
==== Front
J Anim Sci
J Anim Sci
jansci
Journal of Animal Science
0021-8812
1525-3163
Oxford University Press US

39126407
10.1093/jas/skae230
skae230
Environmental Animal Science
AcademicSubjects/SCI00960
Feeding or pen surface application of clinoptilolite with different particle sizes: impact on nitrogen utilization and manure ammonia emissions in feedlot cattle
Myers Cheyanne A Department of Animal, Veterinary, and Food Sciences, University of Idaho, Moscow, ID 83843, USA

de Haro Marti Mario E University of Idaho Extension, Gooding County, Gooding, ID 83330, USA

https://orcid.org/0000-0003-4434-5249
Chahine Mireille Twin Falls Research and Extension Center, University of Idaho Extension, Twin Falls, ID 83301, USA

https://orcid.org/0000-0002-9207-583X
Chibisa Gwinyai E Department of Animal, Veterinary, and Food Sciences, University of Idaho, Moscow, ID 83843, USA

Corresponding author: gchibisa@uidaho.edu
Cheyanne A. Myers Current address: University of Idaho Extension, Canyon County, Caldwell, ID 83605, USA

2024
10 8 2024
10 8 2024
102 skae23014 5 2024
08 8 2024
05 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.

Abstract

This study investigated the effects of feeding clinoptilolite (CLN; 2.5% of diet dry matter) with a particle size of either 30- or 400-µm on ruminal fermentation characteristics, measures of nitrogen (N) utilization, and manure ammonia-N (NH3) emissions in feedlot cattle. The impact of directly applying 30- or 400-µm CLN to the pen surface (2,250 kg/ha) on manure NH3–N emissions was also evaluated. Six beef heifers were used in a replicated 3 × 3 Latin square design with 21-d periods. Dietary treatments were 1) finishing ration with no supplement (CON), 2) CON + 30-µm CLN (CLN-30), and 3) CON + 400-µm CL (CLN-400). Intake was measured daily. To evaluate fermentation characteristics, ruminal fluid was collected on day 19. Indwelling pH loggers were used to measure ruminal pH from days 15 to 21. Blood was collected 3-h post-feeding on day 21 for metabolite analysis. Fecal grab and urine spot samples were also collected from days 19 to 21 to measure nutrient digestibility, route of N excretion, and in vitro NH3 emissions. There was no diet effect (P ≥ 0.12) on nutrient intake and apparent total tract digestibility, and ruminal short-chain fatty acid profile and pH. Ruminal NH3 concentration, which was lower (P = 0.04) for CLN-30 than CON heifers, did not differ between CON and CLN-400 heifers. Although there was no diet effect (P = 0.50) on plasma urea-N (PUN) concentration, proportion of urea-N excreted in urine was lower (P = 0.01) for CLN-30 than CON and CLN-400 heifers. Urinary NH3–N excretion, which was greater (P ≤ 0.04) for CLN-400 than CON heifers, did not differ between CLN-30 and CLN-400 heifers. Feeding CLN also increased (P ≤ 0.02) fecal excretion of potassium (K) and iron (Fe) and reduced (P = 0.01) urinary excretion of calcium (Ca). There was a treatment × time interaction (P = 0.01) for NH3 emission rate, which was greatest within the first 36 h of incubation and was lower for manure from CLN-400 compared to CON and CLN-30 heifers and pen surface application treatments. Cumulative NH3 emissions were lower (P < 0.01) for manure from CLN-400 compared to CON and CLN-30 heifers and the pen surface application treatments. Although surface application was ineffective, feeding 400-µm CLN to finishing cattle could result in a beneficial decrease in manure NH3 emissions. However, changes in fecal and urine excretion of minerals like K and Ca, which suggest a decrease in bioavailability, need to be considered when feeding CLN in finishing cattle diets.

Limiting manure ammonia emissions is one of the keys to ensuring environmental sustainability of the beef industry. Feeding finishing cattle diets containing 2.5% clinoptilolite (DM) with a particle of 400- but not 30-µm resulted in dramatic reductions in the manure ammonia emission rate and cumulative emissions; however, pen surface application of clinoptilolite (2,250 kg/ha) was ineffective.

clinoptilolite particle size
feedlot cattle
manure ammonia emissions
nitrogen utilization
==== Body
pmcIntroduction

Only 10% to 40% of feed nitrogen (N) is captured and converted into meat or milk N in cattle (Calsamiglia et al., 2010). This is problematic in part because up to 65% of N fed to feedlot cattle is lost to the atmosphere as ammonia (NH3), which together with its secondary products like ammonium (NH4+) and nitrates (NO3-) can compromise both air and water quality (VandeHaar and St-Pierre, 2006; Flesch et al, 2007). One of the major reasons for the low efficiency of N use in cattle is the indiscriminate breakdown of dietary protein in the rumen (Calsamiglia et al., 2010). The resultant NH3, if not captured for microbial protein synthesis (MPS), is lost into the hepatic portal system. It is ultimately detoxified by hepatocytes forming urea-N that is released into blood (Hristov, 2011a; Hristov et al., 2011b). The bulk of hepatocyte-derived urea-N (60% to 90%) ends up excreted as urinary urea-N (UUN) (Bristow et al., 1992), which unlike fecal N, is environmentally labile as it is rapidly hydrolyzed to release NH3 (McGinn et al., 2007). Therefore, limiting its loss from the rumen into blood could be effective in reducing manure NH3 emissions. A potential strategy to achieve this is the feeding of silicate minerals like zeolites that have a high affinity for cations, including NH4+ (Li et al., 2008). Mumpton and Fishman (1977) suggested that clinoptilolite (CLN), a natural zeolite, could reversibly bind and act as a reservoir that slowly releases NH4+ in the rumen, thus allowing more time for efficient MPS. Following both in vivo and in vitro experiments, White and Ohlrogge (1974) reported that zeolites like natural CLN could bind up to 15% of ruminal NH4+, which could limit its loss into blood and, subsequently, UUN excretion.

Feeding supplemental CLN (1.2% to 5.0% of diet DM) in high-concentrate diets resulted in a decrease in ruminal NH3 (CLN purity = 88%, particle size < 300µm; McCollum and Galyean, 1983) and plasma urea-N (PUN) concentrations (purity and particle size not reported; Sadeghi and Shawrang, 2006), and N loss from excreted manure (purity and particle size not reported; Eng et al., 2003). On the other hand, feeding CLN (1.4% to 3.0% of diet DM) in high-forage diets had no impact on ruminal NH3 concentration (CLN purity = not reported, particle size approximately 297 µm; Galyean and Chabot, 1981), PUN concentration and UUN excretion (CLN purity = 90%, particle size = 30 and 400 µm; Klaeui et al., 2020), and milk urea-N concentration (CLN purity and particle size not reported; Dschaak et al., 2010). The different responses across studies could be due to factors that influence the cation exchange properties of CLN including purity, particle size, and conditions like pH (Papaioannou et al., 2005). Although not always reported in in vivo feeding trials, purity and particle size are critical. For instance, Leung et al. (2006) reported a 50% increase in in vitro NH4+ adsorption over a 24 h period for CLN with a particle distribution of 50 to 250 µm compared to 250 to 500 µm. In addition, the 24 h NH4+ adsorption rate was also greater for CLN with a 90% compared to 80% purity (Leung et al., 2006). In vitro tests (Kithome et al, 1999; Leung et al., 2007) also revealed pH-related effects, with the NH4+ adsorption and cation resorption capacity of CLN increasing and decreasing, respectively, as pH increases. Moreover, the equilibrium between uncharged NH3 and ionic NH4+ in an aqueous environment as in the rumen is also pH dependent, with the proportion of NH3 increasing as pH rises above 5.5 (Sigurdarson et al., 2018). This could have implications for beef cattle since dietary fermentable carbohydrate (CHO) supply and, in turn, ruminal pH can be variable. Since this information is lacking, we evaluated the effects of feeding CLN with a particle size of 30 or 400 µm on ruminal fermentation characteristics, measures of N utilization, and manure NH3 emissions in feedlot cattle. The impact of particle size on NH3 emissions was also determined when CLN was applied to the pen surface. We hypothesized that fed or pen surface applied CLN would bind NH4+ and, ultimately, reduce NH3 emissions, with particle size impacting the magnitude of the changes.

