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Effects of copper and zinc source on performance and humoral immune response of newly received, lightweight beef heifers¹

Department of Animal and Food Sciences, Texas Tech University, Lubbock 79409

	Abstract

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Two experiments were conducted to evaluate the effects of Cu and Zn source on performance, morbidity, and humoral immune response in lightweight, newly received beef heifers. A 2 x 2 factorial arrangement of treatments was used in both experiments, with either a sulfate or a polysaccharide mineral complex (SQM) source of both Cu and Zn as the factors. Supplemental Cu and Zn were included in the receiving diet at concentrations designed to provide 10 mg of Cu/kg and 75 mg of Zn/kg (DM basis). In Exp. 1, 219 newly received beef heifers (British x Continental, average initial BW = 208 kg) were given ad libitum access to a 65% concentrate diet for 35 d to determine treatment effects on DMI, ADG, G:F, and bovine respiratory disease (BRD) morbidity. In Exp. 2, 24 heifers (average initial BW = 272 kg) were fed a diet with no supplemental Cu or Zn for 35 d, followed by fasting-refeeding-fasting stress, after which the same treatment diets used in Exp. 1 were fed for 21 d to examine the effects on humoral immune response (plasma IgG titer determined by ELISA on d 7, 14, and 21) to an ovalbumin (OVA) vaccine given on d 0 and 14. Copper source x Zn source interactions were not detected in either experiment. In Exp. 1, neither Cu nor Zn source affected (P > 0.10) DMI, ADG, G:F, or BRD morbidity. In Exp. 2, d 14 (P = 0.02) and 21 (P = 0.06) OVA titers were greater for heifers that received SQM Zn compared with heifers receiving ZnSO₄, but heifers receiving CuSO₄ had greater OVA titers than did heifers on the SQM Cu treatment on d 14 (P = 0.01) and 21 (P = 0.001). In summary, neither supplemental Cu nor Zn source affected performance or morbidity of lightweight, newly received heifers; however, source of both Cu or Zn affected the humoral immune response to OVA, although source effects were not consistent for the two minerals.

Key Words: Beef Cattle • Copper • Immune Response • Performance • Zinc

	Introduction

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Lightweight, newly received cattle often have a high incidence of bovine respiratory disease (BRD) as a result of being exposed to infectious agents during a time when they are also stressed by weaning and transportation (Nagaraja et al., 1998). Deficiencies in minerals such as Cu and Zn can decrease an animal’s immune response, thereby increasing susceptibility to disease (Suttle and Jones, 1989). Nonetheless, research on the effects of supplemental Cu and Zn on calf health has yielded conflicting results, and the mechanisms by which these minerals work in conjunction with the immune response are not understood fully (Galyean et al., 1999). Organic complexes of minerals might be more bioavailable than their inorganic counterparts; therefore, during stress and periods of low feed intake, an organic mineral might be beneficial in preventing or correcting deficiencies (Greene, 1995). We conducted two experiments, the first of which was designed to evaluate health and performance responses of newly received heifers to dietary supplementation of Cu and Zn from inorganic (sulfate) and organic (polysaccharide mineral complex) sources. Our second experiment was conducted to determine the effects of these same mineral sources on the humoral immune response to a foreign antigen.

