Experimental design
Prior to the initiation of the experiment, all animal use, handling, and sampling techniques described herein were approved by the Colorado State University Animal Care and Use Committee (IACUC approval #18-7819A). The overall project lasted 553 d with the animals housed in pastures and feedlot pens, depending on time of year (described below).
Thirty-five days prior to the initiation of the experiment, 54 multiparous commercial (Angus & Angus x Hereford) beef cows and 54 nursing calves purchased from a local cow-calf producer in Grand County, CO. Cows were stratified based on age, BW, and initial liver Mo and Cu status, and then randomly assigned to one of six treatments (n = 9 cow-calf pairs per treatment). The study was initiated in June. Cows calved each year in February and March, and calves were weaned in October. Treatments consisted of: 1) negative control (NC; basal diet with no supplemental Mo or Cu); 2) positive control (PC: NC + Cu; 3 mg of supplemental Cu/kg diet DM from CuSO4·5H2O); 3) NC + 500 µg Mo/L from Na2MoO4·2H2O supplied in drinking water (Mo 500-water); 4) NC + 1000 µg Mo/L of Na2MoO4·2H2O supplied in drinking water (Mo 1000-water); 5) NC + Mo 1000-water + 3 mg of supplemental Cu/kg diet DM from CuSO4·5H2O (Mo 1000-water + Cu); and 6) NC + 3.0 mg of supplemental Mo/kg diet DM from Na2MoO4·2H2O (3.0 Mo-diet). Total calculated intake of Cu and Mo (water and feed) expressed on a mg/kg diet DM (Cu/Mo) basis for each treatment were as follows: NC 6.8/2.6; PC 9.8/2.6; Mo 500-water 6.8/4.3; Mo 1000-water 6.8/6.0; Mo 1000-water + Cu 9.8/6.1 and; 3.0 Mo-diet 6.8/5.6
All procedures described below were repeated following the weaning of the first calf crop, except where noted. Cows remained on the same treatment for the duration of the experiment, while calves were removed from treatments at the time of weaning. All cows and calves received standard vaccinations (Bovi-Shield Gold; Zoetis, Kalamazoo, MI and Covexin 8; Merck, Omaha, NE) and were dewormed (Eprinex; Boehringer Ingelheim, St. Joseph, MO) yearly per recommendations of the USDA APHIS – Veterinary Services Foreign Animal Disease Preparedness and Response Plan [14] in consultation with the local veterinarian in Grand County, CO. Additionally, all cows and calves were tested for bovine viral diarrhea virus by obtaining an ear notch from each animal prior to the initiation of the experiment. Bovine viral diarrhea virus was not detected in any animal used in this experiment.
Animal housing
Each year, animals were housed in two different locations. In the summer and early fall months (northern hemisphere), animals were housed in one of 6 pastures (≈ 1.2 hectares per pasture) by treatment. Pastures were located in Grand County, CO and had no standing water and minimal naturally occurring forage cover. Each pasture contained four-1650 L water tanks. Each water tank was fenced to allow only cows access to water tanks. Water utilized in this experiment was transported from the Williams Fork River to the water tanks using a water truck. Each pasture was also equipped with a creep feeder that contained two-265 L water tanks only accessible to calves within that pen. This allowed determination of water intake for cows and calves separately within a pasture. Each pasture also contained one round bale feeder that was placed in an empty water tank to limit calves from consuming hay from the round bale feeder. Feed troughs were placed in each creep feeder to allow calves access to hay. This allowed for feed intake determination for cows and calves independently while housed together within a pasture. In year one, cow/calf treatment groups were rotated to a new pasture location once. In year two, pasture rotation occurred approximately every 28 d so that all cows were exposed to each pasture location. For each rotation, all waterers, feeders and loose salt feeders were moved to the appropriate pasture in order to maintain treatment integrity.
During the winter and early spring months, all animals were transported to the Agriculture, Research, Development, and Education Center (ARDEC) feedlot in Fort Collins, CO and housed in feedlot pens (7 m x 40 m) containing 3 cow-calf pairs from the same treatment per pen (3 replicates per treatment). Each pen was equipped with a concrete feed bunk, a 3 m x 7 m concrete bunk pad, and a 1050 L water tank. Feed was delivered daily in amounts that allowed ad libitum access to feed for cow and calves.
