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Effect of copper status, supplementation, and source on pituitary responsiveness to exogenous gonadotropin-releasing hormone in ovariectomized beef cows^1,2,3

J. K. Ahola^*,4, T. E. Engle^* and P. D. Burns^,5

^* Department of Animal Sciences, Colorado State University, Fort Collins 80523; and and Department of Biological Sciences, University of Northern Colorado, Greeley 80639

	Abstract

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The effect of Cu status, supplementation, and source on pituitary responsiveness to exogenous GnRH was evaluated using nine multiparous, nonpregnant, nonsuckling, ovariectomized Angus cows (7.1 ± 3.3 yr; 622.9 ± 49.8 kg; BCS = 6.0 ± 0.5). Cows were considered Cu-deficient based on liver Cu concentrations (<30 mg of Cu/kg of DM) after receiving a low-Cu, forage-based diet supplemented (DM basis) with 5 mg of Mo/kg and 0.3% S for 216 d. Copper-deficient cows were stratified based on age, BW, BCS, and liver Cu concentration and assigned randomly to repletion-phase treatments. Treatments included 1) control (no supplemental Cu); 2) organic (ORG; 100% organic Cu); and 3) inorganic (ING; 100% inorganic CuSO₄). Treatments were formulated to meet all NRC recommendations, except for Cu, which was supplemented to ORG and ING cows at 10 mg of Cu/kg of dietary DM. During the 159-d repletion phase, Cu status was monitored via liver biopsy samples, and all cows received exogenous progesterone. A controlled intravaginal drug-release device (replaced every 14 d) was used to maintain luteal phase progesterone as a means to provide negative feedback on the hypothalamic-pituitary axis. During the repletion phase, liver Cu concentrations did not differ between ORG and ING cows at any time. By d 77 of the repletion phase, all supplemented cows were considered adequate in Cu, and liver Cu concentrations were greater in supplemented than in nonsupplemented control cows on d 77 (P < 0.05) and throughout (P < 0.01) the repletion phase. Beginning on d 99, exogenous GnRH was administered to all cows at low (0, 3, and 9 µg; Exp. 1) and high doses (0, 27, and 81 µg; Exp. 2) at six different times. Cows were catheterized every fifth day, and blood samples were collected every 15 min for 1 h before and 4 h after GnRH administration and analyzed for LH concentration. In Exp. 1, Cu status and supplementation did not affect basal or peak LH concentrations, but total LH released tended (P < 0.07) to be greater in Cu-supplemented vs. control cows when 3 µg of GnRH was administered. In Exp. 2, there was no effect of Cu supplementation or source on basal, peak, or total LH released, regardless of GnRH dose. Pituitary LH concentrations did not differ across treatments. In conclusion, Cu status, supplementation, and source did not affect GnRH-induced LH secretion or pituitary LH stores in ovariectomized, progesterone-supplemented cows in this experiment.

Key Words: Beef Cattle • Copper • Gonadotropin-Releasing Hormone • Pituitary • Reproduction

	Introduction

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Copper is essential for normal reproduction in mammals (Underwood and Suttle, 1999); however, mechanisms of action have not been identified. Copper supplementation (in an organic form) to young beef cows resulted in a higher 30-d pregnancy rate compared with nonsupplemented controls in the second year of a 2-yr experiment, although 60-d pregnancy rates did not differ in either year (Muehlenbein et al., 2001). Other researchers also have reported positive effects of trace mineral supplementation (Kropp, 1993; Ahola et al., 2004) and source (Kropp, 1990; Stanton et al., 2000) on reproductive measurements in beef cows, but because several trace minerals were supplemented simultaneously, interpretation of the results is difficult.

Early studies show that Cu administration induced ovulation in rabbits (Fevold et al., 1936; Suzuki and Bialy, 1964). More recently, GnRH increased LH and FSH release from rat pituitaries when Cu was present in the portal blood (Kochman et al., 1992), possibly by influencing GnRH receptor binding (Kochman et al., 1997) and/or intracellular Ca activity (Hazum, 1983; Schvartz and Hazum, 1986) in anterior pituitary cells. In Cu-deficient dairy heifers, Phillippo et al. (1987a) reported that supplemental Mo (used to induce Cu deficiency) caused a lower preovulatory LH surge compared with a Cu deficiency induced by Fe supplementation, leading to the conclusion that Mo supplementation, and not Cu deficiency, directly affected the hypothalamic-pituitary axis.

Based on the importance of Cu in reproduction and reported differences in Cu availability by source (Nockels et al., 1993), we hypothesized that pituitary responsiveness to GnRH would vary with Cu status, supplementation, and source. Therefore, our objectives were to examine the effects of Cu status and supplemental source (inorganic vs. organic) in ovariectomized beef cows on pituitary responsiveness to GnRH and pituitary content of LH.

