (Cu) and zinc (Zn) in samples were estimated in atomic absorption spectrophotometer using air-acetylene flame. For estimation of Ca and Mg in samples, acid extracts were suitably diluted with 0.1% lanthanum chloride to avoid interference from phosphates and other minerals. Copper and zinc in feed and fecal samples were analyzed either directly from acid extract without any further dilution or diluted with deionized water, if required. Phosphorus in feed, feces and plasma samples was analyzed by colorimetric method [14] using stannous chloride as a reducing agent. Mineral standards (National Institute of Standards and Technology certified) were run in each analysis to ensure the accuracy of estimation. Estimation of Cu/Zn-superoxide dismutase enzyme activity Cu-and Zn-dependent enzyme, (Cu/Zn-superoxide dismutase, Cu/Zn-SOD) activity was determined in liver tissue and blood
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plasma. Liver homogenate preparation was performed on the day of enzyme analysis. Homogenate was centrifuged at 800g for 10 min, supernatant removed and the pellet resuspended in 0.25 mol/L sucrose buffer to a final concentration of 250 g/L. Homogenate was diluted to a final concentration of 0.5 g/L for analysis of Cu/Zn-SOD activity. The Cu/Zn-SOD activity in liver tissue and plasma was determined by a modified method of pyrogallic acid autoxidation [15]. The change in absorbance/minute in control (without sample) and test was recorded to calculate the SOD activity. One unit of SOD activity was expressed as amount of enzyme required to inhibit the pyrogallol autoxidation by 50%, and activity was expressed as units of enzyme per mg of fresh tissue. Bioavailability estimation of Cu and Zn Bioavailability of Cu and Zn from organic sources was determined from gut absorption, Cu/Zn-SOD activity as well as from Cu and Zn content of liver tissue. Regression procedure was used to calculate the bioavailability of Cu- and Zn-methionine relative to that for feed grade Cu-and Zn-sulfate as per the method described by Cao et al. [9].
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the means was compared using standard Fisher (F)-values and compared statistically at 1% and 5% level of significance. Relative bioavailability values were determined by slope ratio comparison from multiple linear regression [17] using copper sulfate and zinc sulfate as standard sources.
Results Feed intake and weight gain Dietary ingredient and nutrient composition of basal diet is presented in Table 1. The para grass contained 8.67 mg Cu and 76.4 mg Zn per kg DM. The Cu and Zn content of drinking water was negligible and hence was not accounted for calculating the intake. The concentrate mixture used in this study contained 16.82% crude protein and 14.66 mg Cu and 94.17 mg Zn per kg DM. The average daily feed intake and daily weight gain did not differ amongst the groups. However, ewes supplemented with organic source of Cu and Zn consumed 7.5% less feed over 30–60 d period at targeted body weight gain, resulting in better (P40.05) feed conversion efficiency (Table 2).
Statistical analysis The study was conducted as a completely randomized design with ewe as the experimental unit. The effect between the subjects was dietary sources of Cu and Zn. For plasma minerals and Cu/Zn-SOD activity, the study was conducted as a completely randomized design in a factorial arrangement of treatments with repeated measurements, where effect between the subjects was dietary sources of Cu and Zn and effect within subjects was sampling time. The interaction was dietary source and time. The data of various parameters were subjected to one-way ANOVA using the dietary source as a variable [16]. The difference between
Plasma mineral profile Plasma Cu and Zn concentration was significantly higher (P o0.06) in ewes that consumed Cu-Meth+ Zn-Meth compared to those supplemented with Cu-Sulf+ Zn-Sulf sources. The source and period interaction for plasma Cu and Zn was significant and was significantly higher (Po0.05) in ewes fed Cu-Meth+ Zn-Meth, than those supplemented Cu-Sulf+ Zn-Sulf during 30 and 60d of supplementation (Fig. 1). Calcium, phosphorus and magnesium concentrations in blood plasma were statistically (P40.05) similar between inorganic (Cu-Sulf+ Zn-Sulf) and organic (Cu-Meth+ Zn-Meth) Cu and Zn supplemented group throughout the experimental period.
Table 2 Body weight, average daily gain and feed: gain of ewes.
Nutrient utilization and gut absorption Attributes
Cu-Sulf + Zn-Sulf
Cu-Meth+ Zn-Meth
SD
P-value
Initial body weight (kg) Final body weight (kg) Body weight gain (kg) Average daily gain (g/d) Feed intake (g/d) Feed: gain
15.17 22.85 7.68 36.60 412.70 11.8:1
15.00 22.77 7.77 37.00 381.81 10.3:1
2.97 3.46 1.31 6.25 60.35 2.61
0.928 0.969 0.919 0.918 0.401 0.349
4.00
Cu-Sulf & Zn-Sulf Cu-Meth & Zn-Meth
2.50
Cu-Sulf + Zn-Sulf Cu-Meth + Zn-Meth
3.50 Plasma Zn (mg/L)
Plasma Cu (mg/L)
3.00
No significant difference was observed in intake and utilization of macronutrients and macro minerals on supplementing either inorganic or organic source of Cu and Zn. However, the gut absorption of Cu and Zn was significantly (Po0.01) higher in ewes fed diets with organic Cu and Zn compared to inorganic source, resulting in reduced fecal excretion of these minerals (Table 3).
2.00 1.50 1.00 0.50
3.00 2.50 2.00 1.50 1.00 0.50
0.00
0.00 0
1
2
3
4
5
6
7
Period of collection (Month)
8
0
1 2 3 4 5 6 7 Period of collection (Month)
Fig. 1. Effect of dietary supplementation of Cu- and Zn-methionine on plasma Cu and Zn level in ewes.
