Effect of ammonium sulfate fertilization on bahiagrass quality and copper metabolism in grazing beef cattle 1,2

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1 Effect of ammonium sulfate fertilization on bahiagrass quality and copper metabolism in grazing beef cattle 1,2 J. D. Arthington* 3, J. E. Rechcigl* 4, G. P. Yost, L. R. McDowell, and M. D. Fanning 5 University of Florida, *Range Cattle Research and Education Center, Ona and Department of Animal Sciences, Gainesville and Southwest Florida Research and Education Center, Immokalee ABSTRACT: To assess the impact of S fertilization on bahiagrass (Paspalum notatum) quality and Cu metabolism in cattle, two studies were conducted during the summer grazing season (1999 and 2000). Pasture replicates (16.2 ha; n = 2/treatment) received the same fertilizer treatment in each growing season, consisting of 1) 67 kg N/ha from ammonium sulfate (AS), 2) 67 kg N/ha from ammonium nitrate (AN), and 3) control (no fertilizer; C). Forage sampling was conducted at 28-d intervals following fertilization by the collection of whole plants (four samples/pasture) in randomly distributed 1-m 2 grazing exclusion cages and analyzed for CP, in vitro organic matter digestibility, S, P, Ca, K, Mg, Na, Fe, Al, Mn, Cu, and Zn. To determine the effect of fertilizer treatment on liver trace mineral concentrations in grazing cattle, random liver tissue samples were collected (n = 12; four/treatment) at the start and end of the study period in Ammonium sulfate fertilization increased (P < 0.001) forage S concentration in both years. Plant tissue N concentrations were increased by N fertilization, regardless of source, in 2000, but not in Cows grazing AS pastures had lower (P < 0.05) liver Cu concentrations at the end of the study period in 2000 compared to AN and C. In Exp. 2, 37 Cu-deficient heifers grazing AS fertilized pastures were obtained from the same location and allocated to one of two treatments, consisting of supplements providing 123 mg/d of either inorganic (Cu sulfate; n = 12) or organic (Availa-Cu; n = 15) Cu. Treatments were delivered for 83 d. Liver Cu increased over time in all heifers regardless of treatment; however, heifers supplemented with Availa-Cu tended (P = 0.09) to have higher mean liver Cu concentrations than those receiving Cu sulfate. The results of these studies indicate that AS fertilization of bahiagrass increases forage S concentrations. When provided free-choice access to a complete salt-based trace mineral supplement, cows grazing AS-fertilized pastures had lower liver Cu concentrations than cows grazing pastures fertilized with AN; upon removal from high-s pastures, cattle were able to respond to Cu supplementation. Key Words: Cattle, Copper, Liver, Paspalum notation, Sulfur 2002 American Society of Animal Science. All rights reserved. J. Anim. Sci : Introduction The importance of S as a plant nutrient has been has been reviewed previously (Coleman, 1966; Martin and Walker, 1966). Concerns over S deficiency in Florida bahiagrass (Paspalum notatum), the predominant for- 1 This research is supported by the Florida Agric. Exp. Stn. and is approved for publication as R Correspondence: 3401 Experiment Station (phone: , fax: ; jdarthington@mail.ifas.ufl.edu). 3 Appreciation is expressed to Zinpro Corp., Eden Prairie, MN and Honeywell, Hanover, PA for partial funding support of this study. The use of trade names and(or) products in this publication does not imply endorsement or criticism of those products. 4 Present address: th St. E., Bradenton, FL Present address: AgriLogic, Inc., P.O. Box 9990, College Station, TX Received September 28, Accepted March 22, age grass for grazing cattle in Florida, have arisen (Rechcigl et al., 1989). In the past, fertilizer impurities provided forages with S, but with the refinement of modern fertilizer manufacturing processes, S contamination is uncommon. Therefore, the effect of S on bahiagrass quality and the effect of increased forage S on the Cu status of grazing cattle is an important issue. Rechcigl (1991) reported on a 3-yr study investigating the effect of ammonium sulfate fertilization of bahiagrass at a site in south-central Florida. In their study, the application of S via ammonium sulfate resulted in higher forage S concentrations (0.23 and 0.30% for applications of 86 and 174 kg of S/ha, respectively) compared to bahiagrass fertilized with ammonium nitrate (0.10% S). Forage Mo is a commonly recognized contributor to Cu deficiencies in cattle, but adequate dietary S is required in this antagonism. Molybdenum combines with S to form a thiomolybdate complex. Thiomolybdates 2507