	Materials and Methods

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Experimental Animals
Before the initiation of this experiment, all animal use, handling, and sampling techniques described herein were approved by the Colorado State University Animal Care and Use Committee. Twelve mature, cycling, multiparous, nonpregnant, and nonsuckling purebred Angus cows (7.1 ± 3.3 yr; 622.9 ± 49.8 kg; BCS = 6.0 ± 0.5) from the Colorado State University (CSU) Beef Improvement Center (Saratoga, WY) were used for this experiment. Cows were transported to the CSU Agricultural Research, Development, and Education Center and placed into one 7 x 40 m feedlot pen equipped with an automatic waterer and concrete bunk. Immediately after arrival, initial BW (average BW collected over 2 d) and BCS (1 = emaciated to 9 = obese; Richards et al., 1986) were collected, and Cu status of each cow was determined via the collection of a liver biopsy and blood sample. The liver biopsy sample was collected using the true-cut technique described by Pearson and Craig (1980), as modified by Engle and Spears (2000). Immediately after collection, samples were rinsed with a 0.01 M PBS solution, placed into acid-washed polypropylene tubes, capped, placed on ice for approximately 1 h, transported to the laboratory, and stored at –20°C. Liver samples were analyzed for Cu concentration as described by Engle et al. (1997). At the same time that liver biopsy samples were collected, blood samples were collected via jugular venipuncture into heparinized, trace mineral-free Vacutainer tubes (Becton Dickinson Co., Franklin Lakes, NJ). Once collected, blood samples were placed on ice for approximately 1 h, transported to the laboratory, centrifuged at 2,000 x g for 15 min at room temperature, and plasma was transferred to acid-washed polypropylene storage vials and stored at –20°C. Plasma Cu concentrations were determined as described by Ahola et al. (2004).

All cows were ovariectomized via a standing flank procedure (Youngquist et al., 1995) 13 d after arrival to the feedlot to eliminate the effects of ovarian hormones on hypothalamic and pituitary responsiveness to GnRH (Martin et al., 1988). Throughout the experiment, cow BW and BCS data were collected monthly. The BCS scores were assigned by the same technician throughout the experiment.

Depletion Phase
In an attempt to attain Cu deficiency (liver <30 mg of Cu/kg of DM, Mills, 1987), a diet was developed to decrease Cu availability from the forage. To accomplish this, a low-Cu, forage-based basal diet (47.6% ground alfalfa hay, 46.0% ground corn stalks, DM basis; average DMI = 8.0 kg•cow^–1•d^–1; Table 1) and a corn-based supplement (DMI = 0.54 kg•cow^–1•d^–1; 6.4% of diet, DM basis), containing 5 mg of Mo/kg of dietary DM and 0.3% S (DM basis), were used to decrease Cu status. It has been reported that feeding elevated concentrations of Mo and S to mature ruminants causes the formation of thiomolybdates in the rumen, which can dramatically decrease the availability of Cu in the gastrointestinal tract (Suttle, 1991). On a daily basis, the corn-based supplement was fed in the concrete feed bunk approximately 1 h before delivery of the forage-based diet to ensure individual animal intake of the supplement. Basal forage and water trace mineral concentrations were determined using samples collected from ground alfalfa hay, ground corn stalks, and water sources. Mean trace mineral concentrations (±SEM) in the basal diet were alfalfa hay (6.5 ± 2.9 mg of Cu/kg of DM, 0.3 ± 0.1% S, and 3.4 ± 2.2 mg of Mo/kg of DM) corn stalks (2.6 ± 0.1 mg of Cu/kg of DM, 0.07 ± 0.01% S, and <1.0 mg of Mo/kg of DM), and water (0.01 µg of Cu/L, 0.02% S, and <0.02 µg of Mo/L). Mean trace mineral concentrations (±SEM) in the depletion-phase corn-based supplement were 19.1 ± 8.1 mg of Cu/kg of DM, 5.9 ± 0.2% S, 105.8 ± 23.7 mg of Mo/kg of DM, and 243.3 ± 14.0 mg of Fe/kg of DM.

After cows received the depletion diet for 216 d, nine of the original 12 cows were classified as Cu-deficient based on liver Cu concentrations. At that time, the depletion phase was terminated and the repletion phase was initiated. The three cows that failed to reach a Cu-deficient status were removed from the experiment and their depletion phase data were not included in the analyses or results of this experiment.