8
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Table 3 Effect of dietary supplementation of Cu- and Zn-methionine on intake and gut absorption of Cu and Zn in ewes. Attributes
Cu-Sulf +Zn-Sulf
Cu-Meth+Zn-Meth
P-value, significance
Copper Total intake (mg/d) Excreted (mg/d) Gut absorption (mg/d) Gut absorption (%)
8.48 6.63 1.85 21.88
8.10 5.42 2.65 33.11
0.69 0.97 0.62 8.19
0.323, NS 0.022n 0.019n 0.009nn
Zinc Total intake (mg/d) Excreted (mg/d) Gut absorption (mg/d) Gut absorption (%)
45.30 31.67 13.62 30.12
42.33 25.24 17.10 40.40
5.07 5.02 3.07 6.71
0.334, NS 0.010nn 0.034n 0.000nn
81.05 381.27
68.77 304.20
9.47 53.53
Fecal Cu and Zn Fecal Cu (ppm) Fecal Zn (ppm) NS, non-significant; nP o0.05;
Tissue
Cu-Sulf + Zn-Sulf
0.016nn 0.005nn
P o0.01.
nn
Table 4 Effect of Cu- and Zn-methionine supplementation on tissue Cu and Zn concentrations (mg/g) in ewes.
n
SD
Cu-Meth+Zn-Meth
SD
P-value, significance
Copper Livern Kidney Heart Muscle Skin
230.00 17.66 23.00 6.73 10.60
325.00 17.33 23.50 6.07 10.43
91.365 0.797 1.864 1.097 3.594
0.048n 0.496, NS 0.664, NS 0.266, NS 0.940, NS
Zinc Livern Kidney Heart Muscle Skin
111.67 108.33 92.67 120.67 49.83
128.33 106.50 97.17 120.17 49.00
13.554 10.706 12.325 27.401 10.094
0.023n 0.782, NS 0.553, NS 0.977, NS 0.894, NS
Table 5 Cu and Zn concentrations (mg/g) of wool in ewes fed on Cu- and Zn-methionine sources. Attribute
Cu
Zn
Effect of source Cu-Sulf + Zn-Sulf Cu-Meth+ Zn-Meth Significance P-value
12.20 11.72 NS 0.377
115.73 117.40 NS 0.691
10.97a 11.22ab 11.70abc 12.83bc 13.08c
108.17a 109.67a 110.00a 123.33b 131.67b
n
nn
Effect of periodn 0d 60 d 120 d 180 d 240 d Significance P-value SEM
0.037 0.271
0.000 2.070
Po 0.05 Significance level nP o0.05,
Copper and zinc content in liver and wool The Cu and Zn content in liver was significantly (P o0.05) higher in ewes fed diets containing organic sources (Cu and Zn-methionine). No significant differences were observed in Cu and Zn concentrations in other tissues like heart, kidney, muscle and skin (Table 4). The content of Cu and Zn in wool was not influenced by the dietary supplementation of Cu-methionine and Zn-methionine, but when compared with the values of Cu and Zn on day 0, accumulation of these trace minerals was quicker and significantly (P o0.05) higher in ewes supplemented with organic forms than the inorganic forms (Table 5).
Activity of Cu/Zn-superoxide dismutase and relative bioavailability The activity of Cu/Zn-SOD in liver homogenate was significantly (Po0.01) higher in ewes fed Cu and Zn-methionine and was positively correlated with Cu and Zn content in liver (Fig. 2). The bioavailability values for Cu and Zn were higher in ewes fed Cu and Zn-methionine compared to CuSO4 and ZnSO4 and found to be 151% and 134%, respectively. Relative bioavailability estimate based on multiple regression slope ratios of Cu/Zn-SOD regressed on plasma level of Cu and Zn was found to be 150% and 132%, respectively. Bioavailability of Cu and Zn determined from the liver Cu and Zn content and liver SOD activity was estimated at 151% and 133%, for Cu and Zn, respectively. It was found that
nn
Po 0.01.
the bioavailability determined from gut absorption, enzyme activity and tissue deposition of Cu and Zn were similar (Table 6, Figs. 2 and 3).
Discussion The ewes supplemented with organic source of Cu and Zn showed better feed conversion efficiency. Similar observations have been reported by other workers [18,8,19], suggesting better nutrient utilization. Higher plasma levels of Cu and Zn in ewes fed Cu-and Zn-methionine was attributed to better gut absorption of these minerals. Absorption and transportation of Zn-methionine in intact form following oral dosing and slower rate of decline in plasma level has been reported by Spears [20]. Higher gut absorption and lower fecal excretion of Cu and Zn from organic source with similar intake in the present study is in agreement with the finding of Power and Horgan [23] and it could be believed that the mineral ions from inorganic source in gut are released and possibly recombine with other digesta components forming insoluble complexes, thus reducing their gut absorption, while the organic minerals use amino acid uptake mechanism across the mucosa and absorbed to blood completely. Mondal et al. [24] also reported lower excretion of Cu through Cu-proteinate as compared to CuSO4. The higher Cu and Zn levels in the present study for initial period (60–90 d) followed by comparable levels to that of ewes
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250000
y = 267.98x + 81382
200000 150000 100000 50000 100 150 200 250 300 350 400 Liver Cu (mg/kg) Cu-Sulf + Zn-Sulf
300000 250000
y = 1057.5x + 34985
200000 150000 100000 50000 90
100
110
120
130
Liver SOD activity (Unit)
Cu-Sulf + Zn-Sulf 300000
93
Cu-Meth + Zn-Meth
300000
y = 406.79x + 81240
250000 200000 150000 100000
50000 100 150 200 250 300 350 400 450 Liver Cu (mg/kg)
Liver SOD activity (Unit)
Liver SOD activity (Unit)
Liver SOD activity (Unit)
D.T. Pal et al. / Journal of Trace Elements in Medicine and Biology 24 (2010) 89–94
Cu-Meth + Zn-Meth
300000 250000
y = 1408x + 32748
200000 150000 100000 50000 90 100 110 120 130 140 150 160
Liver Zn (mg/kg)
Liver Zn (mg/kg)
Fig. 2. Relationship between liver Cu and Zn and liver SOD activity in ewes fed Cu and Zn from sulfated and methionine-chelated sources.