Feed, supplement, and water delivery: For both years, grass hay was purchased from local hay producers in Grand County, CO. All hay bales were sampled and analyzed for nutrient composition (Table 1). While cattle were on pasture in Grand County, CO, all hay was weighed prior to being placed in the round bale feeders or feed troughs within the creep feeders. Loose white salt was also provided to all cattle while on pasture. For a given pen, loose white salt was placed in rubber troughs and hung on the outside of the creep feeder at a height to only allow cow access. Loose white salt was also placed in a rubber tub within the creep feeder to allow calves access to white salt. Supplemental Cu (as CuSO4∙5H2O) or molybdenum (Na2MoO4·2H2O) was added to the loose salt supplement for those cows and calves receiving dietary Cu or Mo treatments (Table 2). Weekly intake of white salt was determined for cows and calves. Molybdenum and Cu concentrations of the loose salt supplement, as well as the Cu:Mo ratio for each treatment was calculated using the actual intake of water, feed, and supplement consumed by cows and calves.
Table 1
Nutrient composition of grass hay (dry matter basis).
Nutrient | Mean | Standard deviation |
Dry matter, % | 86.45 | 5.69 |
Crude Protein, % | 6.27 | 0.72 |
Acid detergent fiber, % | 35.01 | 1.78 |
Neutral detergent fiber | 57.91 | 3.29 |
Net energy for lactation, Mcal/kg | 0.53 | 0.07 |
Net Energy for gain, Mcal/kg | 1.19 | 0.13 |
Net energy for maintenance, Mcal/kg | 1.06 | 0.08 |
Total digestible nutrients, % | 53.47 | 2.69 |
Digestible energy, Mcal/kg | 2.39 | 0.12 |
Metabolizable energy, Mcal/kg | 2.09 | 0.14 |
Calcium, % | 0.42 | 0.08 |
Phosphorus, % | 0.14 | 0.04 |
Potassium, % | 1.81 | 0.14 |
Magnesium, % | 0.15 | 0.03 |
Sodium, % | 0.03 | 0.01 |
Sulfur, % | 0.15 | 0.06 |
Cobalt, mg/kg | 0.24 | 0.06 |
Copper, mg/kga | 6.84 | 2.91 |
Iron, mg/kg | 99.23 | 14.42 |
Manganese, mg/kg | 180.11 | 20.12 |
Molybdenum, mg/kg | 2.58 | 0.26 |
Selenium, mg/kg | < 1.50 | --- |
Zinc, mg/kg | 20.54 | 1.02 |
aCopper inclusion to copper containing treatments was adjusted based on the copper concentration of each hay source. Samples (n = 525) were included in this analysis. |
Table 2
Molybdenum (Mo) and copper (Cu) concentrations of water, white salt, and dried distiller’s grains (DDG) utilized to deliver experimenal treatmtens (mean ± SD).
| Treatment |
Item | Negative Controla | Positive Controlb | 500 µg Mo/L H2Oc | 1000 µg Mo/L H2Od | 1000 µg Mo/L H2O + dietary Cue | Mo dietf |
Water | | | | | | |
Mo, µg/L | < 10.0 | < 10.0 | 531.4 ± 22.47 | 1037.0 ± 117.62 | 1087.3 ± 121.12 | < 10.0 |
Cu, µg /L | < 10.0 | < 10.0 | < 10.0 | < 10.0 | < 10.0 | < 10.0 |
White Saltg | | | | | | |
Mo, mg/kg DM | < 0.01 | < 0.01 | < 0.01 | < 0.01 | < 0.01 | 458.21 ± 20.34 |
Cu, mg/kg DM | < 0.01 | 441.43 ± 30.31 | < 0.01 | < 0.01 | 445.71 ± 29.27 | < 0.01 |
DDGh | | | | | | |
Mo, mg/kg DM | 0.91 ± 0.07 | 0.89 ± 0.06 | 0.94 ± 0.10 | 0.87 ± 0.14 | 0.92 ± 0.13 | 161.11 ± 10.18 |
Cu, mg/kg DM | 6.21 ± 0.14 | 158.12 ± 12.20 | 6.29 ± 0.18 | 6.18 ± 0.12 | 160.18 ± 10.25 | 6.07 ± 0.19 |
Cu:Mo ratioi | 2.63:1 | 3.79:1 | 1.56:1 | 1.14:1 | 1.59:1 | 1.21:1 |
aNegative Control: no supplemental Mo or Cu added to the diet or water. bPositive Control: 3.0 mg Cu/kg DM from CuSO4·5H2O added to the basal diet. c500 µg Mo/L H2O: Negative Control diet + 500 µg Mo/L from Na2MoO4·2H2O supplied in the drinking water. d1000 µg Mo/L H2O: Negative Control diet + 1000 µg Mo/L of Na2MoO4·2H2O supplied in the drinking water. eMo 1000-water plus 3 mg Cu/kg DM from CuSO4·5H2O added to the basal diet. fMo Diet: Negative Control diet plus 3.0 mg Mo/kg DM from Na2MoO4·2H2O added to the basal diet. gTarget intake was 0.7% of DM intake. hDried distillers grains; supplemented at 0.25 kg∙animal− 1∙d− 1 or 1.9% of diet DM. iCalculated based on the actual intake of water, feed, and supplement consumed by cows in this experiment. |
Due to the low nutrient quality of the hay being fed, a custom molasses-based protein hard lick-tub supplement (30% CP on a DM basis; 16% equivalent CP units from urea and 14% CP units from soybean meal) was formulated to supply the appropriate amount of protein and vitamins A, D, and E for gestating and lactation beef cattle with a targeted intake of 0.34–0.57 kg∙animal-1∙day-1). Supplemental protein contained no added Cu and was provided to all cattle in tubs throughout the duration of the experiment. Protein intake was quantified weekly by weighing the protein tub. All cows and calves within a pen had ad libitum access to the same protein tub.