Repletion Phase
The nine cows were stratified based on age, BW, BCS, and liver Cu concentration, and assigned randomly to one of three treatments: 1) control (no supplemental Cu; n = 3); 2) organic (ORG; 100% organic Cu; n = 3); and 3) inorganic (ING; 100% inorganic CuSO₄; n = 3). A similar corn-based supplement was used to deliver each treatment, and all cows were fed to meet NRC (1996) requirements for all trace minerals (fed as inorganic), with the exception of Cu. Animals assigned to the control treatment did not receive any supplemental Cu, whereas inorganic Cu was provided from CuSO₄ and organic Cu was provided from a commercially available mineral proteinate source (10% Cu; Bioplex trace mineral, Alltech Inc., Nicholasville, KY). Cows receiving either the ING or ORG treatments received Cu at the NRC (1996) recommended concentration of 10 mg of Cu/kg of dietary DM. Trace mineral concentrations (DM basis) of the three corn-based supplements were 1) Control = 29.1 mg of Cu/kg, 0.287% S, 620.0 mg of Zn/kg, <1.0 mg of Mo/kg, and 146.0 mg of Fe/kg; 2) ORG = 209.0 mg of Cu/kg, 0.271% S, 649.0 mg of Zn/kg, <1.0 mg of Mo/kg, and 147.0 mg of Fe/kg; and 3) ING = 184.0 mg of Cu/kg, 0.256% S, 676.0 mg of Zn/kg, <1.0 mg Mo/kg, and 111.0 mg of Fe/kg.

The basal forage-based diet of 47.6% ground alfalfa and 46.0% ground corn stalks (DM basis) was fed for the first 66 d of the repletion phase. On d 66 of the repletion phase, the basal diet was changed to 93.7% ground alfalfa (DM basis; average DMI = 8.0 kg•cow^–1•d^–1). The corn-based supplement that carried each of the three repletion-phase treatments was maintained at a DMI of 0.54 kg•cow^–1•d^–1.

For the first 42 d of the repletion phase, all treatments included supplemental S (from CaSO₄) at NRC (1996) recommended concentrations. After collection and analysis of the first liver biopsy sample for Cu concentration, and upon observing that liver Cu concentrations in cows receiving the ORG and ING treatments were not responding to the supplemental Cu (most likely due to the presence of elevated S concentrations in the total diet), supplemental S was removed from the three treatments and new batches were reformulated to meet NRC (1996) recommended concentrations for all minerals, with the exception of S. Treatments were analyzed for trace mineral concentrations as described previously. Although no supplemental S was included in the reformulated corn-based supplements, the total dietary S concentration was 0.3% S.

Copper status of each animal was monitored during the repletion phase via the collection and analysis of liver biopsies and blood samples. After receiving the repletion diet for 77 d, cows receiving the ORG and ING treatments were considered adequate in liver Cu concentrations, whereas cows receiving the Control treatment were still classified as deficient in Cu (Table 2). Immediately after liver biopsy samples collected on d 77 of the repletion phase were analyzed, and cows were classified as adequate or deficient in Cu, the responsiveness of the anterior pituitary to exogenous GnRH was evaluated in two experiments beginning on d 99.

Because cows had been ovariectomized, no endogenous ovarian hormones were present to negatively feedback on the hypothalamic-pituitary axis and alter the release of GnRH or LH. However, in the absence of negative feedback from ovarian hormones, circulating concentrations of LH increase dramatically (Reeves et al., 1972). Therefore, 19 d before the initiation of the repletion phase, inserts containing exogenous progesterone (1.38 g of progesterone/insert; Eazi Breed controlled intravaginal drug-releasing device (CIDR), Pharmacia-UpJohn, Kalamazoo, MI) were inserted vaginally into all cows to provide a constant source of circulating progesterone and standardize the endocrine milieu. Inserts were replaced every 14 d with new inserts throughout the repletion phase, and were removed from all cows approximately 2 h before euthanasia at the end of the experiment.

Experiment 1
In a Latin square design, two low doses of GnRH (3 and 9 µg; Cystorelin, Merial, Iselin, NJ) and one saline control (sterile water; no GnRH) were administered randomly (i.v.) to all cows once every 5 d throughout an 11-d period beginning on d 99 of the repletion phase. Doses of GnRH were assigned randomly to cows, so that all doses of GnRH were administered to all treatments on each day that cows were challenged. Gonadotropin-releasing hormone from stock solution (50 µg/mL gonadorelin diacetate tetrahydrate; Cystorelin) was diluted with sterile water (Pro Labs Ltd., St. Joseph, MO) to create the appropriate doses of 3 and 9 µg of GnRH. The 0 µg of GnRH dose contained 100% sterile water (Pro Labs Ltd.). The total volume administered, regardless of GnRH dose, was 5 mL.