Table 6 Relative bioavailability values (RBV) of Cu and Zn estimated based on multiple regression slope ratios and from gut absorption. Attribute
Liver Cu Zn
RBV (%)n
Slope Cu-Sulf + Zn-Sulf
Cu-Meth+Zn-Meth
267.98 1057.5
406.79 1408.0
151.79 133.14
Plasma Cu Zn
11.675 10.447
17.522 13.836
150.08 132.44
Gut absorption (%) Cu Zn
21.88 30.12
33.11 40.40
151.32 134.13
n
Assumed that bioavailability of Cu and Zn from Cu- and Zn-sulfate was 100%.
fed organic source indicated the deposition of these minerals in soft tissues as a part of the homeostatic mechanism. This is evident from the higher Cu and Zn content in liver of ewes fed organic source. Further significantly higher activity of Cu/Zn-SOD in liver tissue substantiates the better bioavailability of Cu/Zn from organic source and also explains the Cu and Zn dependent activity of SOD. Andrewartha and Caple [21] also reported similar relationship of SOD with serum Cu. Suttle and Mc Murray [22] recorded a temporal relationship of Cu level with SOD of RBC and hence could be of diagnostic significance, while evaluating the Cu status. In studies with cattle [2,3], pig [5], rat [6] and ewes [8], the liver Cu and Zn contents were higher in organic Cu and Zn group. Wright and Spears [10] also reported higher Zn concentration in liver and kidney of calves fed Zn-proteinate and Zn-methionine compared to ZnSO4. These findings support the higher gut absorption and bioavailability of trace minerals from organic sources. When compared to Cu and Zn values of wool at day 0, accumulation of these minerals was significantly (Po0.05) higher in ewes supplemented with Cu and Zn-methionine. Similar findings have been reported by Ryan et al. [25]. Eckert et al. [26] reported the improvement in wool quality in terms of wool length due to feeding of Cu-proteinate.
Bioavailability indicates the absorption and utilization of a nutrient. Mile and Henry [27] used absorption of a mineral as an indicator of its bioavailability. In some cases certain metabolically essential compounds or enzymes have been used as criteria for assessing mineral bioavailability [28,29]. The results of relative bioavailability of Cu and Zn from gut absorption, plasma Cu and Zn levels, Cu and Zn content in liver and Cu/Zn-SOD activity, indicated similarities, suggesting that these parameters can be used for determining the bioavailability of Cu and Zn. Cao et al. [9] reported the Zn bioavailability values of 130%, 110% and 113% for Zn-proteinate, Zn-amino acid chelate and Zn-methionine, respectively, as compare to zinc sulfate in lambs. In the present study, the ewes were fed a diet where corn (which contains high phytate) was one of the major feed ingredients and para grass as fibre source. Hence, there might be the possibility for interaction of minerals especially, Zn with phytate when supplemented through inorganic sources in the digestive tract and thus reduced the bioavailability of Cu and Zn. Higher phytate and fibre contents in the diet known to reduce the bioavailability of trace minerals by interacting negatively [30] whereas, the above minerals when supplemented through organic sources such negative effects of mineral interaction did not happen as minerals were chelated with amino acid and remained inert in any chemical reactions and thus improved the bioavailability of Cu and Zn.
Conclusions Supplementation of Cu and Zn through organic sources (Cu-methionine, Zn-methionine) as compared to inorganic sources (CuSO4, ZnSO4) improved gut absorption, higher activity of Cu/Zn-SOD and tissue retention of Cu and Zn, suggested better bioavailability through organic/chelated sources. Further, the results have shown that mineral-dependent enzymes like Cu/Zn-SOD can be used as a biomarker for assessing the status of Cu and Zn. Higher bioavailability of trace minerals through organic sources effectively reduces their dietary requirement and hence, is an environment friendly approach in reducing the fecal excretion of minerals.
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Cu-Sulf + Zn-Sulf
Cu-Meth + Zn-Meth
y = 11.675x + 7.9193
25
SOD activity (Unit)
SOD activity (unit)
30 20 15 10 5 0 0
0.5
1
1.5
2
40 35 30 25 20 15 10 5 0
y = 17.522x + 0.2389
0
0.5
Plasma Cu (mg/L) Cu-Sulf + Zn-Sulf
25 20 15 10 5 0 0
0.5 1 1.5 Plasma Zn (mg/L)
2.5
Cu-Meth + Zn-Meth
y = 10.447x + 8.9212
SOD activity (Unit)
SOD activity (Unit)
30
1 1.5 2 Plasma Cu (mg/L)
2
45 40 35 30 25 20 15 10 5 0
y = 13.836x + 4.8113
0
0.5
1 1.5 2 Plasma Zn (mg/L)
2.5
3
Fig. 3. Plasma Cu and Zn level and Cu/Zn-SOD activity in ewes fed on Cu- and Zn-methionine.
Acknowledgements This work has been carried out under the All India Coordinated Research Project (AICRP) of Indian Council of Agricultural Research (ICAR), New Delhi. The facilities provided by Director, National Institute of Animal Nutrition and Physiology (NIANP), Bangalore for conducting this research is thankfully acknowledged. References [1] Spears JW. Organic trace minerals in ruminant nutrition. Anim Feed Sci Technol 1996;58:151–63. [2] Nockels CF, Debonis J, Torrent J. Stress induction affects copper and zinc balance in calves fed organic and inorganic copper and zinc sources. J Anim Sci 1993;71:2539–45. [3] Rojas LX, McDowell LR, Martin FG, Wilkinson NS, Johnson AB, Njeru CA. Relative bioavailability of zinc methionine and two inorganic zinc sources fed to cattle. J Trace Elem Med Biol 1996;10(205-209):34. [4] Spears JW. Trace mineral bioavailability in ruminants. J Nutr 2003;133: 1506S–1509S. [5] Apgar GA, Kornegay ET, Lindrmann MD, Notter DR. Evaluation of copper sulfate and a copper lysine complex as growth promoters for weanling swine. J Anim Sci 1995;73:2640–6. [6] Du ZR, Hemken W, Jackson JA, Trammel DS. Utilization of copper in copper proteinate, copper lysine and cupric sulfate using the rat as experimental model. J Anim Sci 1996;74:1657–63. [7] Rojas LX, McDowell LR, Cousins RJ, Martin FG, Wilkerson NS, Johnson ABq, et al. Relative bioavailability of two organic and two inorganic sources fed to sheep. J Anim Sci 1995;73:1202. [8] Hatfield PG, Swenson CK, Kott RW, Ansotegui RP, Roth NJ, Robinson BL. Zinc and copper status in ewes supplemented with sulfate- and amino acid complexed forms of zinc and copper. J Anim Sci 2001;79(1):261–6. [9] Cao J, Henry PR, Guo R, Holwerda RA, Toth JP, Littell RC, et al. Chemical characteristics and relative bioavailability of supplemental organic zinc sources for poultry and ruminants. J Anim Sci 2000;78:2039–54. [10] Wright CL, Spears JW. Effects of zinc source and dietary level on zinc metabolism in Holstein bull calves. J Anim Sci 2001;79(suppl. 1):86–92. [11] Gowda NKS, Ramana JV, Prasad CS, Singh K. Micronutrient content of certain tropical conventional and unconventional feed resources of Southern India. Trop Anim Health Prod 2004;36:77–94. [12] ICAR. Indian Council of Agricultural Research. Nutrient Requirements of Livestock and Poultry 1998. 2nd ed., ICAR, DIPA, Krishi Bhavan, New Delhi, India. [13] AOAC. Officials methods of analysis (14th ed.). Association of Official Analytical Chemists. Washington D C.1990. [14] Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Biol Chem 1925;66:375–400.