Materials and Methods

All procedures used in this experiment were pre-approved by the Institutional Animal Care and Use Committee at the University of Idaho (Protocol 2019-68).

Animals, experimental design, and treatments

Six ruminally-cannulated (10 cm diameter, Bar Diamond, Inc., Parma, ID) British crossbred beef heifers (initial BW ± SD; 620.8 ± 30.15 kg) were used in a repeated 3 × 3 Latin square design with 21 d periods. Heifers were housed in individual pens at the University of Idaho Beef Center and were fed a total mixed ration (TMR) once daily at 0600 hours for ad libitum intake. Dietary adaptation was over the first 14 d of each period. Dietary treatments were 1) typical finishing ration with no CLN (CON), 2) CON + 30-µm CLN (CLN-30), and 3) CON + 400-µm CLN (CLN-400) (Table 1). Clinoptilolite (90% purity; Ida-Ore Zeolite, Nampa, ID), which was supplemented at 2.5% of diet dry matter (DM), partially replaced corn grain. A dietary inclusion rate of up to 5% was previously reported to be effective in reducing ruminal NH3 in cattle fed a finishing diet (McCollum and Galyean, 1983). Therefore, we opted for an intermediate inclusion level (2.5%). The mineral content of CLN was as follows (% DM): Silica, 71.5%; Aluminum, 11.3%; Potassium (K), 4.55%; Iron (Fe), 2.05%; Sodium (Na), 1.24%; Calcium (Ca), 1.22%, and Barium, 0.15%. Diets were formulated to be isonitrogenous and to exceed the nutrient requirements of animals gaining 1.5 kg/d (NASEM, 2016). At each feeding time, CLN was mixed with 1 kg of alfalfa silage and offered to heifers. Any supplement that was not consumed within 30 minutes was subsequently dosed via the ruminal cannula.

Table 1. Dietary ingredient and chemical composition of experimental diets

	Diet1	
Item	CON	CLN-30	CLN-400	
Ingredient, % of DM	
 Corn grain, dry-rolled	75.9	73.4	73.4	
 Corn DDGS2	13.0	13.0	13.0	
 Alfalfa silage	10.0	10.0	10.0	
 Mineral–vitamin mix3	1.15	1.15	1.15	
 Zeolite (clinoptilolite)4	—	2.50	2.50	
Chemical analyses	
 Dry matter, %	83.1 ± 3.2	83.3 ± 3.1	83.2 ± 3.1	
 Organic matter, % of DM	96.8 ± 0.04	94.6 ± 0.04	94.6 ± 0.04	
 Acid detergent fiber, % of DM	7.90 ± 0.31	7.83 ± 0.30	7.83 ± 0.30	
 Neutral detergent fiber, % of DM	18.8 ± 1.8	18.5 ± 1.7	18.5 ± 1.7	
 Indigestible NDF, % of DM	3.17 ± 0.46	3.15 ± 0.45	3.15 ± 0.45	
 Crude protein, % of DM	12.2 ± 0.5	12.0 ± 0.5	12.0 ± 0.5	
 Starch, % of DM	62.5 ± 3.1	60.5 ± 2.9	60.5 ± 2.9	
1CON, control diet; CLN-30, CON + 30-µm mesh Clinoptilolite (2.5% of diet DM); CLN-400, CON + 400-µm mesh Clinoptilolite (2.5% of diet DM).

2Dried distillers’ grain with solubles.

3Supplement DM contained CP, 51.87%; CP as non-protein-N, 46.7%; crude fat, 1.12%; salt, 12.45%; Ca, 20.75%; P, 0.16%; Mg, 0.77%; K, 0.22%; S, 0.21%; Fe, 14.03 ppm; Mn, 1,244.64 ppm; Zn 2,074.19 ppm; organic Zn, 1,037.26 ppm; Cu, 623.16 ppm; organic Cu, 207.04 ppm; Co, 31.54 ppm; I,177.31 ppm; Se, 13.69 ppm; selenium yeast, 4.56 ppm; vitamin A, 61,170.27 IU/kg; vitamin D, 5,881.76 IU/kg; vitamin E, 207.04 IU/kg; and monensin90 (Elanco Animal Health, Greenfield, IN), 1,141.30 g/ton.

4The oxide content of clinoptilolite (90% purity) used was as follows (% DM): Si, 71.5%; Al, 11.3%; K, 4.55%; Fe, 2.05%; Na, 1.24%; Ca, 1.22%; and Ba, 0.15%).

Measurements

Heifers were weighed prior to morning feeding on back-to-back days at the beginning of each experimental period and at the end of the study. To determine dry matter intake (DMI), the amount of TMR offered and orts were recorded daily. Alfalfa silage samples collected on back-to-back days each week were dried (55 °C for 72 h), with the DM content used to adjust its inclusion level when it deviated by more than 5% from the average. Individual feed ingredients collected daily during the last week were composited for each period. Orts collected daily during the last week of each period and were composited by heifer and period. All samples were dried (55 °C for 72 h) and sequentially ground through a 4- and 2-mm screen (Retsch Cutting Mill SM 200, Retsch, Haan, Germany) in preparation for analysis.

To measure fermentation characteristics, 250 mL of digesta were collected from each of the four regions of the rumen (cranial ventral, caudal ventral, central, and cranial dorsal) on day 19 at 0, 2, 3, 4, 6, 8, and 12 h post-feeding. After straining through polyester monofilament fabric (350 µm mesh opening; ELKO Filtering Co, LLC, Fort Lauderdale, FL), two 5-mL ruminal fluid aliquots were collected. One aliquot was mixed with 1 mL of metaphosphoric acid (H2PO4; 25% wt./vol) for later analysis of short-chain fatty acid (SCFA) and the other aliquot was mixed with 1 mL of 1% sulfuric acid (H2SO4) for later NH3 analysis. Collected samples were immediately placed on dry ice before storage at −80 °C. Ruminal pH was measured continuously (every minute) from days 15 to 21 of each period using indwelling pH loggers (LRCpH; DASCOR, Inc., Escondido, CA). Standardization of loggers was done using buffers of pH 4 and 7 before and after each use.

To determine apparent total tract nutrient digestibility and nutrient excretion, fecal grab and urine spot samples were collected on days 19 (0600, 1200, and 1800 hours), 20 (2400, 1500, and 2100 hours), and 21 (0300 and 0900 hours). At each sampling time, two 200-g fecal subsamples, one for chemical composition analysis and the other for manure NH3, nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) emissions measurement, were collected and immediately placed on dry ice to reduce loss of volatile compounds. Collected subsamples were composited by heifer during each period and were stored at −80 °C. To conduct chemical composition analysis, frozen fecal samples were thawed, dried (55 °C for 72 h), and ground through a 2-mm screen (Retsch Cutting Mill SM 200, Retsch). As for collected urine, a 50-mL subsample was immediately acidified with 3 mL of 2M H2SO4 and placed on dry ice to prevent the loss of NH3. A 1-mL aliquot of the acidified urine was then diluted 1:10 with distilled H2O, composited by animal for each period, and frozen (−80 °C) for later analysis of total N, urea-N, creatinine, and purine derivatives (PD). An additional 200-mL urine subsample collected at each sampling time was snap-frozen using dry ice, composited by animal for each period, and frozen (−80 °C) for later measurement of manure NH3–N and greenhouse gas (GHG) emissions.