Approximately 24 h before each GnRH administration, all cows were nonsurgically fitted with indwelling jugular catheters. Jugular catheter insertion sites were clipped of hair, scrubbed three times with povidone iodine solution (Agri Laboratories, Ltd., St. Joseph, MO) and 70% ethyl alcohol (Agri Laboratories, Ltd.), and the area was locally anesthetized with 3 mL of 2% lidocaine hydrochloride solution (Pro Labs Ltd.). Catheter tubing (1.016 mm i.d., 1.778 mm o.d., 270 cm long; Tygon tubing; Saint-Gobain Performance Plastics, Akron, OH) was inserted through a 12-gauge needle inserted into the jugular vein, and the tubing was threaded 25 cm into the jugular vein. Blood samples were obtained (5 mL/collection; via the catheter) at 15-min intervals for a period of 5 h. Patency was maintained throughout the 5-h blood collection period with sterile 3.5% (wt/vol) sodium citrate in saline. Five blood samples were collected from each cow at 15-min intervals for 1 h before GnRH administration (–60, –45, –30, –15, and 0 min) in order to determine baseline basal LH concentrations. Immediately after the 0-min sample was collected, one of the three randomly assigned doses of GnRH (0, 3, or 9 µg) was administered slowly (over approximately 20 to 30 s) through the catheter (i.v.) before refilling the catheter with sodium citrate to maintain patency. Beginning 15 min after GnRH infusion, 16 blood samples were collected at 15-min intervals for 4 h (Reeves et al., 1970). Each blood sample was collected using a 12-mL syringe after sodium citrate was completely removed from the catheter and discarded. Immediately after each blood sample was taken, new sodium citrate was added to the catheter to maintain patency and blood samples were transferred to prelabeled disposable borosilicate glass culture tubes (13 x 100 mm; Fisher Scientific, Pittsburgh, PA), placed on ice for approximately 1 h and allowed to clot, and centrifuged on-site at 3,000 x g for 20 min at 4°C. Serum was then transferred into polystyrene tubes (12 x 75 mm; Fisher Scientific), placed on ice for approximately 4 h, transferred to the laboratory, and stored at –20°C. After the last blood sample was collected on each day, cows were released from the AI palpation chutes and restrained in a squeeze chute for removal of catheters before returning to their individual pens for access to the daily diet and supplement. New catheters were inserted into cows on alternating sides of the neck using the procedure described previously 4 and 9 d after the first catheters were removed (d 103 and 108 of the repletion phase). A 5-d period between GnRH challenges was selected to enable pituitary responsiveness and LH stores to return to normal and to avoid possible carryover effects of GnRH dose between challenges. In addition, rectal temperatures were obtained on all cows during both experiments, and no cows had an elevated body temperature at any time.

Experiment 2
In a Latin square design as described in Exp. 1, two relatively high doses of GnRH (27 and 81 µg) and one saline control (sterile water; no GnRH) were administered (i.v.) to all cows once every 5 d throughout an 11-d period beginning 6 d after the completion of Exp. 1 (d 115, 120, and 125 of the repletion phase). Doses of GnRH were assigned randomly to cows, so that all doses were administered to all treatments at each GnRH administration. All catheterization, blood collection, and blood analysis procedures performed in Exp. 2 were identical to those described for Exp. 1, except that greater doses of GnRH were administered; however, the same total volume (5 mL) administered in Exp. 1 also was used in Exp. 2.

Pituitary Tissue Collection
Thirty-four days after the completion of Exp. 2 (d 159 of the repletion phase), all cows were transported to the CSU commercial packing facility (10 km) and humanely slaughtered via captive bolt followed by exsanguination. Immediately following exsanguination (approximately 15 min), the anterior pituitary gland tissue was harvested from each cow. Briefly, the pituitary gland from each cow was collected, trimmed, hemisected mid-sagitally, and each half was wrapped in aluminum foil and immediately frozen in liquid N₂. After approximately 6 h in liquid N₂, frozen pituitary samples were transported back to the laboratory and stored at –80°C. Before determination of pituitary LH concentrations, samples were thawed at room temperature, and a 250-mg sample of chilled wet tissue was weighed and homogenized in 750 µL of chilled PBS (0.15 M) with a hand-held homogenizer (PowerGen 125; Fisher Scientific) in a 1.5-mL microcentrifuge tube (Eppendorf, Brinkman Instruments, Inc., Westbury, NY). Homogenized samples were then centrifuged at 12,000 x g for 15 min at 4°C. The supernatant fraction was removed from each sample and stored at –80°C. The supernatant fraction was then thawed at room temperature before determination of LH concentration. Quantity of pituitary LH stores was reported on a wet-weight basis (of pituitary tissue).

Assays for LH Concentration
Concentrations of LH in serum and pituitary samples were determined via a double-antibody radioimmunoassay (Niswender et al., 1969). Serum LH concentration was determined from 100 µL of sample diluted in 400 µL of 1% (wt/vol) PBS gel in polypropylene tubes (12 mm x 75 mm; Becton Dickinson). Pituitary LH concentration was determined from 100 µL of diluted supernatant (diluted 1:100,000 in 1% PBS gel) in 400 µL of 1% PBS gel.