[15] Percival SS. Cu/Zn superoxide dismutase activity does not parallel copper levels in copper supplemented LH-60 cells. Biol Trace Elem Res 1993;38:63–72. [16] Snedecor G.W., Cochran W.G. Statistical Methods. 8th Ed. (Oxford and IBH Publishing Co., Calcutta) Iowa State University Press Ames, Iowa, USA. 1994. [17] Littell RC, Lewis AJ, Henry PR. Statistical evaluation of bioavailability assays. In: Ammerman CB, Baker DH, Lewis AJ, editors. Bioavailability of Nutrients for Animals: Amino Acids, Minerals, and Vitamins. San Diego, CA: Academic Press; 1995. p. 5–33. [18] Ahola JK, Baker DS, Burns PD, Mortimer RG, Enns RM, Whittier JC, et al. Effect of copper, zinc and manganese supplementation and source on reproduction, mineral status and performance in growing beef cattle over a two-year period. J Anim Sci 2004;82:2375–83. [19] Wright CL, Spears JW. Effect of zinc source and dietary level on zinc metabolism in Holstein calves. J Dairy Sci 2004;87:1085–91. [20] Spears JW. Zinc methionine for ruminants: relative bioavailability of zinc in lambs and effects of growth and performance of growing heifers. J Anim Sci 1989;67:835–43. [21] Andrewartha KA, Caple IW. Effects of changes in nutritional copper on erythrocyte cuperoxidase dismutase activity in sheep. Res Vet Sci 1980;28(1):101–4. [22] Suttle NF, McMurray CH. Use of erythrocyte copper zinc superoxide dismutase activity and hair or fleece copper concentrations in the diagnosis of hypocuprosis in ruminants. Res Vet Sci 1983;35:47–52. [23] Power R, Horgan K. Biological chemistry and absorption of inorganic and organic trace metals. In: Lyons TP, Jacques KA, editors. Biotechnology in the Feed Industry. Nottingham, UK: Nottingham University Press; 2000. p. 277–91. [24] Mondal MK, Biswas P, Roy B, Mazumdar D. Effect of copper sources and levels on serum lipid profiles in Black Bengal (Capra hircus) kids. Small Rum Res 2007;67:28–35. [25] Ryan JP, Kearns P, Quinn T. Bioavailability of dietary copper and zinc in adult Texel sheep: a comparative study of the effect of sulphate and Bioplex supplementation. Ir Vet J 2002;5(5):221–4. [26] Eckert GE, Greene LW, Carstens GE, Ramsey WS. Copper status of ewes fed increasing amounts of copper from copper sulfate or copper proteinate. J Anim Sci 1999;77:244–9. [27] Miles RD, Henry PR. Relative trace mineral bioavailability. Cienc Anim Bras 2000;1(2):73–93. [28] Ammerman CB, Henry PR, Miles RD. Supplemental organically-bound mineral compounds in livestock nutrition. In: Garnsworthy PC, Wiseman. J, editors. Recent Advances in Animal Nutrition. Nottingham, UK: Nottingham University Press; 1998. p. 67–91. [29] Ammerman C.B., Henry P.R., Miles R.D. Bioavailability of organic forms of the microminerals. Proceedings of the 9th Annual Florida Ruminant Nutrition Symposium. Gainesville, Florida 1998b; p. 33-49. [30] Wedekind KJ, Lewis AJ, Giesmann MA, Miller PS. Methodology of assessing zinc bioavailability: efficacy estimates for zinc-methionine, zinc sulfate, and zinc oxide. J Anim Sci 1994;70:178–87.