On day 21, blood samples (10 mL) were collected 3 h post-feeding via coccygeal venipuncture. For plasma, samples were collected into a 10-mL vacutainer tube containing 158 IU Lithium heparin (Becton Dickinson, Franklin Lakes, NJ). For serum, samples were collected into a 10-mL vacutainer tube (Becton Dickinson) and immediately stored on ice. Samples were centrifuged (3,000 × g for 25 min at 4 °C) within 2 h of collection. Harvested plasma was stored (−80 °C) for later PUN analysis whereas serum was also stored (−80 °C) for later analysis of albumin, alkaline phosphate, aspartate aminotransferase, Ca, creatine kinase, gamma-glutamyl transferase, globulin, magnesium (Mg), phosphorous (P), and total protein using a blood analyzer (Abaxis Vet Scan 2; Abaxis, Inc., Union City, CA).

Laboratory analyses

Ground TMR, orts, and fecal samples were analyzed for DM (AOAC, 1990; method 930.15), organic matter (OM) (AOAC, 1990; method 942.05), crude protein (CP) using the Kjeldahl procedure (Foss Analytics; Hillerød, Denmark; AOAC, 1990; method 976.05), and acid detergent fiber (ADF), and neutral detergent fiber (NDF), with amylase and sodium sulfite used during NDF determination (AOAC, 2005; method 2002.04). The indigestible NDF (iNDF) content was also determined as described by Valente et al. (2011). Briefly, samples (0.6 g) were weighed in duplicate into filter bags (F57, Ankom Technology, Macedon, NY) and incubated for 288 h in the rumen of 2 cows. After incubation, residues were then analyzed for NDF as described previously.

Acidified urine samples were thawed and analyzed for total N using the Kjeldahl procedure (AOAC, 1990; method 976.05). Urine creatinine and urea-N, and PUN were analyzed using commercial kits (Arbor Assays; Ann Arbor, MI). A method adapted from Stentoft et al. (2014) was used for urine allantoin and uric acid analysis. Briefly, quantification was carried out using a HPLC/MS (Waters Corporation, Milford, MA) fitted with a reversed-phase column (C18, 5 µm particle size, 2 mm × 250 mm; Phenomenex, Torrance, CA) using a 5% methanol mobile phase. Ruminal fluid samples preserved with H2SO4 were thawed before centrifugation (10,800 × g for 20 min at 4°C), with the supernatant analyzed for NH3–N using a phenol-hypochlorite assay (Broderick and Kang, 1980). Ruminal fluid samples preserved with H2PO4 were thawed and centrifuged (12,000 × g for 10 min at 4 °C). The supernatant was collected and centrifuged a second time (16,000 × g for 10 min at 4 °C), with the resultant supernatant filtered through a 0.2-µm Nylon filter and diluted 1:1 with distilled water. A gas chromatograph with a flame-ionization detector (GC-FID; 6890 Series, Hewlett-Packard; Palo Alto, CA) was then used to quantify SCFA as described by Coats et al. (2012).

Emissions of NH3, N2O, CO2, and CH4 from the manure-soil surface were quantified using a vented, non-steady state, closed chamber system (76.2 × 38.1 × 7.62 cm; L × W × H) as described by Dungan et al. (2017). Briefly, aluminum chambers (3.2 mm wall thickness) with an air inlet and outlet port and fitted over anchors were used. The air inlet port was attached to a zeolite-charcoal filter to reduce contamination from the surrounding air. The air outlet/sampling port was connected to a twelve-sampling port multiplexer (eosMX/MX-P Recirculating Multiplexer, Eosense, Dartmouth, CA) using Polytetrafluoroethylene translucent tubing (Cole Parmer, Vernon Hills, IL). A sampling line connected the multiplexer to a Fourier transform infrared (FTIR) gas analyzer (DX40015; Gasmet Tech. Oy, Vantaa, Finland). To simulate feedlot pen conditions, virgin soil (Palouse silt loam; Table 2) was collected and air-dried for a week before placement into six individual utility trays (91.4 × 61.0 × 20.3 cm; Shape Products, Menomonie, WI) to a depth of 10 cm. Prior to measurements, subsamples (200 g) of collected virgin soil were used to determine soil characteristics at a commercial lab (Analytical Sciences Laboratory [ALS]; Moscow, ID). For emission measurements, trays were placed in an environmentally controlled lab (22.1°C ± 0.22), with air temperature and relative humidity (RH) continuously monitored (every 5 min; Table 3) using a logger (RHT10; Extech, Nashua, NH).

Table 2. Characteristics of the soil used for measurement of in vitro ammonia and greenhouse gas emissions

	Soil composition	
Variable	Average	Mean ± SD	
pH	6.93	0.076	
Dry matter, %	89.2	2.69	
Total carbon, %	3.62	0.538	
Total nitrogen, %	0.35	0.039	
Nitrate- + nitrite-nitrogen, ppm	82.2	10.77	
Ammonia-N, ppm	7.90	2.184	

Table 3. Ambient temperature and relative humidity in the laboratory during measurement of in vitro ammonia and greenhouse gas emissions

	Lab temperature, °C	Lab relative humidity, %	
Date	Average	Mean ± SD	Average	Mean ± SD	
18 December to 24 December	22.1	0.21	24.0	2.74	
07 January to 13 January	21.9	0.29	24.2	2.05	
15 January to 21 January	22.2	0.22	23.3	2.87	
23 January to 29 January	21.9	0.18	26.2	2.49	
31 January to 06 February	22.1	0.29	21.7	2.18	
8 February to 14 February	22.5	0.27	22.3	3.24	

Initial tray weights were recorded before anchors were placed to a depth of 7.62 cm and leveled. Thereafter, 1% of daily urine and fecal excretion (Cole et al., 2005) were then applied to the soil in each of the trays as follows: 1) urine and feces from heifers not fed CLN (CON), 2) urine and feces from heifers fed 30-µm CLN (CLN-30), and 3) urine and feces from heifers fed 400-µm CL (CLN-400). A fourth tray that did not contain urine or fecal samples (soil CON) was added to each run for background correction of soil NH3, N2O, CO2, and CH4 emissions. To evaluate the potential impact of applying CLN to the pen surface on emissions, urine and feces from heifers not fed CLN were used, with 30-µm (PAZ-30) or 400- µm CLN (PAZ-400) applied at a rate of 2,250 kg/ha. The manure surface application rate was the lowest amount shown to be effective in limiting emissions by Cole et al. (2007). A zero-calibration was performed before the first and after the final measurement using ultra-high purity grade N gas (UHP; 99.999%, Norco, Twin Falls, ID). A flowmeter (Flowmeter 0-5 LPM; Concoa, Virginia Beach, VA) and regulator (Regulator 213 series; Norlab-Norco, Boise, ID) were used to achieve the targeted 2 L/min flow rate. After the addition of urine, feces, and CLN to appropriate trays, chambers were then fitted to an anchor in each tray before sealing with a water channel. Gases emitted from each chamber were sampled and analyzed at 34 min intervals, with 4 min for stabilization (1 min pre-valve delay; 3 min post-valve delay) and 30 min for measurement. Each chamber was sampled continuously every 2.5 h for the first 72 h (8 measurements a day), and then every 2.5 h for up to 15 h each day from 72 to 156 h (5 measurements a day). To reduce headspace gas accumulation, chambers were left open and only closed during each 34-min measurement period, an approach used by others (Lee et al., 2012; Dungan et al., 2017). After the 156-h measurement period, trays were weighed, and remaining media was thoroughly mixed. A subsample was then collected, weighed, and dried (55°C for 24 h) to determine DM content. Another subsample was collected and immediately stored (-20°C) for later characterization of soil properties at a commercial laboratory (Analytical Sciences Laboratory [ALS]). Subsamples of urine and feces used for emission measurement were also collected for characterization of manure properties at a commercial laboratory (Agro-One Soils Laboratory; Ithaca, NY).