	Materials and Methods

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Experiment 1
Two loads of mixed-breed heifers (primarily British and British x Continental; 109 and 110 heifers in Loads 1 and 2, respectively; average initial BW = 210.8 kg [SD = 12.54] and 205.2 kg [SD = 16.4] for Loads 1 and 2, respectively) were purchased by a cattle buyer for Cactus Feeders, Ltd., from auction barns in South Texas and shipped approximately 11 h from Gilmer, TX, to the Texas Tech University Burnett Center located near New Deal. Processing occurred immediately after arrival and included an individual BW measurement, an individual ear tag, vaccinations (Bovishield-4 + Lepto and Fortress 7; Pfizer Animal Health, Lee’s Summit, MO), and treatment for internal and external parasites (Dectomax, Pfizer Animal Health). After processing, the heifers were randomly assigned to soil-surfaced pens (4.88 m x 30.48 m) with a 4.88-m feed bunk at one end of the pen and a water trough (shared with another pen) located at the opposite end. Within each load, there were three pens per treatment (nine to 10 heifers per pen). Treatments were arranged in a 2 x 2 factorial, consisting of 65% concentrate diets with the following supplemental Cu and Zn concentrations (DM basis): 1) 10 mg/kg of supplemental Cu from CuSO₄ + 75 mg/kg of supplemental Zn from ZnSO₄; 2) 10 mg/kg of supplemental Cu from CuSO₄ + 75 mg/kg of supplemental Zn from a polysaccharide mineral complex (SQM) source Zn; 3) 10 mg/kg of supplemental Cu from SQM Cu + 75 mg/kg of supplemental Zn from ZnSO₄; and 4) 10 mg/kg of supplemental Cu from SQM Cu + 75 mg/kg of supplemental Zn from SQM Zn. The SQM sources of Zn and Cu were polysaccharide complexes manufactured by Quali Tech, Inc. (Chaska, MN). Dietary concentrations of supplemental Cu and Zn were chosen to meet the NRC (1996) recommendations for lightweight, stressed beef cattle, ignoring any contribution from the basal dietary ingredients. Heifers were allowed ad libitum access to the 65% concentrate diet (Tables 1 and 2) for 35 d, and long-stemmed alfalfa hay was offered in the feed bunk in addition to the concentrate diet from d 0 through 6. Weekly feed bunk samples of the diets were obtained, composited, and analyzed for DM, ash, CP, Ca, P, Zn, and Cu using AOAC (1990) procedures. Values are shown in Table 2. Individual BW was measured on d 0, 14, 28, and 35 using a single-animal squeeze chute (C & S, Garden City, KS). Feed bunks were cleaned, and feed refusals were removed, weighed, and sampled for DM analysis at each weigh date immediately before the cattle were weighed. On d 14, heifers received a booster injection of Bovishield-4 + Lepto and were implanted (Ralgro, Schering-Plough Animal Health, Union, NJ).

Approximately 10 mL of blood were collected from 10 heifers per treatment (two to four per pen) from Load 1, and from 12 heifers per treatment (four heifers per pen) from Load 2 to measure serum Cu and Zn concentrations. Blood was collected via jugular puncture on d 0, 14, and 28 in evacuated serum-separator tubes (Fisher Scientific, Houston, TX), and the serum obtained following centrifugation (1,000 x g) was analyzed for Cu and Zn concentration using an atomic absorption spectrophotometer (model 2380, Perkin-Elmer, Norwalk, CT). Serum used for Cu analysis was diluted 2:1 (serum:deionized water), and standards were prepared in 15% (vol/vol) glycerin solution. Serum for Zn analysis was diluted 1:2 (serum:deionized water), and standards were prepared in 10% (vol/vol) glycerin solution.

Dry matter intake, ADG, and G:F were analyzed as a randomized block design using the Mixed procedure of SAS (SAS Inst., Inc., Cary, NC). Pen was the experimental unit, and the model included Cu source, Zn source, the Cu source x Zn source interaction, and the random effects of load and load x Cu source x Zn source. Serum Cu and Zn data were analyzed using the same model as that used for performance data (GLM procedure of SAS). Morbidity and retreatment data were analyzed with the CATMOD procedure of SAS, with individual animal as the experimental unit. A preliminary model with pen, Cu source, Zn source, and load was used to determine that pen effects were not present (P > 0.10); thereafter, a model including load, Cu source, Zn source, and their interactions was used.

Experiment 2
In Exp. 2, 24 heifers (average initial BW = 272 kg; SD = 26.5) from Exp. 1, Load 1, were trained to use individual Calan gates (American Calan, Northwood, NH) for a 35-d period. During this period, the heifers were fed a 65% concentrate diet (Table 1) that was devoid of supplemental Cu or Zn. Following the 35-d training period, heifers were subjected to a fasting-refeeding-fasting stress that consisted of 24 h of withholding feed and water, 24 h of ad libitum access to feed and water, and a final 24-h period in which feed and water were again withheld. Following the fasting-refeeding-fasting stress, heifers were allowed ad libitum access to their randomly assigned treatment diets. Treatment diets consisted of the same 2 x 2 factorial arrangement of Cu and Zn sources used in Exp. 1 (Tables 1 and 2).

