ORGANIC TRACE MINERALS: BIOAVAILABILITY AND FUNCTIONAL EFFECTS IN ANIMALS

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1 ORGANIC TRACE MINERALS: BIOAVAILABILITY AND FUNCTIONAL EFFECTS IN ANIMALS Dennis Nuzback, Gavin R. Bowman, Mercedes Vázquez-Añón and James D. Richards Novus International, Inc. 20 Research Park Dr. St. Charles, MO Introduction Trace minerals such as zinc, copper, manganese and selenium are essential in all animals for a wide variety of physiological processes. Several hundred enzymes require the presence of one or more of these minerals for their activity. For example, collagen and keratin synthesis both require zinc, and crosslinking of both requires copper. Overall, immune development and response, hoof and bone development and integrity, and protection against oxidative stress are just a few examples of mineral-dependent processes (Underwood and Suttle, 1999; Leeson and Summers, 2001). As such, most diets are supplemented with inorganic and/or organic forms of trace minerals. Inorganic trace minerals (ITM) such as mineral sulfates and oxides form the bulk of trace mineral supplementation, but these forms are more prone to dietary and environmental antagonisms and under such circumstances are therefore less bioavailable than organic trace minerals (OTM). Indeed, use of OTMs has been shown to enhance mineral uptake and reduce mineral excretion (Predieri, et al., 2005; Yan and Waldroup, 2006). Nevertheless, traditional bioavailability assays, as well as more recently developed gene expression assays for mineral bioavailability demonstrate that not all OTMs are equally bioavailable. These assays indicate that MINTREX organic trace minerals (zinc, copper or manganese, each chelated in a 2:1

2 stoichiometry by the methionine hydroxy analogue) provide a highly bioavailable source of trace minerals. Stability and Antagonisms Historically, zinc, copper and manganese have been supplemented in animal diets using inorganic salts such as oxides and sulfates. However, use of inorganic salts can result in poor bioavailability of the mineral, primarily because of the numerous nutrient and ingredient antagonisms that impair absorption (Underwood and Suttle, 1999). Antagonisms can occur between one mineral and another. For example, high levels of molybdenum and iron reduce the availability of copper (Underwood and Suttle, 1999). Other dietary components such as sulfates and fiber can also reduce mineral availability. The common denominator in these antagonisms is the dissociation of the metal cation from its corresponding anion in the rumen or in the relatively low ph of the abomasum. When the mineral reaches the higher ph of later gut segments it can bind to a number of minerals, nutrients and non-nutritive components of the digesta, such as fiber, that render it insoluble. Insoluble forms of minerals are excreted and therefore lost to the animal. The advantage of organic trace minerals (OTM) is that the binding of the organic ligand(s) to the mineral should provide stability of the complex in the upper gastrointestinal system, thereby avoiding mineral losses to antagonists and allowing the complex to be delivered to the absorptive epithelium of the small intestine for mineral uptake (Leeson and Summers, 2001). In other words, the OTM is in the mineral delivery business; it serves solely to deliver the mineral in protected form to the mineral transporters in the intestinal epithelium for absorption into the cells of the animal. All physiological effects of OTM supplementation should be considered to be the result of increased mineral delivery, rather than as a direct function of the 2

3 OTM complex per se. It should be noted that different organic trace minerals are not equally stable at low ph, and therefore will not necessarily increase the bioavailability of a given mineral to the same extent (Brown and Zeringue, 1994; Cao, et al., 2000; Guo, et al., 2001; Cao, et al., 2002). Bioavailability of OTMs Given their stability in the upper GI tract, one would predict that OTMs would provide a more bioavailable form of trace minerals than ITMs. Indeed, tissue mineral and slope-ratio type experiments have demonstrated the increased availability of organic forms compared to inorganic forms (Fly, et al., 1989; Paik, et al., 1999; Cao, et al., 2000; Guo, et al., 2001; Leeson, 2005; Predieri, et al., 2005; Yan and Waldroup, 2006). Tissue mineral experiments generate useful data, and can be very informative. However, as with any approach they do have limitations. First, tissue mineral experiments measure only a fraction of the mineral that is taken up by the animal. Minerals are absorbed by the small intestine, and then distributed via the bloodstream to other tissues. Therefore, tissue mineral levels only measure the mineral that is distributed to those particular tissues, and as such may not reflect total mineral uptake. A second shortcoming is that tissue mineral levels actually represent a storage pool of mineral, rather than the total amount of mineral delivered to that particular tissue. Liver minerals, for example, measures the amount of mineral that has entered the liver, less the amount of mineral that has left. Ideally, a true measure of mineral absorption would reflect just the amount of minerals that have been delivered to a particular tissue. One solution to this problem is to measure the expression of mineral-responsive biomarkers in the animal, preferably in the small intestine where minerals are absorbed. For example, the expression levels of several genes and proteins increase or decrease rapidly 3