Calculations

Ruminal pH data for each heifer were summarized by period as daily minimum, mean, and maximum. The duration (h/d), area under the curve (pH × h), and acidosis index (pH × h/kg DMI) were calculated using pH thresholds of 5.8 (mild acidosis; Zebeli et al., 2012), 5.5 (subacute acidosis; Schwaiger et al., 2013), and 5.2 (acute acidosis; Owens et al., 1998).

Fecal DM output was calculated by dividing iNDF intake (kg/d) by fecal iNDF concentration. Apparent total tract digestion of DM, OM, CP, NDF, and ADF were then calculated as follows:

100−100 (iNDFconcentration in feediNDF concentration in feces ×nutrient concentration in fecesnutrient concentration in feed)

Urine output (kg/d) was estimated using urine creatinine concentration (mg/dL), BW (kg), and the creatinine constant of 29 mg/kg BW/d (Valadares et al., 1999) according to the following equation:

Urine output=29 × BW0.96creatinine concentration

Apparent N balance was calculated by difference (N intake—N excreted in urine and feces).

The excretion of allantoin and uric acid (nmol/d) were used to estimate the total absorption of PD (Chen and Gomes, 1992) according to the following equation:

PDexcreted=0.85(PDabsorbed)+(0.385 BW0.75),

where 0.85 is the recovery of absorbed purines as PD and 0.385 BW0.75 is representative of purine excretion from endogenous sources. The flow of microbial N (g/d) was then calculated using the following equation:

Microbial N=70(PDabsorbed)(0.116 × 0.83 × 1,000),

where 70 represents the N content of purines, 0.83 is the digestibility of purines, and the ratio of purine-N:total N in rumen microbes is 11.6:100.

The increase (linear or nonlinear) in concentration within the chamber headspace was used to calculate gas fluxes over time (Hutchinson and Mosier, 1981) using the GAS FLUXES function in R (R Core Team, 2020). The FORECAST function in Microsoft Excel 2020 (Microsoft Corporation, Redmond, WA) was used to generate estimates of daily gas emissions between sampling times as described by Dungan et al. (2017).

Statistical analysis

Nutrient intake, ruminal fermentation characteristics, nutrient digestibility, and nutrient excretion were analyzed as a replicated 3 × 3 Latin square using the MIXED procedure of SAS (SAS 9.4; SAS Inst. Inc., Cary, NC). As for emissions data, analysis was as a replicated 5 × 5 Latin square. The model for both was as follows:

Yijkl=μ+Si+Pj+Ck(i)+Tl+STil+Eijkl,

where Yijkl is the dependent variable, μ is the overall mean, Si is the fixed effect of square i, Pj is the fixed effect of period j, Ck(i) is the random effect of cow k (within square i), Tl is the fixed effect of treatment l, STil is the interaction between square i and treatment l, and Eijkl is the error term assumed to be normally distributed, with mean = 0 and constant variance. Temporal ruminal NH3 and emission rate data were analyzed accounting for repeated measures through the inclusion of an additional term for time (hour) and treatment × time interaction in the model described previously. For each response variable, the variance–covariance structure of the repeated measures was modeled separately, and an appropriate structure was fitted using the lowest values of the fit statistics based on the Bayesian information criteria. Residual distributions were evaluated for normality and homoscedasticity prior to analysis. Manure GHG emissions data was transformed to achieve normality. Data are presented as least square means. Significance was declared at P < 0.05 and trends at 0.05 < P ≤ 0.10.

Results

Nutrient intake and digestibility

There was no diet effect (P ≥ 0.65) on DM, OM, ADF, NDF, or CP intake (Table 4). In addition, there was no diet effect (P ≥ 0.13) on apparent total tract nutrient digestibility.

Table 4. Nutrient intake and apparent total tract nutrient digestibility for heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM

	Diet			
Variable	CON	CL-30	CL-400	SEM	P value	
Intake, kg/d	
 Dry matter	19.6	21.6	19.5	1.87	0.68	
 Organic matter	18.5	19.8	17.8	1.91	0.74	
 Acid detergent fiber	1.57	1.72	1.54	0.156	0.65	
 Neutral detergent fiber	3.69	3.98	3.55	0.427	0.77	
 Crude protein	2.28	2.49	2.19	0.277	0.74	
Apparent total tract nutrient digestibility, %	
 Dry matter	73.5	74.3	78.8	4.54	0.29	
 Organic matter	74.0	77.0	80.8	3.86	0.13	
 Acid detergent fiber	35.3	36.1	35.1	11.28	0.98	
 Neutral detergent fiber	42.3	53.6	47.4	6.77	0.14	
 Crude protein	61.9	57.6	60.9	7.34	0.44	

Ruminal fermentation

Feeding supplemental CLN had no impact (P ≥ 0.28) on mean, minimum, or maximum pH (Table 5). Similarly, the duration, area, and acidosis index for pH thresholds of 5.8, 5.5, and 5.2. did not differ (P ≥ 0.12) across treatments. Feeding CLN also had no impact (P ≥ 0.21) on the ruminal SCFA profile.

Table 5. Ruminal fermentation characteristics for heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM

	Diet			
Variable	CON	CLN-30	CLN-400	SEM	P value	
pH	
 Mean	5.15	4.97	4.99	0.151	0.52	
 Minimum	4.58	4.52	4.56	0.083	0.86	
 Maximum	6.16	5.68	5.74	0.228	0.28	
pH < 5.8	
 Duration, min/d	1,227	1,354	1,336	71.6	0.31	
 Area, pH units × min/d	1,013	1,222	1,196	205.3	0.60	
pH < 5.5	
 Duration, min/d	1,068	1,266	1,269	106.6	0.29	
 Area, pH units × min/d	665	827	804	186.3	0.68	
pH < 5.2	
 Duration, min/d	873	1,071	1,100	159.5	0.24	
 Area, pH units × min/d	369	470	444	151.4	0.82	
Acidosis index1	
 <5.8	50.0	56.6	70.3	12.93	0.12	
 <5.5	32.7	38.1	47.1	10.49	0.25	
 <5.2	18.0	21.2	25.7	7.70	0.57	
SCFA2	
Total, mM	145	129	136	11.1	0.21	
 Acetate, mol/100mol	46.7	47.5	48.1	2.70	0.85	
 Propionate, mol/100mol	34.9	35.8	32.2	3.83	0.77	
 Butyrate, mol/100mol	14.2	11.1	15.1	3.14	0.59	
 Isobutyrate, mol/100mol	0.47	0.56	0.64	0.070	0.30	
 Valerate, mol/100mol	2.36	3.67	3.18	0.537	0.26	
 Isovalerate, mol/100mol	1.09	1.19	1.57	0.459	0.74	
 Acetate:propionate	1.43	1.34	1.68	0.251	0.53	
 Total BCFA3, mol/100 mol	1.56	1.75	2.20	0.480	0.64	
1Area under curve/kg of DMI.