Analysis of serum samples was completed in four assays for Exp. 1 and five assays for Exp. 2. All pituitary samples were analyzed in one assay. The standard used in all assays was NIH LH-B8. The sensitivities of the assays were 0.15 and 0.28 ng per tube for Exp. 1 and 2, respectively. The intra- and interassay CV were 5.9 and 9.7% for Exp. 1, and 4.4 and 14.4% for Exp. 2, respectively. Analysis of all count data was performed using RIANAL software (Duddleson et al., 1972).

Once LH concentrations were determined for each serum sample, basal LH concentrations were subtracted from individual values, and the maximum concentration of LH at peak during the 4-h after GnRH administration was determined. In addition, the total quantity of LH released during the 4-h period following GnRH administration (area under curve) was calculated using the trapezoid rule in SigmaPlot (SPSS Inc., Chicago, IL).

Statistical Analyses
Copper status (liver and plasma Cu concentrations) and cow performance data (BW and BCS change) were assessed using a REML-based, mixed-effects model, repeated-measures analysis (Proc Mixed; SAS Inst., Inc., Cary, NC). Initial models for Cu status and performance contained the fixed effects of treatment, time, and the treatment x time interaction; animal within treatment was included as a random effect. A spatial power covariance structure was used in the analysis, and the containment approximation was used to calculate denominator degrees of freedom. All LH concentration data (compiled via analysis of serum and pituitary tissue) were log-transformed before statistical analysis and determination of P-values; however, calculated raw means for LH concentrations (with standard errors calculated using nontransformed data) are reported in tables. Basal LH concentration data and data characterizing response to GnRH (peak LH release and area under the curve) were analyzed using Proc Mixed of SAS. Serum LH concentration data from Exp. 1 were analyzed and reported separately from Exp. 2. Initial models for response to GnRH included the fixed effects of treatment, dose sequence, and the interaction, with animal within treatment included as a random effect. Animal was used as the experimental unit for all analyses. When an interaction was not significant, it was removed from the model and the model was reduced. Differences among means were determined using preplanned single-df contrasts, including 1) Control vs. supplemented cattle; and 2) ORG vs. ING.

	Results and Discussion

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Depletion Phase
Liver and Plasma Mineral Status.
Liver Cu concentrations at the start of the depletion phase were higher than anticipated (130.0 ± 10.3 mg of Cu/kg of DM). Because mean initial liver Cu concentration was greater than 100 mg of Cu/kg of DM, cows were fed the depletion diet for a period of 216-d, until liver Cu concentrations were considered deficient (<30 mg of Cu/kg of DM liver Cu; Mills, 1987). Throughout this 216-d period, mean loss of Cu from the liver averaged 0.49 ± 0.12 mg of Cu•kg^–1•d^–1.

Previously reported rates of liver Cu loss during a depletion phase have been variable. Similar to the current experiment, liver Cu concentrations decreased approximately 0.27 mg of Cu•kg^–1•d^–1 (from approximately 55 to 38.9 mg of Cu/kg of DM) during a 60-d period when Mo (from Na₂MoO₄) was supplemented at 1.5 times the concentration of Cu present in the forage (forage Cu = 1.54 mg of Cu/kg of DM) and S was supplemented at 0.3% of the dietary DM in pregnant Hereford x Angus heifers (Arthington et al., 1995). In contrast, in young Hereford x Holstein heifer calves, supplementation of 5 mg of Mo/kg of diet (from Na₂MoO₄) alone decreased liver Cu approximately 1.88 mg of Cu•kg^–1•d^–1 (from approximately 124.7 to 19.5 mg of Cu/kg of DM) over a 56-d period, and approximately 0.26 mg of Cu•kg^–1•d^–1 throughout the next 56 d, resulting in liver Cu concentrations of 4 mg of Cu/kg of DM after 224 d of depletion via Mo supplementation (Phillippo et al., 1987b). Phillippo et al. (1987b) also reported that these calves were fed a low-Cu diet before initiation of the experiment, which began when calves were 91 to 133 d of age and may have affected the rate of liver Cu loss. Additionally, calves also had a small body mass compared with cattle in the current experiment. A substantial rate of liver Cu loss also was reported in young Hereford x Holstein heifer calves (122 to 175 d old), in which liver Cu decreased approximately 1.37 mg of Cu•kg^–1•d^–1 (from approximately 104.8 to 28.3 mg Cu/kg DM) during a 56-d period followed by a drop of 0.14 mg of Cu•kg^–1•d^–1 during the next 168 d (resulting in liver Cu concentration of less than 4 mg of Cu/kg DM) due to supplementation of 5 mg of Mo/kg diet only (Mo source not reported; Humphries et al., 1983). In older dairy steers, supplementation of 10 mg of Mo/kg diet (from ammonium molybdate; (NH₄)₆Mo₇O₂₄•4H₂O) led to a decrease of approximately 1.34 mg of Cu•kg^–1•d^–1 (from over 100 to less than 25 mg of Cu/kg DM) throughout a 56-d period, followed by a drop in liver Cu of 0.05 mg of Cu•kg^–1•d^–1 over the subsequent 112 d to less than 20 mg of Cu/kg of DM (Xin et al., 1993).