4 depending on the mineral status of the animal. Metallothionein (MT) is such a biomarker, because its expression is regulated by zinc status (Davis and Cousins, 2000). When zinc is absorbed by a cell, it must be bound up quickly into protein, because free zinc can be toxic. The cell therefore responds to zinc uptake by synthesizing MT mrna (as an intermediate) and then MT protein. The MT protein is then able to bind to the zinc, until it is needed by other enzymes in the cell (McCormick, et al., 1981; Jacob, et al., 1998; Davis and Cousins, 2000). Furthermore, research has shown that in many tissues from a wide variety of species, MT mrna and protein expression increase when more zinc is taken up, and decrease when less zinc is taken up. As such, MT mrna or protein expression is widely used as an indicator of the zinc status of humans and animals, and to evaluate the bioavailability of different zinc sources (McCormick, et al., 1981; Lu, et al., 1990; Reeves, 1995; Rojas, et al., 1995; Sullivan, et al., 1998; Blanchard, et al., 2001; Cao, et al., 2002; Martinez, et al., 2004). Novus International, Inc. has developed patent-pending, real-time polymerase chain reaction (RT-PCR) assays to measure the expression of MT mrna, as a marker for both zinc status and bioavailability. In a recent broiler trial, the bioavailability of three zinc sources was compared to each other and to a zinc-deficient control diet by measuring small intestinal MT as the marker for bioavailability. First, all birds were fed a zinc-deficient milo-soy control diet for 20 days. The birds were then switched to corn-soy treatment diets which consisted of a zinc-deficient control (29ppm Zn from ingredients), or that same control diet formulated to contain an additional 70 ppm Zn from either zinc oxide (ZnO), a zinc amino acid complex (ZnAAC) or MINTREX Zn. MT expression was measured two days after the switch to treatment diets. Whereas zinc oxide and the ZnAAC only numerically increased MT expression versus the control, animals fed MINTREX Zn expressed significantly greater MT than all other treatments (Figure 1). This 4

5 result indicates that more zinc from MINTREX Zn than from the other sources was being absorbed by the small intestine. Tibia zinc was also measured in these animals after two weeks 30 Figure 1 b on treatment diets. The results from this assay MT mrna (relative units) a a supported the MT results, in that the birds supplemented with MINTREX Zn contained significantly or 5 0 a Control Zn Oxide Zn AAC MINTREX Zn numerically more tibia zinc than the birds receiving the other diets (data not shown). Together, these results indicate that MINTREX Zn was more bioavailable than the other zinc sources in this experiment. Mineral bioavailability was also evaluated in a recent dairy trial (Thering, et al., 2007). Multiparous midlactating Holstein cows (n=30) were placed on basal diets containing 47ppm Zn, 11ppm Cu and 43ppm Mn. After three weeks on the basal diet, livers biopsies were collected to measure MT and liver copper (timepoint = week 0). During the following four weeks, diets were top-dressed with either a rice-hull carrier (control) or one of two OTM blends plus biotin, providing 320mg Zn, 150mg Cu 130mg Mn, 3.79mg Se and 20 mg biotin per day. One blend was supplied as MINTREX organic trace minerals and the other (AA-complex) as methionine or lysine complexes of trace minerals, selenium yeast and biotin. After one week on the experimental diets (timepoint = week 1), liver biopsies were taken for MT analysis, and after 4 weeks biopsies were taken for Cu analysis. MT expression was significantly (P<0.05) increased 5