2Short-chain fatty acid.

3Branched chain fatty acid = isobutyrate + isovalerate.

Serum metabolite, enzyme, and mineral profile

There was no diet effect (P ≥ 0.20) on serum albumin, globulin, or total protein concentrations (Table 6). Similarly, serum enzyme and mineral composition did not differ (P ≥ 0.21) across diets.

Table 6. Serum metabolite, enzyme, and mineral composition for heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM

	Diet			
Variable	CON	CLN-30	CLN-400	SEM	P value	
Compounds	
 Albumin, g/dL	3.98	3.85	3.82	0.097	0.20	
 Globulin, g/dL	3.95	4.00	3.95	0.184	0.91	
 Total protein, TP; g/dL	7.92	7.83	7.75	0.146	0.52	
Enzymes	
 Alkaline phosphate, U/L	54.3	46.2	53.7	5.19	0.21	
 Aspartate aminotransferase, U/L	112	184	119	41.4	0.28	
 ɣ-glutamyl transpeptidase, U/L	63	47	104	25.1	0.26	
 Creatine kinase, U/L	189	201	213	27.3	0.69	
Ions	
 Calcium, mg/dL	9.82	9.72	9.53	0.209	0.38	
 Magnesium, mg/dL	2.65	2.63	2.75	0.157	0.39	
 Phosphorus, mg/dL	6.38	6.38	6.62	0.388	0.87	

Nitrogen utilization

There was no diet effect (P ≥ 0.24) on N intake and fecal excretion of DM, N, and N as a proportion of N intake and total excreted N (Table 7). Similarly, total urinary excretion of N and N as a proportion of N intake and total excreted N did not differ (P ≥ 0.24) across diets. However, UUN excretion tended to be lower (P = 0.07) for CLN-30 heifers than CON and CLN-400 heifers. The proportion of total urine N that was excreted as urea-N was lower (P = 0.01) for CLN-30 compared to CON and CLN-400 heifers. Urinary excretion of creatinine, allantoin, uric acid, total PD, and estimated microbial N flow did not differ (P ≥ 0.50) across diets. Excretion of manure N and manure N as a proportion of N intake and apparent N retention also did not differ (P ≥ 0.23) across diets. Ruminal NH3 concentration, which was lower (P = 0.04) for CLN-30 than CON heifers, did not differ between CLN-30 and CLN-400 heifers. Additionally, there was a tendency (P = 0.08) for a diet × time interaction for ruminal NH3–N concentration (Figure 1). However, PUN concentration did not differ (P = 0.50) across diets.

Table 7. Measures of nitrogen (N) utilization for heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM

	Diet			
Variable	CON	CLN-30	CLN-400	SEM	P value	
N intake, g/d	364.5	397.6	349.9	44.38	0.74	
Fecal excretion	
 Dry matter, kg/d	5.36	5.47	4.23	1.07	0.39	
 N, g/d	139.3	133.8	106.6	30.07	0.26	
 N, % of N intake	38.1	35.6	30.4	7.34	0.39	
 N, % total N excretion	53.1	53.6	46.7	6.23	0.24	
Urinary excretion	
 Total output, kg/d	12.8	12.5	11.9	2.38	0.82	
 N, g/d	117.1	111.0	114.9	11.70	0.88	
 Urea-N, g/d	85.1	61.9	81.1	11.5	0.07	
 Urea-N, % of total urine N	72.0a	55.6b	69.0a	5.58	0.01	
 Total N, % of N intake	34.2	28.1	35.6	4.58	0.47	
 Total N, % total N excretion	46.9	46.4	53.3	6.23	0.24	
 Allantoin, mmol/d	57.0	62.4	63.4	6.47	0.65	
 Uric acid, mmol/d	27.4	30.6	30.9	4.86	0.50	
 Total purine derivatives, mmol/d	84.4	92.9	94.4	10.13	0.62	
 Microbial N flow, g/d	25.0	31.6	33.8	8.46	0.63	
 Creatinine, mg/dL	144.9	158.1	166.7	28.79	0.56	
 Urine:creatinine	0.12	0.11	0.11	0.038	0.85	
Total N excretion	
 g/d	256.4	244.8	221.5	30.48	0.23	
 % of N intake	72.3	63.8	66.0	7.39	0.70	
Apparent N retention, g/d	108.2	152.9	128.4	36.21	0.70	
Ruminal ammonia-N, mg/dL	5.03a	3.61b	4.54ab	0.473	0.04	
Plasma urea-N, mg/dL	13.2	12.4	14.0	1.15	0.50	
a,bMeans with different superscripts differ (P < 0.05).

Figure 1. Diurnal changes in ruminal ammonia–N concentration for heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm Clinoptilolite (CLN-30), and CON + 400-µm Clinoptilolite (CLN-400) at 2.5% of diet DM. Feeding time was 0600 hours. Diet, P = 0.04; time, P < 0.01; diet × time interaction, P = 0.08. The error bars represent the SEM associated with time.

Manure characteristics

There was no diet effect (P ≥ 0.13) on fecal and urine pH (Table 8). Fecal DM content, which tended to be greater (P = 0.10) for CLN-30 than CLN-400, did not differ between CON and CLN-400 heifers. Fecal C (g/d), Fecal N and total ammoniacal N content, and fecal N and C excretion did not differ (P ≥ 0.15) across treatments. Fecal C:N ratio did not differ (P = 0.36) across treatments. There was no diet effect (P ≥ 0.84) on urinary total N excretion and urea-N content and excretion. However, urinary total N content tended (P = 0.06) to be greater for CLN-400 compared to CLN-30 and CON heifers. Urinary NH3–N concentration was lowest, intermediate, and greatest (P ≤ 0.04) for CON, CLN-30, and CLN-400 heifers, respectively. Fecal concentration and excretion of Ca, Mg, Na, P, Zinc, Copper, Manganese, and Molybdenum (Mo) and urinary concentration and excretion of P, K, Na, and Mo did not differ (P ≥ 0.18) across dietary treatments (Table 9). Feeding supplemental CLN resulted in an increase (P ≤ 0.02) in fecal concentration and excretion of K and Fe. Fecal excretion of Fe, which was greater (P < 0.01) for CLN-30 than CON heifers, did not differ between CON and CLN-400 heifers. Feeding supplemental CLN also resulted in a decrease (P ≤ 0.02) in urinary Ca excretion. Although urinary Mg concentration tended to be greater (P = 0.08) for CLN-400 than CON heifers, it did not differ between CON and CLN-30 heifers.