The substantial rates of liver Cu loss during depletion phases discussed above (in excess of 1.5 mg of Cu•kg^–1•d^–1) during the first 56-d were not observed in the current experiment. The differences in rate of liver Cu loss that exist between our data and previous research may be due to one or more factors, including composition of the basal diet, initial animal liver Cu concentrations, source of Mo, presence of other antagonists (e.g., S) at elevated concentrations, physiological status of the animal (i.e. growing, gestating, lactating, etc.), breed (dairy vs. beef), age, body size, and/or nutritional and trace mineral history of each animal.

With the exception of depigmentation and defective keratinization, other commonly reported clinical (bone disorders, connective tissue disorders, and diarrhea) and subclinical (growth retardation) symptoms of Cu deficiency (Underwood and Suttle, 1999) were not readily observed. At the end of the depletion phase, extremely wavy, harsh, and slightly depigmented hair coats were observed in all cows. Humphries et al. (1983) reported limited clinical signs of Cu deficiency, including loss of hair pigment and texture, skeletal changes, and a "stilted" gait in young heifer calves that received 5 mg of Mo/kg of diet for 20 wk. Bone and growth symptoms due to Cu deficiency commonly observed in growing cattle were not detected in the current experiment, most likely because cows were mature. Interestingly, once the repletion phase was initiated, and supplemental Mo/S was removed from the diet, the hair coat symptoms described above dissipated in a matter of weeks, including those in cows receiving the control treatment that remained deficient in Cu for the remainder of the experiment. This finding indicates a possible effect of Mo and S supplementation on hair coat appearance, rather than the absence of Cu.

Plasma Cu concentrations remained above Cu concentrations considered deficient in all cows throughout the experiment (data not shown); however, plasma Cu concentrations were inconsistent with liver Cu concentrations, even when collected simultaneously. Inconsistencies between liver and plasma Cu concentrations in cattle have been reported. Holstein cows supplemented with 40 mg of Cu/kg of DM had twofold greater liver Cu concentrations but numerically lower plasma Cu concentrations compared with cows receiving no supplemental Cu (Engle et al., 2001). In addition, when liver Cu concentrations decreased nearly threefold in Angus heifers from 144.8 to 48.3 mg of Cu/kg of DM during a 149-d experiment, plasma Cu concentrations did not differ (0.97 to 0.99 3g of Cu/L; Mullis et al., 2003).

Cow BW and BCS
No treatment x time interaction was present for BW (P = 0.75); however, there was a time effect (P < 0.01). During the depletion phase, initial BW did not differ from final BW (Figure 1). Relative to BCS, there was no treatment x time interaction (P = 0.63), and no time effect (P = 0.21). Additionally, initial BCS was not different from final BCS during the depletion phase (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 1. Least squares means for cow BW change in nine ovariectomized cows during the 216-d Cu depletion phase due to Mo (5 ppm) and S (0.3%) supplementation (pooled SEM = 17.8; n = 3 cows per treatment).

Repletion Phase
Liver Mineral Status.
At the initiation of the repletion phase, liver Cu concentrations were not different across treatments (Table 2). As expected, during the 159-d repletion phase, there was a treatment x time interaction (P < 0.01) for liver Cu concentration. By d 77 of the repletion phase, all supplemented cows reached liver Cu concentrations considered adequate (liver >30 mg of Cu/kg DM, Mills, 1987) and Cu concentrations were greater (P < 0.05) in supplemented than nonsupplemented control cows. Supplemented cows also had greater liver Cu concentrations at the initiation of Exp. 1 (d 99; P < 0.01), at the initiation of Exp. 2 (d 155; P < 0.01), and at the end of the repletion phase (d 159; P < 0.01) compared with controls. Liver Cu concentrations did not differ between ORG and ING treatments at any point during the repletion phase. When liver Cu concentrations within each treatment at the initiation of Exp. 1 were compared with those of Exp. 2, no differences were found; however, in ORG cows, liver Cu concentrations in Exp. 1 were less (P < 0.01) than in Exp. 2, and liver Cu concentrations in Exp. 1 tended (P = 0.08) to be lower than those in Exp. 2 in ING cows. These differences were expected because Cu was being provided during this time frame. Results of Exp. 1 and 2 were not combined because liver Cu concentrations were not the same throughout the two experiments.