2 2508 Arthington et al. bind with Cu to form an insoluble complex, rendering Cu unavailable for absorption (Mason, 1990; Suttle, 1991). Impaired immune competence is the likely result of Cu deficiency in cattle (Boyne and Arthur, 1986; Xin et al., 1991). Sulfur-molybdenum-induced Cu deficiency has a direct impact on the ability of cattle to mount a normal response to viral infection (Arthington et al., 1996). This alteration in immune competence may result in failure to respond to vaccination along with increased energy losses during disease exposure. The objectives of this study were to determine the effect of agronomic levels of ammonium sulfate fertilization on bahiagrass S concentrations and determine whether an increase in forage S affects the Cu status of cows consuming this forage. Materials and Methods Pastures, Fertilizer Treatments, and Forage Sampling: Exp. 1. This study was conducted over 2 yr (1999 and 2000). Pasture replicates (16.2 ha; n = 2/treatment) consisted of established bahiagrass grown on a Myakka soil (sandy siliceous, hyperthermic, Aeric Alaquod). Each pasture received the same fertilizer treatment in each growing season. The treatments consisted of, 1) 67 kg N/ha from ammonium sulfate (AS), 2) 67 kg N/ha from ammonium nitrate (AN), and 3) control (no fertilizer; C). Ammonium sulfate contains 24% S; therefore, ASfertilized pastures received 77 kg of S/ha. Date of fertilizer application varied each year as dictated by spring precipitation (May 7 and March 2 for 1999 and 2000, respectively). Sampling of forage regrowth was conducted at 28-d intervals by the collection of whole plants (four samples/pasture) in randomly distributed 1-m 2 grazing exclusion cages. Additionally, a 1.08-m 2 area outside of, but adjacent to, each cage was sampled for an estimate of forage availability. Forage samples were cut using a Sensation mower (Sensation Corp., Omaha, NE) at a stubble height of 7.5 cm, dried in a forced-air oven at 50 C, and ground to pass a 1-mm screen. Forage N and in vitro organic matter digestibility (IVOMD) analysis were conducted at the Forage Evaluation Support Laboratory, University of Florida, Gainesville. Briefly, for N analysis, samples were digested using a modification of the aluminum block digestion procedure of Gallaher et al. (1975). Sample weight was 0.25 g, catalyst used was 1.5 g of 9:1 K 2 SO 4 :CuSO 4, and digestion was conducted for at least 4hat375 C using 6 ml of H 2 SO 4 and2mlofh 2 O 2. Nitrogen in the digestate was determined by semiautomated colorimetry (Hambleton, 1977). For IVOMD analysis, a modification of the two-stage technique (Tilley and Terry, 1963) was used as previously described (Moore and Mott, 1974). Rumen fluid was obtained from donor cows consuming bermudagrass (Cynodon dactylon) hay and provided with 450 g of soybean meal 1 h prior to collection. Forage mineral analysis was performed by A&L Southern Agricultural Laboratories, Pompano Beach, FL, using Minerals Table 1. Mineral composition of free-choice supplement (DM basis) in Exp. 1 a Amount Macromineral, % Ca P K 0.89 Mg 1.01 Na 8.75 S 1.18 Micromineral, mg/kg Co Cu 2, Fe 10, Mn 1, Mo 6.88 Zn 1, a Continuous supply of mineral was offered to ensure that animals were allowed to consume mineral on an ad libitum basis. Intake was not recorded. atomic absorption methods previously described (Wolf, 1982). Animals: Exp. 1. In 2000, the effect of fertilizer treatment on cow liver Cu concentrations was evaluated. The animals utilized in these experiments were cared for by acceptable practices as outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching Consortium, (1988). Liver biopsy collections were performed