After transporting cattle to the ARDEC facility in Fort Collins, CO, dried distillers grains (DDG) were used as the carrier for all Mo and Cu dietary treatments. Dried distillers grains were added to each pen daily (0.25 kg animal/d) at the time of hay delivery. Cattle on treatments not receiving supplemental Mo or Cu were fed the same amount of DDG without additional Mo or Cu (Table 2). The same hay fed to cattle in pastures was fed at the ARDEC facility. Water intake was monitored twice a week by measuring the disappearance of water over a given time period as described by Kistner et al. [12]. Sodium molybdate dehydrate (Na2MoO4·2H2O) was added to each water tank at the appropriate concentration, via a dilution of a 65,000 µg of Mo/L stock solution. Water Mo and Cu concentrations for each treatment over the entire experiment are shown in Table 2.
Animal sampling
For all cows and calves, BW and jugular blood samples were obtained approximately every 28 d. Blood samples were collected into three 7 ml vacutainer tubes [1) heparinized trace-mineral-free vacutainer tubes; 2) vacutainer tubes containing EDTA; and 3) vacutainer tube containing no additive; Becton Dickinson Co., Franklin Lakes, NJ]. Once collected, blood samples were placed on ice and transported back to the laboratory (approximate time from collection until processing was 6 h). Heparinized trace-mineral-free vacutainer tubes and vacutainer tubes with no additive were centrifuged at 2,000 × g for 15 min at room temperature and plasma or serum was then transferred to acid-washed storage vials and stored at − 20°C. One ml of red blood cells (RBC) from each tube was lysed in 4 mL of cold deionized H2O and stored at − 80ºC until superoxide dismutase activity analysis could be performed. Vacutainer tubes containing EDTA were submitted to the Colorado State University Diagnostic Laboratory for complete blood count (CBC) determination. Whole blood was also analyzed for pH, pCO2, and pO2 using an I-STAT blood chemistry device.
Milk samples (approximately 50 mL) were obtained from all cows at approximately 3 month post calving, for both years, by placing each cow in a squeeze chute and manually hand milking each cow. Liver biopsies (approximately 150 mg wet weight) were obtained from all cows at the beginning of the experiment, prior to transport to the ARDEC facilities, prior to transport back to pastures in Grand County, CO, and prior to transport back to ARDEC in the second year of the experiment [15, 16]. Liver samples (approximately 100g wet weight) were also obtained from the right lobe of the liver from all cows at the time of slaughter. Calf liver biopsies were obtained from all calves at approximately 3 months of age for year 1 calves and 1.5 months of age for year two calves.
Once weaned, all calves were removed from their respective treatments, commingled by sex, and fed a standard feedlot finishing diet for approximately 220 d. On the day of slaughter, cattle were transported to a commercial abattoir, and individual carcass data and liver samples were collected. Hot carcass weight was determined at the time of slaughter. Carcasses were allowed to chill for approximately 36 h. Standard carcass data measurements were collected by trained personnel. After weaning the second calf crop, all cows were slaughtered as describe above. Kidney, muscle (longissimus dorsi), pancreas, spleen, and subcutaneous fat were obtained post slaughter and analyzed for Mo and Cu concentrations.