The magnitudes of response to Cu supplementation by treatments (±SEM), based on liver Cu concentrations (mg of Cu/kg of DM) were: control = –0.3 ± 19.7; ORG = 69.2 ± 13.2; and ING = 75.6 ± 6.2. Among these liver concentrations, supplemented cows had greater (P < 0.01) magnitudes of response to Cu supplementation than nonsupplemented controls, whereas ORG and ING cows did not differ. During the 159-d repletion phase, the rate of Cu repletion (mg of Cu•kg^–1•d^–1) was as follows: Control = –0.002 ± 0.124; ORG = 0.44 ± 0.08; and ING = 0.48 ± 0.04.

Arthington et al. (1995) reported that by d 45 after initiation of a Cu repletion phase (10 mg of Cu/kg of dietary DM supplemented via either an organic or inorganic form) in pregnant beef heifers, Cu-supplemented heifers had greater liver Cu concentrations than controls not supplemented with Cu but still receiving supplemental Mo at 1.5 times that of the forage Cu concentration. Unlike the current experiment, however, no treatment was included that had all supplemental Cu, Mo, and S removed. The authors reported that the overall magnitude of response to Cu supplementation throughout the 45-d repletion phase did not differ by Cu source (organic vs. inorganic). Arthington et al. (1995) also reported that liver Cu concentrations were adequate, but maximum concentrations of liver Cu (48.9 and 67.7 mg of Cu/kg of dietary DM in the inorganic and organic Cu treatments, respectively) were numerically lower than those observed in the current experiment.

Several experiments have evaluated the availability of Cu, but results have been variable. In young Holstein calves receiving a Cu-depleting diet via the consumption of hay with an elevated Mo concentration (5 mg of Mo/kg of dietary DM), organic Cu had greater availability than inorganic Cu in the presence of Mo (Kincaid et al., 1986). In contrast, in older beef steers depleted of Cu (via the supplementation of 10 mg of Mo/kg of dietary DM as ammonium molybdate) no difference in Cu availability between sources (organic vs. inorganic) was reported (Wittenberg et al., 1990). In pregnant beef heifers, when no supplemental Mo or S was provided, no difference in Cu availability by source was observed (Arthington et al., 1995).

In the present study, the rate of liver Cu repletion was slower than has previously been reported in beef cattle after a depletion phase. In pregnant heifers over a 45-d period, Arthington et al. (1995) reported repletion rates of 0.95 (first 14 d) to 1.45 (next 3 d) mg of Cu•kg^–1•d^–1 in heifers receiving 10 mg Cu/kg of dietary DM (in either organic or inorganic forms) after becoming Cu deficient (average liver Cu concentration = 38.9 mg Cu/kg DM) due to Mo and S supplementation. However, the authors also reported liver Cu gains of 0.1 (first 14-d period) to 2.1 (next 31-d period) mg of Cu•kg^–1•d^–1 in heifers supplemented with 8 mg Cu/kg of dietary DM inorganic Cu after beginning the supplementation period with an adequate liver Cu status (128 mg of Cu/kg DM). It is unknown why rates of liver Cu repletion between the current experiment and previously reported research are inconsistent; however, variables including animal size, age, physiological status (gestating, growing, lactating), diet consumed, and previous nutritional history may affect the rate of Cu repletion into the liver.

Plasma Mineral Status.
Plasma Cu concentrations at the start of Exp. 1 were 0.83, 0.66, and 0.99 mg/L for Control, ORG, and ING cows, respectively (SEM = 0.144). Plasma Cu concentrations did not differ between supplemented and nonsupplemented cows or between ORG and ING cows. It has been documented that plasma Cu concentrations are poorly correlated (r = 0.28) with liver Cu concentrations in beef cattle (Vermunt and West, 1994). For this reason, liver Cu concentration is considered to be the standard method of assessing Cu status in cattle (McDowell, 1992; Wikse et al., 1992). In the current experiment, liver Cu concentrations were used as the primary method of determining Cu status and deficiency.

Cow BW and BCS.
Body weight and BCS data collected during the repletion phase are presented in Table 3. During the repletion phase, there were no treatment x time interactions for cow BW or BCS; however, a time effect was present (P < 0.01) for BW but not for BCS. Initial repletion phase BW and BCS did not differ across treatments. In addition, repletion phase initial BW and BCS were not different from final BW and BCS within the Control or ING treatments; however, BW increased (P < 0.05) during the repletion phase in the ORG cows, whereas BCS did not differ. At the start of Exp. 1, neither BW nor BCS was different between supplemented and nonsupplemented controls, or between ORG and ING cows. Similarly, at the start of Exp. 2, neither BW nor BCS was affected by Cu status or source, and final BW and BCS (collected on d 159 of the repletion phase) did not differ between supplemented and nonsupplemented control cows or between ORG and ING cows. There has been little research on BW and BCS changes due to Cu repletion after cattle were deficient in Cu due to the feeding of known Cu antagonists.