by a trained technician using techniques previously described (Arthington and Corah, 1995) and approved by the University of Florida, Institutional Animal Care and Use Committee (Project #A603). Random liver tissue samples were collected (n = 12; four/treatment) at the start and end of the study period (117 d) in Liver tissue samples were collected, handled, and analyzed for mineral concentrations using methods previously described (Arthington et al., 1996). Assay variation was controlled using bovine liver standards (National Institute of Standards and Technology, Gaithersburg, MD). Differences in available forage due to fertilizer treatment were expected, and therefore stocking rates were applied based on predicted forage availability as shown by a previous study (Rechcigl, 1991) such that available forage would not be limiting in any treatment. Pastures were initially stocked (Period 1) at 2.0, 2.7, and 4.1 ha/ cow for AS, AN, and C, respectively, during the first 61 d of grazing. During the final 56 d of grazing (Period 2), stocking rates were increased to reflect greater forage availability (1.8, 2.3, and 3.2 ha/cow for AS, AN, and C, respectively). Group cow body weights were collected for each pasture at the start and end of each grazing period. Each pasture was provided a single mineral box providing free-choice access to a balanced salt-based trace mineral supplement containing 0.25% Cu from Cu sulfate (Table 1). Mineral intake was not monitored. The absence of this information is important when considering the effect of fertilizer treatment on cow mineral

3 Forage and cattle responsese to sulfur fertilization 2509 Table 2. Nutrient composition of grain supplement and free-choice limpograss hay in Exp. 2 Feed source CP TDN a Sulfur Mo Cu % ppm Grain supplement b c Limpograss hay d a TDN values for grain supplement derived from NRC, TDN value for limpograss hay calculated as [(0.49 IVOMD)+32.2] b Grain supplement consisted of a corn-cottonseed meal blend fed three times weekly at a rate of 2.48 kg DM/d. c Copper derived from basal diet. An additional 123 mg of Cu/d was included in individual Cu treatments CuSO 4 (25.5% Cu) and Availa-Cu (10% Cu). d Limpograss hay offered free-choice. status. Therefore, a second controlled-mineral feeding experiment was conducted. Exp. 2. Copper status was assessed by liver biopsy sampling of 48 pregnant Brangus heifers grazing ASfertilized pastures at the ranch location of Exp. 1. Heifers were consuming an average of 141 mg of Cu/d for at least 90 d via a free-choice, salt-based trace mineral supplement. Average liver Cu concentration for all 48 heifers was 74.0 ppm. Thirty-seven heifers with the lowest concentration of liver Cu were chosen to be transported to the University of Florida, Range Cattle Research and Education Center, Ona. Following calving, heifers were assigned to one of two Cu repletion treatments. Treatments were delivered for 83 d to pens (114 m 2 ) housing two or three heifers. Two 14-d enrollment periods were utilized to account for calving dates over a 28-d period. Within each period, and after calving, heifers were allocated to receive 123 mg/d of supplemental Cu from either inorganic (Cu Sulfate; n = 12) or organic (Availa-Cu, Zinpro Corp., Eden Prairie, MN; n = 15) sources. Copper treatments were formulated into a corn/cottonseed meal carrier and were offered three times weekly (2.48 kg/heifer daily) in conjunction with free-choice access to long-stem limpograss (Hemarthria altissima) hay (Table 2). Liver tissue and jugular blood were collected on d 0, 14, 28, 56, and 84. Liver samples were analyzed as described for Exp. 1. Blood was collected into heparin-coated, evacuated tubes. Plasma was harvested from blood for analysis of ceruloplasmin concentration using colorimetric procedures previously described (Demetriou et al., 1974). Statistical Analysis. Analysis of variance was performed using the general linear model procedure of SAS (SAS Inst. Inc., Cary, NC) for a completely randomized model. Forage data were analyzed as a split-plot in time with plot as the whole-plot treatment and harvest as the subplot. The model included the effect