Animals were injected (s.c.) before feeding on d 0 and 14 (booster injection) with 4 mL of an ovalbumin (OVA) vaccine. Ovalbumin was used to elicit an immune response because it is a foreign, yet harmless, protein and one that heifers should not have been previously exposed to. The OVA vaccine comprised 2 mg of crystallized OVA (chicken egg albumin, Grade V, product No. A-5503, Sigma Chemical, St. Louis, MO) suspended in 1 mL of nonsterile PBS). This OVA–PBS (2 mg of OVA/mL of PBS) solution was diluted 1:1 with Freund’s incomplete adjuvant (product No. F-5506, Sigma Chemical), yielding the final OVA vaccine used for injection. The OVA vaccine was emulsified and stored at –4°C until injection on d 0 and 14. Before feeding on d 0, 7, 14 (before the booster injection), and 21, 10 mL of blood were collected via jugular venipuncture in both a heparinized evacuated tube and a serum-separator tube (Fisher Scientific, Houston, TX). Heparinized tubes were centrifuged at 850 x g, and plasma samples were extracted and stored frozen for ELISA analysis of OVA immunoglobulin (IgG) titers. Serum was used to determine Cu and Zn concentrations as described for Exp. 1.

All procedures and acitivities involving animals in both Exp. 1 and 2 were reviewed and approved by the Texas Tech University Animal Care and Use Committee.

ELISA Analysis
Plasma samples were thawed at room temperature (22°C) before use and analyzed for specific IgG titers to the OVA antigen. The ELISA analysis was described previously by Rivera et al. (2002). Optical density was determined using a 96-well plate reader (Titertek Multiskan, Flow Laboratories, McLean, VA) at a wavelength of 405 nm. Binding that occurred on control wells (no OVA antigen) was nonspecific, and the optical density value was subtracted from the corresponding optical density value of the sample well (OVA antigen-coated). Titers for each animal and sampling day were expressed as the inverse of the plasma dilution at the point that the optical density reached the baseline (d 0) optical density.

Titer and serum mineral concentration data were analyzed using the MIXED procedure in SAS. For both titer and serum mineral data, animals were the experimental units, and data were analyzed within sampling day using a model that included Cu and Zn source as the main effects and the Cu source x Zn source interaction. Titer data were transformed to log₂ before statistical analysis, but untransformed means and standard errors are presented.

	Results and Discussion

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Experiment 1
The Cu source x Zn source interaction was not significant (P > 0.25) for any of the performance variables measured in this experiment; therefore, only main-effect means will be discussed. No effects of Cu source or Zn source (P > 0.10) were observed for ADG or DMI (Table 3). Spears et al. (1991) and Galyean et al. (1995) also reported no differences in performance when organic vs. inorganic Zn sources were supplemented to newly received, stressed calves or to growing steers (Spears and Kegley, 2002), and Ward et al. (1993) reported no differences in ADG by steers supplemented with CuSO₄ or copper lysine during a 21-d receiving trial. In the current study, DMI from d 0 to 14 by heifers receiving SQM Cu was 2.88 kg/d, which tended (P = 0.13) to be higher than the 2.71 kg/d of DMI that CuSO₄-supplemented heifers consumed; however, DMI for d 0 to 28 and for the overall 35-d period did not differ between Cu sources. As expected from DMI and ADG results, no differences (P > 0.30) were noted among treatments for G:F throughout the 35-d trial. The G:F values for the period from d 0 to 14 were much lower than in the other periods, reflecting the low DMI and low (in some cases negative) BW gains by the cattle, presumably reflecting the high incidence of morbidity during this period.

Experiment 2
Dry matter intake and BW were not affected by treatment (P > 0.05; data not shown) at any time during the experiment. As noted previously, BW on d 0 averaged 272 kg. On d 21 of the study, BW averaged 321 kg (SD = 25.9). For the 5-d period before the fasting-refeeding-fasting period, DMI averaged 6.37 kg (SD = 0.11), whereas DMI averaged 8.53 kg (SD = 1.29) for the 21-d experimental period after the fasting-refeeding-fasting stress.