Concentrations of basal and naturally occurring pre-ovulatory LH in the circulation of Cu deficient cattle also have been reported. Phillippo et al. (1987a) observed a lower (P < 0.05) peak LH amplitude in young (90 to 130 d old) Cu-deficient Hereford x Holstein heifers supplemented with 5 mg Mo/kg diet (7 ng of LH/mL) vs. Cu-deficient heifers receiving either 500 mg of Fe/kg diet (15 ng of LH/mL) or Cu-adequate heifers receiving no supplemental trace minerals (18 ng of LH/mL). In addition, a decreased pulsatile LH concentration was reported in Cu-deficient heifer calves after 133 d of supplementation with 5 mg of Mo/kg diet vs. Cu-deficient heifer calves receiving either 500 mg of Fe/kg diet or Cu-adequate heifers receiving no supplemental trace minerals. The authors attributed the decrease in LH concentration to the presence of Mo, rather than to a deficiency in Cu. However, in both experiments all Cu-deficient cattle were consuming antagonists (Mo or Fe) when LH concentrations were evaluated, and no treatments were included to enable the specific evaluation of either Cu status or supplementation of an antagonist on LH concentrations.

Dose response curves were created for peak LH and total LH released for Exp. 1 and 2 separately (data not shown) because data from Exp. 1 and 2 were not combined into a single dose response curve, for reasons previously mentioned. It is clear that maximal LH release in response to GnRH apparently occurred when either 27 or 81 µg of GnRH was administered in Exp. 2.

Pituitary LH Concentration.
Least squares means for LH concentrations in anterior pituitaries collected at the end of the repletion phase were: 1.66, 1.31, and 1.41 mg of LH/g of wet tissue for Control, ORG, and ING treatments, respectively (SEM = 0.17). Based on these data, Cu status and supplementation did not affect (P = 0.21) pituitary LH concentration, and pituitary LH concentrations did not differ (P = 0.70) between ORG and ING cows.

Lower (P < 0.09) pituitary LH concentrations were reported in dairy steers receiving 5 mg of Mo/kg of dietary DM compared with control calves receiving 20 mg of Cu/kg of dietary DM (154.6, 213.7, and 213.5 µg of LH/g of wet tissue in Mo-supplemented, nonsupplemented control, and Cu-supplemented calves; Xin et al., 1993). Despite these results, the effect of Cu on pituitary LH concentration reported by Xin et al. (1993) cannot truly be assessed because Cu status was confounded by Mo supplementation. Pituitary LH concentrations could have been affected by Cu deficiency and/ or Mo supplementation because Mo-supplemented cattle also were Cu deficient, whereas both Cu-supplemented and control cattle had adequate liver Cu concentrations (>50 mg Cu/kg DM in nonsupplemented control and >250 mg of Cu/kg of DM in Cu-supplemented cattle).

	Implications

Top Abstract Introduction Materials and Methods Results and Discussion Implications Literature Cited

 
Our results indicate that copper status, supplementation, and source did not alter gonadotropin-releasing hormone-induced luteinizing hormone secretion or storage in the pituitary gland of cows in this experiment. Therefore, in this experiment, mechanism(s) of action by which copper affect(s) reproduction did not seem to occur at the level of the hypothalamic-pituitary axis. Further research is needed in areas where copper may affect reproductive performance in beef cows, including additional development of the model used in this experiment, hypothalamic responsiveness to ovarian hormones, ovarian response to circulating gonadotropins, ovarian activity including follicular and luteal function, uterine involution, and/or biological activity of circulating hormones.

	Footnotes

 
¹ Use of trade names in this publication does not imply endorsement by Colorado State Univ. or criticism of similar products not mentioned.

² This research was supported in part by grants from the Colorado State Univ. Agric. Exp. Stn. and Alltech Inc., Nicholasville, KY. Appreciation also is extended to Alltech Inc. for donation of the Bioplex trace mineral.

³ The authors thank J. E. Bruemmer and D. J. Denniston, Dept. of Anim. Sci., Colorado State Univ., Fort Collins, for performing the ovariectomy procedure.

⁴ Current address: Dept. of Anim. and Vet. Sci., Univ. of Idaho, Caldwell Res. and Ext. Center, Caldwell 83607.

⁵ Correspondence: 2536 Ross Hall, Campus Box 92 (phone: 970-351-2695; fax: 970-351-2335; e-mail: patrick.burns{at}unco.edu).

Received for publication December 3, 2004. Accepted for publication April 15, 2005.

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Top Abstract Introduction Materials and Methods Results and Discussion Implications Literature Cited

 


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