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Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
Nutrition
Effect of copper- and zinc-methionine supplementation on bioavailability, mineral status and tissue concentrations of copper and zinc in ewes D.T. Pal n, N.K.S. Gowda, C.S. Prasad 1, R. Amarnath, U. Bharadwaj, G. Suresh Babu, K.T. Sampath National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore 560 030, India
a r t i c l e in fo
abstract
Article history: Received 9 January 2008 Accepted 30 November 2009
The effect of feeding Cu- and Zn-methionine to ewes was studied in a 240d feeding trial. The plasma and tissue Cu and Zn concentrations and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity were employed to assess the relative bioavailability from Cu- and Zn-methionine. The macro and micronutrient intake, utilization, plasma mineral status, tissue accumulation of Cu and Zn as well as wool concentration of Cu and Zn were studied in ewes (n =12) fed a corn-soybean meal based basal diet with 50% more Cu and Zn supplementation over the basal diet either from Cu- and Zn-sulfate (Cu-Sulf+ Zn-Sulf group) or Cu- and Zn-methionine (Cu-Meth +Zn-Meth group). The average daily feed intake and body weight gain of ewes did not differ due to dietary supplementation of Cu- and Zn-methionine. However, dry matter intake was comparatively lower and thus resulted in better feed: gain in Cu- and Zn-methionine group as compared to ewes fed Cu- and Zn-sulfate. Supplementation of Cu and Zn over the basal diet either from methionine-chelated or sulfate sources resulted in increased plasma Cu and Zn as well as Cu/Zn-SOD activity on d-30, which indicated a positive correlation between plasma Cu and Zn and Cu/Zn-SOD activity. The gut absorption, liver concentrations of Cu and Zn, and liver Cu/Zn-SOD activity were significantly (P o0.01) higher in ewes supplemented with Cu- and Zn-methionine compared to Cu- and Zn-sulfate. Periodical analysis of wool samples indicated no significant difference in Cu and Zn content between Cu-and Zn-methionine and Cu- and Zn-sulfate groups. Feeding of Cu and Zn from methionine-chelated source resulted in reduced (P o 0.01) excretion of Cu and Zn in feces indicating their better utilization, and this will have positive implication on environment. The gut absorption values, plasma and liver tissue concentrations of Cu and Zn supported the hypothesis that Cu- and Zn-methionine supplements have better bioavailability compared to Cu- and Zn-sulfate and Cu- and Zn-dependent enzyme (Cu/Zn-SOD) could be used to determine the bioavailability of Cu and Zn. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Bioavailability Copper-methionine Superoxide dismutase Tissue minerals Zinc-methionine
Introduction Copper (Cu) and zinc (Zn) are critical trace minerals for production and reproduction in farm animals. The inorganic form of trace minerals are not sufficiently absorbed and retained. However, the organic form of these minerals are absorbed efficiently and retained in the tissue to enhance performance, improve immunity, health and reproduction compared to inorganic forms [1]. Limited research has been done concerning the biological availability of organic mineral sources in sheep. In studies with cattle [2–4], pigs [5], rats [6] and ewes [7,8] tissue concentrations of Cu and Zn were found higher for organic than inorganic sources. Lambs supplemented with Zn in the diet from Zn-lysine had higher Zn concentration in different tissues than lambs received Zn-sulfate or Zn-oxide [7,9,10].
Availability of trace minerals from common feeds is variable from poor to levels that can interfere with absorption or utilization of other minerals. Cu and Zn are the most limiting trace minerals under farm conditions [11]. The aim of the present study was to evaluate the effect of supplementing Cu- and Zn-methionine sources on gut absorption, plasma Cu and Zn status, tissue concentrations of Cu and Zn, and relative bioavailability as compared to inorganic source of Cu and Zn (Cu- and Zn-sulfate) in ewes and to ascertain the utility of Cu/Zn-superoxide dismutase (Cu/Zn SOD), as a biochemical marker to determine the bioavailability of Cu and Zn from different sources.
Materials and methods Animals and experimental design
n
Corresponding author. Tel.: + 91 80 25711304; fax: + 91 80 25711420. E-mail address: [email protected] (D.T. Pal). 1 ICAR, Krishi Bhavan, New Delhi 110 001, India.
0946-672X/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2009.11.007
Animal experimental protocol was approved by the Institute Ethical Committee, NIANP, Bangalore, India. Twelve ewes based
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Table 1 Dietary composition of basal diet (%). Ingredients
Concentrate feed
Para grass
Crushed corn Soybean meal Calcium carbonate Sodium chloride Mineral mixturea Vitamin A, D and Eb
55.00 43.50 1.00 0.50 0.2 0.02
– – – – – –
Chemical composition (% DM basis) OM CP Ash AIA Ca P Mg Cu (ppm) Zn (ppm)
94.55 16.82 5.45 0.59 0.56 0.69 0.34 14.66 94.17
89.26 6.05 10.74 4.20 0.57 0.34 0.40 8.67 76.40
a Mineral mixture consists of 25 mg of Fe as FeSO4, 20 mg of Mn as MnSO4 and 0.1 mg of Co as CoCl2 without Cu and Zn salts per kg of diet. b Provided 5000 IU of vitamin A, 500 IU of vitamin D3 and 187 mg of vitamin E per kg of diet.
on their body weight randomly assigned to two groups of six animals each. One group was supplemented with 50% more of Cu and Zn over the basal diet from feed grade inorganic source (copper sulfate and zinc sulfate; Cu-Sulf+ Zn-Sulf group) and the other group was supplemented with 50% more of Cu and Zn over the basal diet from commercially available feed grade organic source (copper-methionine and zinc-methionine; Cu-Meth+Zn-Meth group). The ewes were fed a corn-soybean meal and green para grass based basal diet (9.8 mg of Cu and 39.33 mg of Zn per kg of DM). The diet was formulated to be adequate in protein, energy, vitamins and minerals for this category of sheep [12]. Ingredient composition of concentrate feed is provided in Table 1. All ewes were maintained under uniform managerial conditions by housing them in a wellventilated shed with facilities for individual feeding. The ewes were dewormed with anthelmintic and were vaccinated for common diseases before the experiment. Ewes were individually fed the dry matter (DM) at 3% of their body weight to meet the nutrient requirement as per ICAR [12]. Fresh drinking water was made available at all the time. Water samples were collected at the beginning, middle and end of the study, composited and analyzed for Cu and Zn. Body weight of ewes was recorded at monthly interval till end of the study. A digestibility trial of six days duration was conducted to study the nutrient utilization and gut absorption of minerals in ewes fed organic and inorganic sources of Cu and Zn in the diet.