of treatment, harvest, and the interactions for treatment harvest and treatment plot. Mean comparisons for forage data were made each year to account for the variability associated with weather and initial fertilization date. Cow mineral status in Exp. 1 was evaluated using pasture as the experimental unit. The model included the effect of treatment and pasture and the interaction for treatment pasture. Treatment means for both forage and cow data were compared using single-df orthogonal contrasts. Comparisons made included fertilization vs no fertilizer (AS and AN vs C) and fertilizer source (AS vs AN). In Exp. 2, Cu treatments were delivered to pens, therefore, average liver Cu concentration for the pen was used as the experimental unit. For multiple measures of liver mineral concentration, over time, a splitplot design was used with pen as the whole-plot treatment and time as the subplot. Treatment means were compared using least significant differences using the error associated with time pen (treatment) interaction. Results Exp. 1: Bahiagrass Quality. Compared to unfertilized pastures, plant tissue N was higher (P < 0.05) in both AS- and AN-fertilized pastures in 2000, but not in 1999 (Table 3). Plant tissue S was greater (P < 0.001) in bahiagrass fertilized with AS in both 1999 and 2000 compared to AN and C (Table 3). Nominal increases in forage IVOMD were realized by fertilization regardless of N source in each year (Table 3). Forage K and Mn were increased (P < 0.05) in AS-fertilized pastures compared to AN and C (0.41, 0.31, and 0.32 % for K and 50.8, 45.5, and 45.5 ppm Mn for AS, AN, and C, respectively). Similarly, forage Zn concentrations were greater (P < 0.05) in AS-fertilized pastures compared to C, but not AN (61.0, 55.3, and 50.4 ppm Zn for AS, AN, and C, respectively). Fertilizer treatment did not affect other forage mineral concentrations analyzed for this study (average macromineral concentrations = 0.62, 0.14, 0.19, and 0.05 % for Ca, P, Mg, and Na and average micromineral concentrations = 73.0, 33.2, and 8.1 ppm for Al, Fe, and Cu, respectively). Soil Mn concentrations were lower (P < 0.05) in C pastures compared to AS and AN (2.6, 2.3, and 1.5 ppm for AS, AN, and C, respectively). Soil ph was not affected by fertilizer treatment (avg soil ph = 4.8). All other soil mineral concentrations evaluated in this study were not affected by fertilizer treatment (216.3, 46.7, 11.7, 5.8, 2.7, 0.33, 7.3, and 3.8 ppm for Ca, Mg, K, P, Zn, Cu, Fe, and Na, respectively).

4 2510 Arthington et al. Table 3. Effect of N fertilizer source on bahiagrass nutritive value in Exp. 1 a Pooled AS & AN Item and year AS AN C SEM c vsc ASvsAN % (P-value) IVOMD < Sulfur <0.001 < <0.001 <0.001 Nitrogen < a Four observations/pasture were obtained by the collection of whole plants in randomly distributed 1-m 2 grazing exclusion cages on each of four and five 28-d intervals in 1999 and 2000, respectively. b Fertilizer treatments (AS = 67 kg N/ha from ammonium sulfate; AN = 67 kg N/ha from ammonium nitrate; C = no fertilizer). c Pooled standard error of the mean. Animal Performance and Mineral Status. Cow ADG was not affected by fertilizer source (0.95, 1.16, and 1.13 and 0.45, 0.32, and 0.61 kg/d for AS, AN, and C during Periods 1 and 2, respectively). Average available forage was higher (P < 0.05) during Period 2 than during Period 1 but was not affected by fertilizer treatment (277, 259, and 277 and 321, 473, and 379 kg/cow for AS, AN, and C in Periods 1 and 2, respectively). Cows grazing pastures fertilized with AS had lower (P < 0.05) liver Cu concentrations at the end of the grazing season compared to cows grazing AN and C (Table 4), and cows grazing AN-fertilized pastures had lower (P < 0.05) liver Cu concentrations than cows grazing C. Liver Fe, Mn, Mo, and Zn were not affected by fertilizer source. A random collection of 12 liver samples at the start of the study period revealed an initial liver Cu concentration of 68.4 ± 7.9 ppm. These results suggest that the cows had marginal liver Cu stores when they initially entered the study (McDowell, 1992). Cattle grazing AN and C pastures had normal liver Cu