Table 8. Fecal and urine carbon (C) and nitrogen (N) profiles for heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM

	Diet			
Variable	CON	CLN-30	CLN-400	SEM	P value	
Feces	
 pH	6.08	6.02	6.08	0.190	0.94	
 Dry matter (DM), g/kg	238	251.4	222.8	8.69	0.10	
 Carbon, g/kg as is	111.8a	105.0ab	95.6b	3.87	0.04	
 Carbon, g/kg of DM	469.8a	417.7b	429.5b	5.23	<0.01	
 Carbon, g/d	2,515	2,292	1,808	472.4	0.32	
 Nitrogen (N), g/kg as is	5.31	5.66	4.57	0.354	0.07	
 N, g/kg of DM	22.3	22.5	20.5	1.24	0.47	
 N, g/d	118.9	119.4	85.8	23.26	0.16	
 Ammoniacal1-N, g/kg as is	0.09	0.11	0.07	0.019	0.29	
 Ammoniacal-N, g/kg of DM	0.38	0.43	0.29	0.075	0.46	
 Ammoniacal-N, g/d	1.87	2.16	1.05	0.556	0.15	
 Total N, g/kg as is	10.6	11.3	9.13	0.709	0.07	
 Total N, g/kg of DM	44.7	45.0	41.0	2.480	0.47	
 Total N, g/d	237.8	238.6	171.6	46.53	0.16	
 Carbon:nitrogen ratio	21.2	19.3	21.2	1.28	0.36	
Urine	
 pH	7.40	6.87	6.82	0.250	0.13	
 Total N, g/kg	11.28	11.13	13.23	2.732	0.06	
 Total N, g/d	88.0	86.6	87.0	16.01	1.00	
 Urea-N, g/kg	9.03	11.7	11.4	3.45	0.84	
 Urea-N, g/d	152.2	149.6	147.0	72.4	1.00	
 Ammonia-N, g/kg	0.43b	0.87ab	2.07a	0.41	0.04	
 Ammonia-N, g/d	5.00b	9.63ab	16.4a	2.33	0.02	
1Includes all forms of ammonia (NH3) and ammonium (NH4+).

a,bMeans with different superscripts differ (P < 0.05).

Table 9. Fecal and urine mineral composition for heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM

	Diet			
Variable	CON	CLN-30	CLN-400	SEM	P value	
Feces	
 Calcium, g/kg as is	2.32	2.49	2.30	0.285	0.87	
 Calcium, g/kg of DM	9.91	9.87	10.43	1.235	0.94	
 Calcium, g/d	48.9	55.3	45.2	14.12	0.50	
 Magnesium, g/kg as is	0.615	0.603	0.528	0.0826	0.68	
 Magnesium, g/kg of DM	2.62	2.40	2.38	0.360	0.86	
 Magnesium, g/d	12.5	13.4	10.6	3.18	0.42	
 Sodium, g/kg as is	0.123	0.160	0.167	0.0946	0.67	
 Sodium, g/kg of DM	0.529	0.645	0.783	0.4312	0.59	
 Sodium, g/d	2.42	3.96	2.18	1.578	0.42	
 Phosphorous, g/kg as is	1.51	1.59	1.55	0.209	0.95	
 Phosphorous, g/kg of DM	6.46	6.33	7.13	1.006	0.81	
 Phosphorous, g/d	30.9	35.5	30.2	7.00	0.67	
 Potassium, g/kg as is	0.62b	1.76a	1.78a	0.154	<0.01	
 Potassium, g/kg of DM	2.64b	6.99a	8.14a	0.747	<0.01	
 Potassium, g/d	13.6b	38.4a	35.0a	4.66	0.02	
 Iron, mg/kg as is	96.0b	381.5a	277.7a	52.89	<0.01	
 Iron, mg/kg of DM	404b	1,514a	1,244a	214.5	<0.01	
 Iron, mg/d	2.16b	8.54a	5.32b	2.033	<0.01	
 Zinc, mg/kg as is	31.7	31.7	28.1	3.98	0.76	
 Zinc, mg/kg of DM	137	126	127	18.7	0.89	
 Zinc, mg/d	0.64	0.72	0.55	0.133	0.39	
 Copper, mg/kg as is	6.03	6.28	5.27	0.733	0.58	
 Copper, mg/kg of DM	26.1	25.0	23.7	3.39	0.89	
 Copper, mg/d	0.12	0.26	0.10	0.079	0.18	
 Manganese, mg/kg as is	18.8	22.8	20.0	2.69	0.57	
 Manganese, mg/kg of DM	80.8	90.8	90.3	11.96	0.81	
 Manganese, mg/d	0.37	0.52	0.38	0.103	0.18	
 Molybdenum, mg/kg as is	0.15	0.13	0.11	0.024	0.41	
 Molybdenum, mg/kg of DM	0.60	0.53	0.50	0.091	0.65	
 Molybdenum, mg/d	3,612	2,864	2,757	740.1	0.54	
Urine	
 Calcium, g/kg	0.89a	0.16b	0.22b	0.157	0.02	
 Calcium, g/d	5.78a	1.61b	1.70b	0.900	0.01	
 Phosphorus, g/kg	0.74	0.68	0.65	0.208	0.92	
 Phosphorus, g/d	10.7	8.39	7.91	3.410	0.46	
 Magnesium, g/kg	0.60	0.75	0.83	0.189	0.08	
 Magnesium, g/d	6.79	7.60	8.05	0.934	0.60	
 Potassium, g/kg	4.77	4.02	4.63	0.441	0.34	
 Potassium, g/d	65.3	57.0	54.4	12.95	0.73	
 Sodium, g/kg	0.57	0.29	0.49	0.179	0.37	
 Sodium, g/d	7.27	4.65	7.24	2.840	0.63	
 Molybdenum, g/kg	149	133	179	39.8	0.60	
 Molybdenum, g/d	1.62	1.30	1.31	0.231	0.38	
a,bMeans with different superscripts differ (P < 0.05).

Manure ammonia and greenhouse gas emissions

There was a treatment × time interaction (P = 0.01) for NH3 emission rate, which was greatest within the first 36 h of incubation and was lower for CLN-400 compared to CON and CLN-30 heifers and all post-excretion application treatments (Figure 2). However, there was no treatment × time interaction (P ≥ 0.90) for N2O (Figure 3) and CO2 (Figure 4) emission rates. Cumulative NH3 emissions (0 to 72 h, 72 to 156 h, and 0 to 156 h) were lower (P < 0.01) for manure from CLN-400 compared to CON and CLN-30 heifers and the pen surface application treatments (PAZ-30 and PAZ-400) (Table 10). However, both feeding and pen surface application of CLN with a 30 and 400 µm particle size had no effect (P ≥ 0.22) on cumulative N2O and CO2 emissions. Methane emission rate was negative for all treatments in the current experiment, indicating sequestration by the soil, and is therefore not reported.

Table 10. Cumulative ammonia, nitrous oxide, and carbon dioxide emissions during the 156-h incubation period for manure collected from heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM, and for manure from CON heifers following pen surface application (2,250 kg/ha) of 30-µm (PAZ-30) and 400-µm clinoptilolite (PAZ-400)

	Treatment			
Variable	CON	CLN-30	CLN-400	PAZ-30	PAZ-400	SEM	P value	
Ammonia, mg/m2	
 0 to 72 h	43.5a	85.9a	5.63b	71.4a	44.2a	21.2	<0.01	
 72 to 156 h	4.06a	8.25a	1.30b	8.96a	6.74a	2.139	<0.01	
 0 to 156 h	47.5a	94.2a	6.93b	80.4a	51.0a	22.82	<0.01	
Nitrous oxide, mg/m2	
 0 to 72 h	0.085	0.052	0.041	0.066	0.078	0.0166	0.35	
 72 to 156 h	0.069	0.089	0.051	0.179	0.050	0.0390	0.27	
 0 to 156 h	0.154	0.141	0.092	0.245	0.127	0.0370	0.22	
Carbon dioxide, mg/m2	
 0 to 72 h	623	1,013	1,028	1,241	616	334.1	0.65	
 72 to 156 h	182	241	163	305	131	87.2	0.81	
 0 to 156 h	805	1,254	1,191	1,546	747	384.2	0.74	
a,bMeans with different superscripts differ (P < 0.05).