Sample collection The concentrate feed was provided to ewes at 08.30 h daily, chaffed green para grass (Brachiaria mutica) was offered in the afternoon. Residue if any was weighed next morning to calculate the feed intake by the ewes. Representative samples of green para grass and concentrate feed were collected daily and dried in hot air oven at 105 1C to determine the DM content and calculate the daily DM intake by ewes. Blood samples were collected on d 0 of the experiment and then subsequently at d 30, 60, 90, 120, 150, 180, 210 and 240 of the experimental period via jugular venipuncture in heparinizedtrace mineral free tubes. The whole blood was centrifuged at 800g for 10 min, plasma was aspirated into multiple polyethylene
tubes and frozen at 20 1C until analyzed for minerals and enzyme. Wool samples were collected by clipping 4 4 cm2 area from rib region at d 0, 30, 120 and 240. The dirt and grease of wool samples were removed by thoroughly rinsing the wool first with detergent followed by ethanol. The wool was finally washed several times in bidistilled water, oven dried at 60 1C for 24 h and kept in sealed plastic bags for minerals analysis. At the middle of the experiment (d 160), a digestibility trial was conducted to determine the nutrient utilization in ewes. The feed and fecal samples of the trial were collected and processed daily for DM estimation in hot air oven and then ground in a willy grinding machine to pass through 1 mm mesh and stored in plastic containers for further analysis. All the ewes were slaughtered at the end of the study and tissue samples of liver, kidney, heart, lung and muscle (Biceps femoris) from femoral region and skin from lower front leg were collected and frozen at 20 1C for further mineral and enzyme analysis. Wool was shaved from each skin sample, and visible connective tissue was separated from the skin and preserved at 20 1C for further analysis. Chemical analysis Total ash and crude protein estimation Total ash and acid insoluble ash of the experimental diets and fecal samples were estimated as per the method of A. O. A. C. [13], whereas the protein content in feed and feces was estimated in semi-automatic Kjel Plus systems (Pelican Equipments, Chennai, India). Reference material used was purified casein (Titan Biotech Indian Pvt. Ltd., New Delhi, India) for standardization of protein content in samples and crude protein content in standard casein was 87.30% on dry weight basis. Mineral analysis Samples were analyzed for minerals in atomic absorption spectrophotometer (Perkin Elmer AA Analyst 300, USA). Samples of feeds, fodders and feces were wet digested with 36 N nitric acid and 70% perchloric acid (3:1) in a microwave digestion system (Multiwave 3000, Anton Paar, USA) operated using a software at 15 bar pressure and 190 1C temperature with 10 min ramp and 30 min holding time. Wool and tissue samples (0.2 g) were also wet digested with nitric acid and perchloric acid (3:1) at 15 bar pressure and 190 1C temperature with 8 min ramp and 19 min holding times in the microwave digestion system. After completion of digestion, the clear extract obtained was transferred to volumetric flask and volume made up to 100 mL with deionized water for analysis of macro and micro minerals. Calcium (Ca), magnesium (Mg), copper (Cu) and zinc (Zn) in samples were estimated in atomic absorption spectrophotometer using air-acetylene flame. For estimation of Ca and Mg in samples, acid extracts were suitably diluted with 0.1% lanthanum chloride to avoid interference from phosphates and other minerals. Copper and zinc in feed and fecal samples were analyzed either directly from acid extract without any further dilution or diluted with deionized water, if required. Phosphorus in feed, feces and plasma samples was analyzed by colorimetric method [14] using stannous chloride as a reducing agent. Mineral standards (National Institute of Standards and Technology certified) were run in each analysis to ensure the accuracy of estimation. Estimation of Cu/Zn-superoxide dismutase enzyme activity Cu-and Zn-dependent enzyme, (Cu/Zn-superoxide dismutase, Cu/Zn-SOD) activity was determined in liver tissue and blood
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plasma. Liver homogenate preparation was performed on the day of enzyme analysis. Homogenate was centrifuged at 800g for 10 min, supernatant removed and the pellet resuspended in 0.25 mol/L sucrose buffer to a final concentration of 250 g/L. Homogenate was diluted to a final concentration of 0.5 g/L for analysis of Cu/Zn-SOD activity. The Cu/Zn-SOD activity in liver tissue and plasma was determined by a modified method of pyrogallic acid autoxidation [15]. The change in absorbance/minute in control (without sample) and test was recorded to calculate the SOD activity. One unit of SOD activity was expressed as amount of enzyme required to inhibit the pyrogallol autoxidation by 50%, and activity was expressed as units of enzyme per mg of fresh tissue. Bioavailability estimation of Cu and Zn Bioavailability of Cu and Zn from organic sources was determined from gut absorption, Cu/Zn-SOD activity as well as from Cu and Zn content of liver tissue. Regression procedure was used to calculate the bioavailability of Cu- and Zn-methionine relative to that for feed grade Cu-and Zn-sulfate as per the method described by Cao et al. [9].
91
the means was compared using standard Fisher (F)-values and compared statistically at 1% and 5% level of significance. Relative bioavailability values were determined by slope ratio comparison from multiple linear regression [17] using copper sulfate and zinc sulfate as standard sources.
Results Feed intake and weight gain Dietary ingredient and nutrient composition of basal diet is presented in Table 1. The para grass contained 8.67 mg Cu and 76.4 mg Zn per kg DM. The Cu and Zn content of drinking water was negligible and hence was not accounted for calculating the intake. The concentrate mixture used in this study contained 16.82% crude protein and 14.66 mg Cu and 94.17 mg Zn per kg DM. The average daily feed intake and daily weight gain did not differ amongst the groups. However, ewes supplemented with organic source of Cu and Zn consumed 7.5% less feed over 30–60 d period at targeted body weight gain, resulting in better (P40.05) feed conversion efficiency (Table 2).
Statistical analysis The study was conducted as a completely randomized design with ewe as the experimental unit. The effect between the subjects was dietary sources of Cu and Zn. For plasma minerals and Cu/Zn-SOD activity, the study was conducted as a completely randomized design in a factorial arrangement of treatments with repeated measurements, where effect between the subjects was dietary sources of Cu and Zn and effect within subjects was sampling time. The interaction was dietary source and time. The data of various parameters were subjected to one-way ANOVA using the dietary source as a variable [16]. The difference between
Plasma mineral profile Plasma Cu and Zn concentration was significantly higher (P o0.06) in ewes that consumed Cu-Meth+ Zn-Meth compared to those supplemented with Cu-Sulf+ Zn-Sulf sources. The source and period interaction for plasma Cu and Zn was significant and was significantly higher (Po0.05) in ewes fed Cu-Meth+ Zn-Meth, than those supplemented Cu-Sulf+ Zn-Sulf during 30 and 60d of supplementation (Fig. 1). Calcium, phosphorus and magnesium concentrations in blood plasma were statistically (P40.05) similar between inorganic (Cu-Sulf+ Zn-Sulf) and organic (Cu-Meth+ Zn-Meth) Cu and Zn supplemented group throughout the experimental period.
Table 2 Body weight, average daily gain and feed: gain of ewes.