concentrations at the end of the study period (McDowell, 1992; Table 4). In Exp. 2, heifers assigned to both treatments had marginal initial liver Cu concentrations (56 and 51 ppm liver Cu for Cu sulfate and Availa-Cu treatments, respectively). Liver Cu increased over time (P < 0.01) in all heifers regardless of treatment (Figure 1); however, heifers supplemented with organic Cu tended (P = 0.09) to have higher mean liver Cu values than those receiving inorganic Cu (156 ± 5.7 vs 119 ± 5.7 ppm). Over 83 d of supplementation the rate of liver Cu repletion was 2.20 vs 1.68 ppm/d for Availa-Cu- and Cu sulfate-supplemented heifers, respectively. No treatment differences were detected (P > 0.10) in plasma ceruloplasmin concentrations (19.4 ± 0.6 and 20.3 ± 0.5 mg/100 ml for Availa-Cu and Cu Sulfate, respectively). Discussion Bahiagrass Quality. Ammonium sulfate fertilization resulted in substantially higher (P < 0.001) forage S concentrations compared to unfertilized pastures or pastures fertilized with AN (Table 3). Hardt et al. (1991) made similar conclusions for results obtained from ASfertilized small grains, whereas AS application significantly increased forage S concentration without improving yield. Table 4. Effect of N fertilizer source on liver trace mineral concentrations of grazing cows a Pooled AS & AN Item AS AN C SEM b vsc ASvsAN ppm (P-value) Cu Fe Mn Mo Zn a Fertilizer treatments (AS = 67 kg N/ha from ammonium sulfate; AN = 67 kg N/ha from ammonium nitrate; C = no fertilizer). Final samples collected from 12 cows (two/pasture) at the end of the grazing period; year 2000, Exp. 1. Initial liver mineral concentrations obtained from 12 random cows at the start of the grazing study were 68.4 ± 8, 372 ± 24, 14.7 ± 1.4, 2.6 ± 0.1, and 124 ± 3.7 ppm for Cu, Fe, Mn, Mo, and Zn, respectively. b Pooled standard error of the mean.

5 Forage and cattle responsese to sulfur fertilization 2511 Figure 1. Liver Cu concentrations in postpartum Brangus heifers following supplementation with 123 mg of Cu/d from inorganic (Cu sulfate, ) and organic (Availa- Cu, ) sources. Treatment day interaction was nonsignificant (P = 0.44). Main effect of treatment tended (P = 0.09) to differ (mean liver Cu concentrations pooled over all times = 156 ± 5.7 and 119 ± 5.7 ppm for Availa-Cu and Cu sulfate, respectively). Forage N concentration was greater in fertilized than in unfertilized pastures in 2000, but not in 1999 (Table 3). The growing season in 1999 was late due to spring drought, which may explain the lack of N uptake in the plant. However, the drought conditions also extended into 2000, whereas bahiagrass N concentration did respond to fertilization. On-site measures of rainfall were not collected; however, rainfall at the Range Cattle Research and Education Center (approximately 80 km) was 23.5 and 35.2 cm below a 58- and 59-yr average for the grazing months in 1999 and 2000, respectively. Nominal increases in forage IVOMD were realized by fertilization, regardless of N source in each year (Table 3). Slight improvements in the digestibility of AS-fertilized bermudagrass (Cynodon dactylon; Mathews et al., 1994) and small grains (Hardt et al., 1991) have been reported previously. This improvement in digestibility may be related to increased forage S, which may have a complementary impact on the ruminal microbial environment. Increased digestibility of pangola grass (Digitaria eriantha) has been reported with S supplementation of sheep (Rees et al., 1974). The increases in mineral concentration of AS-fertilized forages may be a result of improved mineral solubility as a result of lower soil ph. Ammonium sulfate application decreases soil ph, which has been associated with an increase in Mn uptake in corn (Zea mays; Miner et al., 1986). In this study, soil ph was 5.7, compared to 6.2 in AS- and AN-fertilized fields, respectively. In the current study, soil ph was only measured in 2000, whereas the soil ph of AS-treated pastures was similar to C (ph = 4.7 and 4.9 for AS and C, respectively), suggesting that other factors may also contribute to the observed increase in mineral concentration in AS-fertilized bahiagrass. Animal