Data for OVA-specific IgG titers for Cu and Zn sources are presented in Table 7. There were no interactions (P > 0.20) between Cu and Zn source detected for any of the sampling days. On d 7, the specific antibody response to OVA did not differ (P > 0.20) among treatments. However, on d 14, heifers receiving supplemental CuSO₄ had higher (P = 0.01) OVA IgG titers than did heifers receiving SQM Cu. Similarly, on d 21, heifers receiving supplemental CuSO₄ had a higher (P = 0.001) OVA IgG titer than did those receiving SQM Cu. In contrast to results with Cu source, in which the sulfate form yielded higher OVA titers, heifers receiving SQM Zn had greater (P = 0.02) OVA IgG titers on d 14 than did heifers receiving ZnSO₄. Likewise, SQM Zn heifers had greater (P = 0.06) titers than did ZnSO₄ heifers on d 21.

Serum Cu concentrations on d 0, 7, 14, and 21 (data not shown) were not affected (P > 0.10) by either Cu or Zn source. Values averaged 0.91, 0.81, 0.93, and 0.83 µg/mL for d 0, 7, 14, and 21, respectively. On d 0, there was a Cu source x Zn source interaction (P = 0.04) for serum Zn concentration. Heifers assigned to the SQM Cu + ZnSO₄ treatment had a d-0 serum Zn concentration of 1.63 µg/mL, which differed (P < 0.05) from those assigned to the CuSO₄ + SQM Zn and SQM Cu + SQM Zn treatments (1.34 and 1.20 µg/mL, respectively), but did not differ from that of heifers assigned to the CuSO₄ + ZnSO₄ treatment (1.38 µg/mL). Despite these differences in serum Zn concentration on d 0, no effects of Cu source (P > 0.10) were noted on d 7, 14, and 21 (average Zn concentrations of 0.95, 1.5, and 1.31 µg/mL, respectively). Similarly, no effects of Zn source (P > 0.30) on serum Zn concentrations were noted on d 7 and 21, but on d 14, heifers receiving supplemental ZnSO₄ had a higher (P = 0.04) serum Zn concentration than those supplemented with SQM Zn (1.61 vs. 1.39 µg/mL, respectively). Serum Cu and Zn concentrations are generally not considered to be accurate indicators of Cu and Zn status unless severe deficiencies exist (Underwood, 1981). In the current study, heifers were fed a diet with no supplemental Cu and Zn for a 35-d period before the fasting-refeeding-fasting stress; however, serum Cu and Zn concentrations were generally greater than those in Exp. 1. This difference between the experiments likely reflects the much greater feed intake by the heifers in Exp. 2 than by those in Exp. 1. For example, DMI averaged 4.55 kg/d for the 35-d period of Exp. 1, which equates to approximately 2.2% of BW. In contrast, DMI by heifers in Exp. 2 averaged 8.53 kg/d for the 21-d experimental period, which equates to 2.9% of BW. Moreover, although the fasting-refeeding-fasting stress imposed in Exp. 2 might have had some effect on mineral concentrations of the heifers, the increases in DMI and BW over the 21-d period by these heifers suggests the stress imposed was fairly minimal.

	Implications

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When added to the diet at concentrations designed to meet the NRC (1996) recommendations for lightweight, stressed cattle, no differences in performance or morbidity of newly received heifers were noted when Cu and Zn were supplied in either sulfate or polysaccharide complex forms. Thus, either source of these minerals should be effective in receiving diets for lightweight beef cattle newly received at feedlots. In heifers subjected to a sequence of fasting-refeeding-fasting, source of Cu and Zn affected humoral immune response to a novel antigen, with the sulfate form increasing plasma titers to the antigen for Cu, but the polysaccharide source increasing titers with Zn. Whether these effects on humoral immune response have practical benefits in lightweight, stressed cattle is yet to be determined.

	Footnotes

 
¹ We thank Quali Tech, Inc., Chaska, MN, for partial financial support of the experiment, Cactus Feeders, Ltd., Amarillo, TX, for supplying the cattle used in the experiments, and Elanco Animal Health, Pfizer Animal Health, and Schering-Plough Animal Health for product support.

² Correspondence: P.O. Box 215, Pratt, KS 67124 (phone: 620-672-5618; fax: 620-672-5564; e-mail: gbsalyer{at}juno.com).

Received for publication January 14, 2003. Accepted for publication April 23, 2004.

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