Nutrient utilization and gut absorption Attributes
Cu-Sulf + Zn-Sulf
Cu-Meth+ Zn-Meth
SD
P-value
Initial body weight (kg) Final body weight (kg) Body weight gain (kg) Average daily gain (g/d) Feed intake (g/d) Feed: gain
15.17 22.85 7.68 36.60 412.70 11.8:1
15.00 22.77 7.77 37.00 381.81 10.3:1
2.97 3.46 1.31 6.25 60.35 2.61
0.928 0.969 0.919 0.918 0.401 0.349
4.00
Cu-Sulf & Zn-Sulf Cu-Meth & Zn-Meth
2.50
Cu-Sulf + Zn-Sulf Cu-Meth + Zn-Meth
3.50 Plasma Zn (mg/L)
Plasma Cu (mg/L)
3.00
No significant difference was observed in intake and utilization of macronutrients and macro minerals on supplementing either inorganic or organic source of Cu and Zn. However, the gut absorption of Cu and Zn was significantly (Po0.01) higher in ewes fed diets with organic Cu and Zn compared to inorganic source, resulting in reduced fecal excretion of these minerals (Table 3).
2.00 1.50 1.00 0.50
3.00 2.50 2.00 1.50 1.00 0.50
0.00
0.00 0
1
2
3
4
5
6
7
Period of collection (Month)
8
0
1 2 3 4 5 6 7 Period of collection (Month)
Fig. 1. Effect of dietary supplementation of Cu- and Zn-methionine on plasma Cu and Zn level in ewes.
8
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Table 3 Effect of dietary supplementation of Cu- and Zn-methionine on intake and gut absorption of Cu and Zn in ewes. Attributes
Cu-Sulf +Zn-Sulf
Cu-Meth+Zn-Meth
P-value, significance
Copper Total intake (mg/d) Excreted (mg/d) Gut absorption (mg/d) Gut absorption (%)
8.48 6.63 1.85 21.88
8.10 5.42 2.65 33.11
0.69 0.97 0.62 8.19
0.323, NS 0.022n 0.019n 0.009nn
Zinc Total intake (mg/d) Excreted (mg/d) Gut absorption (mg/d) Gut absorption (%)
45.30 31.67 13.62 30.12
42.33 25.24 17.10 40.40
5.07 5.02 3.07 6.71
0.334, NS 0.010nn 0.034n 0.000nn
81.05 381.27
68.77 304.20
9.47 53.53
Fecal Cu and Zn Fecal Cu (ppm) Fecal Zn (ppm) NS, non-significant; nP o0.05;
Tissue
Cu-Sulf + Zn-Sulf
0.016nn 0.005nn
P o0.01.
nn
Table 4 Effect of Cu- and Zn-methionine supplementation on tissue Cu and Zn concentrations (mg/g) in ewes.
n
SD
Cu-Meth+Zn-Meth
SD
P-value, significance
Copper Livern Kidney Heart Muscle Skin
230.00 17.66 23.00 6.73 10.60
325.00 17.33 23.50 6.07 10.43
91.365 0.797 1.864 1.097 3.594
0.048n 0.496, NS 0.664, NS 0.266, NS 0.940, NS
Zinc Livern Kidney Heart Muscle Skin
111.67 108.33 92.67 120.67 49.83
128.33 106.50 97.17 120.17 49.00
13.554 10.706 12.325 27.401 10.094
0.023n 0.782, NS 0.553, NS 0.977, NS 0.894, NS
Table 5 Cu and Zn concentrations (mg/g) of wool in ewes fed on Cu- and Zn-methionine sources. Attribute
Cu
Zn
Effect of source Cu-Sulf + Zn-Sulf Cu-Meth+ Zn-Meth Significance P-value
12.20 11.72 NS 0.377
115.73 117.40 NS 0.691
10.97a 11.22ab 11.70abc 12.83bc 13.08c
108.17a 109.67a 110.00a 123.33b 131.67b
n
nn
Effect of periodn 0d 60 d 120 d 180 d 240 d Significance P-value SEM
0.037 0.271
0.000 2.070
Po 0.05 Significance level nP o0.05,
Copper and zinc content in liver and wool The Cu and Zn content in liver was significantly (P o0.05) higher in ewes fed diets containing organic sources (Cu and Zn-methionine). No significant differences were observed in Cu and Zn concentrations in other tissues like heart, kidney, muscle and skin (Table 4). The content of Cu and Zn in wool was not influenced by the dietary supplementation of Cu-methionine and Zn-methionine, but when compared with the values of Cu and Zn on day 0, accumulation of these trace minerals was quicker and significantly (P o0.05) higher in ewes supplemented with organic forms than the inorganic forms (Table 5).
Activity of Cu/Zn-superoxide dismutase and relative bioavailability The activity of Cu/Zn-SOD in liver homogenate was significantly (Po0.01) higher in ewes fed Cu and Zn-methionine and was positively correlated with Cu and Zn content in liver (Fig. 2). The bioavailability values for Cu and Zn were higher in ewes fed Cu and Zn-methionine compared to CuSO4 and ZnSO4 and found to be 151% and 134%, respectively. Relative bioavailability estimate based on multiple regression slope ratios of Cu/Zn-SOD regressed on plasma level of Cu and Zn was found to be 150% and 132%, respectively. Bioavailability of Cu and Zn determined from the liver Cu and Zn content and liver SOD activity was estimated at 151% and 133%, for Cu and Zn, respectively. It was found that
nn
Po 0.01.
the bioavailability determined from gut absorption, enzyme activity and tissue deposition of Cu and Zn were similar (Table 6, Figs. 2 and 3).