Mineral Status. Decreases in circulating plasma Cu are associated with liver Cu concentrations of approximately 40 ppm and lower (Claypool et al., 1975). Once these concentrations are achieved, animals are considered severely deficient because availability of Cu to peripheral tissue sites is compromised. In general, dry liver Cu concentrations below 25 to 75 ppm are considered deficient for cattle (McDowell, 1992). Thus, the cows in the present study may have been Cu-deficient when they initially entered the study: their initial liver Cu concentrations averaged 68.4 ppm. Liver Cu concentrations are the most reliable indicator of Cu status in cattle. Normal liver Cu concentrations in Florida cattle range between 100 and 300 ppm on a DM basis (Ammerman, 1969). At the end of the study in 2000, cows grazing AS pastures were still considered marginally Cu-deficient. In contrast, cows grazing AN and C pastures had liver Cu stores into the adequate range (McDowell, 1992, Table 4). The most likely explanation for the low liver Cu concentrations in cows grazing AS-fertilized pastures is high forage S concentrations. The 2-yr average S concentration for forage samples collected on AS-treated pastures was 0.50%. The antagonistic effect of S on Cu metabolism is well recognized (Mason, 1990). Suttle (1974) reported a 56% reduction in the increase in plasma Cu following Cu sulfate supplementation of Cu-deficient sheep fed a diet containing 0.40 vs 0.10% S. A review of the literature (NRC, 1996) suggests that the maximum limit for potential S toxicity in cattle is 0.40%, and S toxicity in ruminants has been reviewed previously (Kandylis, 1984). Even though this threshold was exceeded in cattle grazing AS pastures, no signs of S toxicity were noted. Excessive dietary S is often associated with a neurological disease in feedlot cattle called polioencephalomalacia (Jeffrey et al., 1994; McAllister et al., 1997). Even though cattle grazing AS pastures consumed large amounts of S, the only indicator of S excess was their failure to respond to Cu supplementation. The absence of mineral intake information in Exp. 1 is a shortcoming of the present study when relating cow Cu status to fertilizer treatment. All cows were offered an equal opportunity to consume free-choice mineral. We are not aware of any previous data suggesting that high-s forages may limit free-choice mineral intake. However, we cannot preclude the potential influence of variable free-choice mineral intake on subsequent liver Cu concentrations reported in this study. To provide further insight, a group of heifers grazing AS-fertilized pastures from the same ranch were assessed. Despite the consumption of 142 mg of Cu/d, these heifers were found to have low liver Cu concentrations (52.7 ppm) prior to the start of Exp. 2. Once removed from the pastures containing high concentrations of S, Cu-deficient heifers were able to rapidly respond to Cu supplementation from both inorganic (Cu sulfate) and organic (Availa-Cu) sources. Heifers receiving an organic Cu source tended (P = 0.09) to have higher mean liver Cu concentrations than heifers sup-

6 2512 Arthington et al. plemented with Cu sulfate (156 ± 5.7 and 119 ± 5.7, respectively). Implications Application of agronomic rates of ammonium sulfate (67 kg N/ha) to bahiagrass pasture results in significant increases in forage sulfur concentrations. Increased forage sulfur can affect copper metabolism in grazing ruminants. Cattle grazing bahiagrass containing 0.50% sulfur had lower liver copper concentrations than cows grazing bahiagrass containing 0.22 and 0.25% sulfur. Upon removal from high-sulfur forages, copper-deficient cattle are able to respond to copper supplementation from both organic and inorganic sources. These data suggest that the application of sulfur-containing fertilizer may affect the copper status of grazing cows. In regions where grazing cattle may be prone to copper deficiency, specific attention to fertilizer source is warranted. Literature Cited Ammerman, C. B Recent developments in cobalt and copper in ruminant nutrition: A review. J. 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