Cow reproductive performance
To determine the effects of treatments on cow reproductive performance, every cow was inseminated once following a modified Select-Synch method [17] with the addition of a controlled internal drug-release insert (CIDR) estrus synchronization protocol as described by Ahola et al. [18]. Cows and calves were then returned to their appropriate pastures. Fourteen days after mass insemination, six Angus bulls, that had passed breeding soundness exams, were housed (1 per pasture) with the cows and calves for 60 d (1 bull per pasture). Pregnancy was determined at 40 d after mass insemination via rectal ultrasonography to classify fetuses as either artificial insemination pregnancies or natural service pregnancies.
Cow and calf performance: Cow body weights and body condition scores (BCS; 1 = emaciated, 9 = obese; [19] were collected at approximately 28-d intervals throughout the experiment. Over both years, body weight gain and 205 d adjusted weaning weights were collected to evaluate calf performance.
Health status, immune parameters, and blood chemistry
Throughout the entire experiment, all animals were visually monitored daily to detect signs of morbidity by trained personnel as described by Caldera et al. [20]. Necropsies were performed on animals that died during the experiment. In order to assess immune function, interferon gamma concentrations, total IgG and IgM concentrations, RBC superoxide dismutase (SOD) enzyme activity, complete blood counts (CBC), pH, pO2, and pCO2, were determined on blood samples collected on d 0 and approximately every 56 d throughout the experiment on all cows and calves.
Analytical procedures: Feed samples were analyzed for: moisture using the AOAC [21] Official Method 950.46 moisture removal process; CP using the AOAC [21] Official Method 992.15 (TruSpec CN, 2004); ash using the ash oven method described in the AOAC [21] Official Method 920.153; and acid and neutral detergent fiber [22]. Feed, water, plasma, milk, and liver samples were wet ashed and analyzed for Mo and Cu concentrations using inductively coupled plasma mass spectrometry (EPA 200.8, rev. 5.4, [23]; PerkinElmer; NexION 2000 B). Water quality was analyzed using standard analytical techniques [24, 25].
Complete blood counts from whole blood were determined using a Siemens Advida 120 Hematology Analyzer (Siemens Healthineers, Erlangen, Germany). Serum ceruloplasmin activity was determined using a spectrophotometric procedure described by Houchin [26]. Total serum IgG and IgM concentrations were determined using single radioimmunodiffusion assay kits (VMRD 240 − 30 and 246 − 30; VMRD, Inc., Pullman, WA) as described by Stabel et al. [27]. Plasma samples were analyzed for INF-ɣ concentrations using an ELISA assay (Biosource KBC1231, Biosource International, Inc., Camarillo, CA). The assay was designed as a qualitative assay but for this study was modified into a quantitative assay. Briefly, a positive control that was supplied with the kit was diluted with a negative control to make standards of known concentrations. A standard curve was created using linear regression to quantitate the INF-ɣ concentrations in the unknown samples. The regression coefficient of the standard curve was 0.991 and samples were reported as log10. Lysed RBC were analyzed for SOD activity using a SOD 525™ Assay Kit (Biotech® 21010; Oxis Health Products, Inc., Portland, OR). Superoxide dismutase activity was expressed as SOD activity per milligram hemoglobin. Hemoglobin concentration was determined using a Total Hemoglobin Assay Kit (Sigma 525-A; Sigma-Aldrich, St. Louis, MO). Blood chemistry (pH, pO2 and pCO2) were analyzed using an i-STAT analyzer (Abbott, North America, Orlando FL).
Statistical analysis
Cow and calf performance data, mineral status, nutrient analysis, and immune measurements were assessed using a restricted maximum likelihood-based, mixed-effects model, repeated-measures analysis (PROC MIXED; SAS Inst., Inc., Cary, NC) where appropriate. Initial cow performance and mineral status models contained fixed effects of treatment, time, and treatment × time interaction. Initial calf performance models included fixed effects of treatment, year, age of dam, age of calf, sex of calf, and all relevant two- and three-way interactions. A spatial power covariance structure was used in the analysis and the containment approximation was used to calculate denominator degrees of freedom. Pen was considered the experimental unit for all response variables measured. Reproductive response data were analyzed using logistic regression (PROC GENMOD of SAS). Initial models for reproductive response contained fixed effects of treatment, BCS, BW, and year, in addition to all relevant two- and three-way interactions. When an interaction was not significant, it was removed from the model. If the interaction of year × treatment was not significant, data were pooled across years; otherwise, data were reported for each year separately. Significance was determined at P ≤ 0.05, and tendencies were determined if P > 0.05 and ≤ 0.10.