Discussion The ewes supplemented with organic source of Cu and Zn showed better feed conversion efficiency. Similar observations have been reported by other workers [18,8,19], suggesting better nutrient utilization. Higher plasma levels of Cu and Zn in ewes fed Cu-and Zn-methionine was attributed to better gut absorption of these minerals. Absorption and transportation of Zn-methionine in intact form following oral dosing and slower rate of decline in plasma level has been reported by Spears [20]. Higher gut absorption and lower fecal excretion of Cu and Zn from organic source with similar intake in the present study is in agreement with the finding of Power and Horgan [23] and it could be believed that the mineral ions from inorganic source in gut are released and possibly recombine with other digesta components forming insoluble complexes, thus reducing their gut absorption, while the organic minerals use amino acid uptake mechanism across the mucosa and absorbed to blood completely. Mondal et al. [24] also reported lower excretion of Cu through Cu-proteinate as compared to CuSO4. The higher Cu and Zn levels in the present study for initial period (60–90 d) followed by comparable levels to that of ewes
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250000
y = 267.98x + 81382
200000 150000 100000 50000 100 150 200 250 300 350 400 Liver Cu (mg/kg) Cu-Sulf + Zn-Sulf
300000 250000
y = 1057.5x + 34985
200000 150000 100000 50000 90
100
110
120
130
Liver SOD activity (Unit)
Cu-Sulf + Zn-Sulf 300000
93
Cu-Meth + Zn-Meth
300000
y = 406.79x + 81240
250000 200000 150000 100000
50000 100 150 200 250 300 350 400 450 Liver Cu (mg/kg)
Liver SOD activity (Unit)
Liver SOD activity (Unit)
Liver SOD activity (Unit)
D.T. Pal et al. / Journal of Trace Elements in Medicine and Biology 24 (2010) 89–94
Cu-Meth + Zn-Meth
300000 250000
y = 1408x + 32748
200000 150000 100000 50000 90 100 110 120 130 140 150 160
Liver Zn (mg/kg)
Liver Zn (mg/kg)
Fig. 2. Relationship between liver Cu and Zn and liver SOD activity in ewes fed Cu and Zn from sulfated and methionine-chelated sources.
Table 6 Relative bioavailability values (RBV) of Cu and Zn estimated based on multiple regression slope ratios and from gut absorption. Attribute
Liver Cu Zn
RBV (%)n
Slope Cu-Sulf + Zn-Sulf
Cu-Meth+Zn-Meth
267.98 1057.5
406.79 1408.0
151.79 133.14
Plasma Cu Zn
11.675 10.447
17.522 13.836
150.08 132.44
Gut absorption (%) Cu Zn
21.88 30.12
33.11 40.40
151.32 134.13
n
Assumed that bioavailability of Cu and Zn from Cu- and Zn-sulfate was 100%.
fed organic source indicated the deposition of these minerals in soft tissues as a part of the homeostatic mechanism. This is evident from the higher Cu and Zn content in liver of ewes fed organic source. Further significantly higher activity of Cu/Zn-SOD in liver tissue substantiates the better bioavailability of Cu/Zn from organic source and also explains the Cu and Zn dependent activity of SOD. Andrewartha and Caple [21] also reported similar relationship of SOD with serum Cu. Suttle and Mc Murray [22] recorded a temporal relationship of Cu level with SOD of RBC and hence could be of diagnostic significance, while evaluating the Cu status. In studies with cattle [2,3], pig [5], rat [6] and ewes [8], the liver Cu and Zn contents were higher in organic Cu and Zn group. Wright and Spears [10] also reported higher Zn concentration in liver and kidney of calves fed Zn-proteinate and Zn-methionine compared to ZnSO4. These findings support the higher gut absorption and bioavailability of trace minerals from organic sources. When compared to Cu and Zn values of wool at day 0, accumulation of these minerals was significantly (Po0.05) higher in ewes supplemented with Cu and Zn-methionine. Similar findings have been reported by Ryan et al. [25]. Eckert et al. [26] reported the improvement in wool quality in terms of wool length due to feeding of Cu-proteinate.
Bioavailability indicates the absorption and utilization of a nutrient. Mile and Henry [27] used absorption of a mineral as an indicator of its bioavailability. In some cases certain metabolically essential compounds or enzymes have been used as criteria for assessing mineral bioavailability [28,29]. The results of relative bioavailability of Cu and Zn from gut absorption, plasma Cu and Zn levels, Cu and Zn content in liver and Cu/Zn-SOD activity, indicated similarities, suggesting that these parameters can be used for determining the bioavailability of Cu and Zn. Cao et al. [9] reported the Zn bioavailability values of 130%, 110% and 113% for Zn-proteinate, Zn-amino acid chelate and Zn-methionine, respectively, as compare to zinc sulfate in lambs. In the present study, the ewes were fed a diet where corn (which contains high phytate) was one of the major feed ingredients and para grass as fibre source. Hence, there might be the possibility for interaction of minerals especially, Zn with phytate when supplemented through inorganic sources in the digestive tract and thus reduced the bioavailability of Cu and Zn. Higher phytate and fibre contents in the diet known to reduce the bioavailability of trace minerals by interacting negatively [30] whereas, the above minerals when supplemented through organic sources such negative effects of mineral interaction did not happen as minerals were chelated with amino acid and remained inert in any chemical reactions and thus improved the bioavailability of Cu and Zn.
Conclusions Supplementation of Cu and Zn through organic sources (Cu-methionine, Zn-methionine) as compared to inorganic sources (CuSO4, ZnSO4) improved gut absorption, higher activity of Cu/Zn-SOD and tissue retention of Cu and Zn, suggested better bioavailability through organic/chelated sources. Further, the results have shown that mineral-dependent enzymes like Cu/Zn-SOD can be used as a biomarker for assessing the status of Cu and Zn. Higher bioavailability of trace minerals through organic sources effectively reduces their dietary requirement and hence, is an environment friendly approach in reducing the fecal excretion of minerals.
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Cu-Sulf + Zn-Sulf
Cu-Meth + Zn-Meth
y = 11.675x + 7.9193
25
SOD activity (Unit)
SOD activity (unit)
30 20 15 10 5 0 0
0.5
1
1.5
2
40 35 30 25 20 15 10 5 0
y = 17.522x + 0.2389
0
0.5
Plasma Cu (mg/L) Cu-Sulf + Zn-Sulf
25 20 15 10 5 0 0
0.5 1 1.5 Plasma Zn (mg/L)
2.5
Cu-Meth + Zn-Meth
y = 10.447x + 8.9212
SOD activity (Unit)
SOD activity (Unit)
30
1 1.5 2 Plasma Cu (mg/L)
2
45 40 35 30 25 20 15 10 5 0
y = 13.836x + 4.8113
0
0.5
1 1.5 2 Plasma Zn (mg/L)
2.5
3
Fig. 3. Plasma Cu and Zn level and Cu/Zn-SOD activity in ewes fed on Cu- and Zn-methionine.
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