6 3 2.5 Figure 2 P = 0.02 in cows fed MINTREX but not MT Fold Induction (Week 1 MT / Week 0 MT) P = 0.96 P = 0.22 in the AA-complex cows or control cows (Figure 2; values expressed are MT 0 expression at week 1 Control AA-complex MINTREX divided by MT expression at week 0. Therefore a value of 1 (as seen in the control cows) means no change in expression. P values indicate significance of the change in expression within a given treatment). Liver copper tended Figure 3 a * (P<0.10) to be higher for cows fed 30 MINTREX and AA- Cu (ppm) ab complex compared to control cows -10 following the b a, b: P<0.05 * = Diff. than zero (P < 0.1) week experimental period. However, Control AA-Complex MINTREX liver copper increased over time (week 0 to week 4) only in the MINTREX cows (P<0.10) (Figure 3; values expressed are the change in liver copper from week 0 to week 4). Therefore, consistent with the broiler data, the MINTREX organic trace minerals were more bioavailable than the other source. 6

7 Functional Effects of Mineral Bioavailability Delivering more bioavailable trace minerals should allow one to formulate with lower mineral inclusion, and/or see enhanced benefits in the animal. In a recent commercial broiler trial with straight run Cobb 700 broilers, diets included twice the NRC recommended levels of inorganic forms of Zn, Cu and Mn, or the minerals at NRC levels but supplied as a 50:50 blend of inorganic and MINTREX minerals. Performance was better in the ITM/OTM birds, and white meat yield in male birds was substantially greater in the ITM/OTM birds than in the 2x NRC ITM birds. These data indicate that trace mineral inclusions can be reduced greatly if OTMs are used, while at the same time improving performance and yield. One reported benefit of OTM supplementation in dairy cows is a reduction in somatic cell shedding (Spears, 1996; Spain, 2005). The use of organic zinc and copper, especially, are believed to help maintain healthy skin via the synthesis of keratin and collagen; by promoting formation of the keratin plug in the streak canal, which protects against pathogen entry into the SCC in milk (x1000) Figure 4: SCC were reduced in MINTREX cows with high SCC MINTREX vs ZnMet 3 months in study >1000 Block by SCC levels * * P < mammary gland; and by enhancing the immune response to pathogens that do establish infection (Spain, 2005 and references within). Recently, a somatic cell shedding trial was conducted in California, comparing the effects of supplementing dairy 7

8 cows with 360mg organic zinc per day, either as MINTREX Zn or as a zinc-amino acid complex. Whereas both OTMs reduced SCC in the dairy cows, the reduction was greater in the cows supplemented with MINTREX Zn. When the cows were blocked by SCC levels, it is clear that the real difference in sources was seen in the cows with the highest somatic cell levels (Figure 4). Summary Trace minerals such as Zn, Cu and Mn play fundamental roles in animal development and health. The majority of these trace minerals are supplemented in inorganic forms, yet ITMs can suffer from high rates of loss due to dietary antagonisms. Use of OTMs can help prevent these losses, due to increased stability in the upper GI tract of the animal. Indeed, a variety of trials have demonstrated greater bioavailability of OTMs, which in turn would allow for lower inclusion rates and reduced excretion. Nevertheless, not all OTMs are equally able to deliver more bioavailable mineral to the animal. Recently, MT expression assays have been designed to measure the Zn that is absorbed by the tissues of the animal. These assays, as well as traditional tissue mineral assays demonstrate greater bioavailability of MINTREX versus other inorganic and organic forms. Greater bioavailability translates into a variety of animal benefits, including improved performance and yield in broilers, and reduced somatic cell counts in dairy cows. REFERENCES Blanchard, R. K., J. B. Moore, C. L. Green, and R. J. Cousins Proc. Natl. Acad. Sci. USA 98: Brown, T. F., and L. K. Zeringue J. Dairy Sci. 77: Cao, J., P. R. Henry, S. R. Davis, R. J. Cousins, R. D. Miles, R. C. Littell, and C. B. Ammerman Anim. Feed Sci. Tech. 101: Cao, J., P. R. Henry, R. Guo, R. A. Holwerda, J. P. Toth, R. C. Littell, R. D. Miles, and C. B. Ammerman J. Anim. Sci. 78:

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10 Underwood, E. J., and N. F. Suttle The mineral nutrition of livestock. 3rd Edition. CABI Publishing, New York. Yan, F., and P. W. Waldroup Int. J. Poult. Sci. 5: MINTREX is a trademark of Novus International, Inc. and is registered in the United States and other countries. 10