Figure 2. Ammonia emission rate during the 156-h incubation period for manure collected from heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM, and for manure from CON heifers following pen surface application (2,250 kg/ha) of 30-µm (PAZ-30) and 400-µm clinoptilolite (PAZ-400). Diet, P = 0.02; time, P < 0.01; diet × time interaction, P = 0.01. The error bars represent the SEM associated with time.

Figure 3. Nitrous oxide emission rate during the 156-h incubation period for manure collected from heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM, and for manure from CON heifers following pen surface application (2,250 kg/ha) of 30-µm (PAZ-30) and 400-µm clinoptilolite (PAZ-400). Diet, P = 0.37; time, P = 0.05; diet × time interaction, P = 0.98. The error bars represent the SEM associated with time.

Figure 4. Carbon dioxide emission rate during the 156-h incubation period for manure collected from heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM, and for manure from CON heifers following pen surface application (2,250 kg/ha) of 30-µm (PAZ-30) and 400-µm clinoptilolite (PAZ-400). Diet, P = 0.75; time, P < 0.01; diet × time interaction, P = 0.90. The error bars represent the SEM associated with time.

Remaining media chemical composition

Ending media pH was lower (P = 0.01) for soil CON than CLN-30 and PAZ-400 treatments; however, it did not differ between soil CON, CON, CLN-400, and PAZ-30 treatments (Table 11). There was no treatment effect (P ≥ 0.19) on ending media DM, total C, total N, and NOx-N concentrations. However, ending media total ammoniacal-N concentration was lower (P < 0.01) for the soil CON compared to the other treatments.

Table 11. Chemical composition of media at the conclusion of the 156-h incubation period for manure collected from heifers fed a typical finishing diet with no supplement (CON), CON + 30-µm clinoptilolite (CLN-30), and CON + 400-µm clinoptilolite (CLN-400) at 2.5% of diet DM, and for manure from CON heifers following pen surface application (2,250 kg/ha) of 30-µm (PAZ-30) and 400-µm clinoptilolite (PAZ-400)

	Treatment			
Variable	CON	CLN-30	CLN-400	Soil CON	PAZ-30	PAZ-400	SEM	P value	
Ending	
 pH	7.02ab	7.08a	7.03ab	6.93b	7.03ab	7.05a	0.030	<0.01	
 Dry matter (DM), %	92.7	92.1	93.5	94.7	92.3	91.5	1.12	0.25	
 Total carbon, %	3.77	4.15	3.83	3.52	3.50	3.73	0.278	0.19	
 Total nitrogen, %	0.370	0.400	0.377	0.343	0.353	0.365	0.0244	0.19	
 Nitrate- + Nitrite-nitrogen, ppm DM	83.8	82.2	82.3	82.2	86.3	84.0	5.18	0.96	
 Nitrous dioxide-nitrogen1, ppm	—	—	—	—	—	—	—	—	
 Ammoniacal-nitrogen2, ppm DM	28.8a	35.5a	29.2a	4.85b	34.0a	33.2a	4.87	<0.01	
1Concentration was below the limit of detection (<0.72 ppm).

2Includes all forms of ammonia (NH3) and ammonium (NH4+).

a,bMeans with different superscripts differ (P < 0.05).

Discussion

Our objective was to determine the effects of feeding supplemental CLN with a particle size of 30- or 400-µm to finishing beef cattle on measures of N utilization and manure NH3 emissions. We also evaluated the effects of applying CLN directly to the manure surface on NH3 emissions. Diets were formulated to be isonitrogenous and based on compositional analysis of collected feed samples, that goal was achieved with dietary CP content ranging from 12.0% to 12.2% (DM basis). Similarly, dietary DM, ADF, NDF, and iNDF content did not differ across diets. However, dietary OM and starch content were 2% lower for the CLN compared to CON diets, which reflected the negligible OM content and lack of starch in CLN, which partially replaced corn grain.

Interest in the use of CLN to limit manure reactive N emissions is due in part to its ability to bind NH4+ in the rumen when fed to cattle. A major contributor to ruminal NHx–N is dietary N intake, which did not differ across diets in the present study. Others (Sherwood et al., 2005, 2006; Sadeghi and Shawrang, 2006) also did not observe changes in N intake when finishing steers were fed 1.2% to 3% CLN (purity and particle size not reported) as diets were isonitrogenous and DMI was not hindered like in the current study. White and Ohlrogge (1974) determined that up to 15% of ruminal NH4+ can be adsorbed by zeolite, with its release a slow process that is influenced by factors like the presence of other cations like Na. In the present study, there was a 28% reduction in average ruminal NH3 concentration for CLN-30 heifers. However, although there was also a reduction for CLN-400 relative to CON heifers, it was not substantial. The larger surface area for CLN-30 than CLN-400 due to its smaller particle size could have led to a greater NH4+ adsorption capacity thereby accounting for the observed decrease in ruminal NH3. This is supported by the documented inverse relationship between CLN particle size and NH4+ adsorption rate under in vitro conditions (Leung et al., 2006; Kotoulas et al., 2019). As in the present study, McCollum and Gaylean (1983) also reported a decrease in ruminal NH3 concentration 3, 6, and 9 h post-feeding in steers fed a high-concentrate diet containing CLN (2.5% and 5% of diet DM; 88% purity; <300 µm). Similarly, Sadeghi and Shawrang (2006) observed a decrease in ruminal NH3 concentration 2.5 but not 5.5 h post-feeding in steers on a high-concentrate diet containing urea (2% of diet DM) with CLN (3% of diet DM; purity and particle size not reported) compared to without. Urías-Estrada et al. (2017) also reported a linear decrease in ruminal outflow of NH3 to the duodenum in finishing steers fed increasing amounts of CLN (1%, 2%, and 3% of diet DM; purity and particle size not reported). On the other hand, adding CLN (3% of diet DM; 500 µm; purity not reported) to a high-forage diet had no impact on ruminal NH3 concentration (Galyean and Chabot, 1981). Klaeui et al. (2020) also made similar observations when CLN (2.5% of diet DM; 30 and 400 µm; 90% purity) was added to a high-forage backgrounding diet. These discrepancies could partially be attributed to differences in particle size and/or purity, major factors that impact cation exchange properties of CLN (Papaioannou et al., 2005). For instance, Leung et al. (2006) reported a faster 24 h NH4+ adsorption rate for CLN with a 90 than 80% purity. However, both particle size and purity are not always reported, which makes it difficult to make meaningful across study comparisons.

Across studies, a reduction in ruminal NH3 concentration has primarily been reported when CLN is fed in high-concentrate, but not high-forage diets. A potential reason for this could be the differences in ruminal pH conditions between cattle fed high-grain and high-forage diets. Using an in vitro approach, Kithome et al. (1999) demonstrated limited NH4+ adsorption by CLN as pH decreased from 7 to 4. This is due to a pH-related decrease in the negative surface charge and, thus, cation exchange capacity of CLN (Oorts et al., 2004; Waldrip et al., 2015). However, it is also important to note that in an aqueous environment like the rumen, a decrease in pH below 7 causes a dramatic shift in the equilibrium between uncharged NH3 and ionic NH4+ towards ionic NH4+, which makes up to 99% of species at pH 5.5 (Sigurdarson et al., 2018). However, even at a low pH, a high NH4+ concentration will increase the probability of its adsorption by CLN (Kithome et al, 1999). In the present study, although there were no differences across diets, the average duration that ruminal pH was below thresholds used for subacute (pH 5.5; Schwaiger et al., 2013) and acute acidosis (5.2; Owens et al., 1998) ranged from 17 to 20 h. Therefore, a greater concentration of NH4+ under acidotic conditions and for an extended period as in this study, could account for the greater effectiveness of fed CLN in impacting ruminal N metabolism compared to when high-forage diets are fed. However, this still needs further evaluation under in vivo conditions. It has also been suggested that because of its affinity for cations, CLN could bind H+ and act as a rumen buffer when fed to cattle (Dschaak et al., 2010). As in the present study, others (Sadeghi and Shawrang, 2006; Urías-Estrada et al., 2017) did not observe a beneficial increase in ruminal pH when CLN was fed in high-concentrate diets. On the other hand, McCollum and Galyean et al. (1983) observed a decrease in ruminal pH in finishing steers fed 2.5% and 5% CLN (DM basis) at 6 and 12 h post-feeding, respectively. It is possible that variation in fermentable CHO intake over time across studies could have contributed to the noted differences in pH. However, overall, there does not appear to be a benefit of feeding CLN on ruminal pH possibly because of the greater affinity of CLN for other cations like NH4+.

Urea derived from hepatocyte detoxification of NH3, whose origin includes the rumen, is released into blood, with some ending up excreted in urine as UUN. In the current study, PUN concentration did not mirror ruminal NH3 data as there were no differences across diets. Similarly, Sallam et al. (2021) observed a decrease in ruminal NH3 but not PUN 3 h post-feeding in lambs fed a high-concentrate diet containing CLN (2% of diet DM; purity and particle size not reported) in combination with urea (1.1% of diet DM). On the other hand, Sadeghi and Shawrang (2006) reported a decrease in ruminal NH3 at 2.5 but not 5.5 h post-feeding and a corresponding decrease in PUN at 3 but not 6 h post-feeding in steers fed CLN in combination with urea. Therefore, the differences across studies could be due to variation in timing and frequency of sampling. In the present study, multiple ruminal fluid samples were collected (0, 2, 3, 4, 6, 8, and 12 h post-feeding) for NH3 analysis, whereas a single blood sample (3 h post-feeding) was collected for PUN analysis. However, UUN excretion, which was measured using 8 samples collected at 3-h intervals to represent a 24-h cycle, mirrored ruminal NH3 data. Absolute UUN excretion was 24% to 27% lower for heifers fed CLN-30 than CON and CLN-400 heifers. Typically, at least 60% of excreted N in urine is urea-N (Whitehead et al., 1989). This was the case for CON (72%) and CLN-400 (69%), but not CLN-30 heifers (56%), and this is suggestive of greater effectiveness of the smaller CLN particle size in ruminal binding of NH4+.

Up to 90% of NH3 emitted from the pen surface in feedlots is from UUN (Stewart, 1970; Waldrip et al., 2015). Therefore, we expected the NH3 emission rate to be lower for manure from CLN-30 compared to CON or CLN-400 heifers. However, this was not the case as both NH3 emission rate, and cumulative emissions were lower for manure from CL-400 compared to CON and CL-30 heifers. The intensity of NH3 emissions is typically greatest within the first 48 to 72 h of storage (Lee et al., 2011). In the present study, 91% of total cumulative NH3 emissions were within 72 h of incubation for manure from CON and CLN-30 heifers compared to 81% for CLN-400 heifers. The high-efficacy of fed CLN-400 in abating emissions as reflected by the 85% to 93% reduction in the total amount of NH3 lost over 156 h compared to manure from CON and CLN-30 heifers offers a potential opportunity to dramatically reduce reactive N emissions from the beef sector. However, it is not clear why 1) the lower UUN for heifers fed the CLN-30 diet compared to CON and CLN-400 heifers did not translate to a decrease in manure NH3 emissions and 2) there was a decrease in NH3 emissions for CLN-400 but not CLN-30 in the current study. Beyond the rumen, there are dramatic shifts in pH, which is around 2 to 3 in the abomasum and duodenum, increases in the jejunum (4 to 7) and ileum (7 to 8; Frey et al., 2010). In cattle fed high grain diets, as in the present study, another decrease in pH could occur in the hindgut due to acidosis. Although there is no pH threshold for it, Sulzberger et al. (2016) demonstrated a substantial decrease in fecal pH (from about 6.4 to 5.5) after a grain challenge in dairy cattle. Besides pH, ionic concentration of digesta also varies in different segments of the gut (Goff, 2018). Using the in vitro approach to mimic changes in pH in the gut, Leung et al. (2007) observed a decrease in NH4+ adsorption and mineral (e.g., Ca, K, and Mg) release by CLN (80% purity) at an acidic than neutral pH (1.5 vs. 7). In the same study, addition of a Na-rich buffer also reduced adsorption of NH4+. Particle release that causes clogging and slows ion exchange rates has also been reported to be greater for CLN with fine compared to coarse particles under certain conditions including low ionic strength environments (Abadzic and Ryan, 2001). Therefore, it is possible that variable digesta pH and ionic concentration along the gut could have resulted in changes and, potentially, differences in the NH4+ sorption capacity of fed CL-30 compared to CL-400 that ended up excreted. However, in vivo studies that evaluate potential changes in cation exchange properties of fed CLN (especially of different particle sizes) as it moves along the gut and implications on manure NH3 emissions are still needed.

Few other studies have evaluated the effectiveness of fed CLN in limiting feedlot NH3 emissions. Using a similar approach to ours, Cole et al. (2007) did not observe changes in manure NH3 emissions and ending soil NHx–N and C content when feedlot steers consumed 1% to 2% potassium zeolite (purity and particle size not reported). Although NH3 emissions were not measured, Eng et al. (2003) reported a decrease in manure N loss during 30 d of storage after finishing cattle were fed supplemental CLN (1.2% of diet DM purity and particle size not reported). In contrast, Sherwood et al. (2005, 2006) did not observe changes in volatile N losses from open lot pens when cattle were fed supplemental CLN (1.2% of diet DM; purity and particle size not reported) over a 168-d finishing period. Compared to when fed, surface-applied CLN (both 30- and 400-µm; rate equivalent to 2,250 kg/ha) had no impact on NH3 emissions in the present study. However, unlike with fed CLN, surface application of CLN at different rates (2,250; 4,500; and 9,000 kg/ha) led to a 49% to 67% decrease in in vitro manure NH3 emissions in the study by Cole et al. (2007). Using an in vitro approach, Waldrip et al. (2015) also reported a decrease in manure NH3 emissions with surface-applied CLN (1% and 5% of manure DM; purity and particle size not reported). Similarly, although not as effective as when fed, surface-applied CLN also resulted in a decrease in manure N loss over a 30-d storage period (Eng et al., 2003). In lab-scale studies (Cole et al., 2007; Waldrip et al., 2015) that are comparable to the present one, feces, urine, soil, and CLN were thoroughly mixed prior to incubation and measurement of emissions. However, to simulate what would happen on-farm, CLN was added without thorough mixing into manure in the present study. Therefore, this could partially account for the differences in responses between the current study and others (Cole et al., 2005; Waldrip et al., 2015). The incomplete reporting of vital experimental information, like soil moisture content and ambient temperature, also complicates across study comparisons. However, it is important to note that in feedlots, feeding CLN is more appealing in part because unlike with slurries (e.g., in the dairy industry), it is not practical to mix surface-applied CLN into solid manure and urine spots, which limits effectiveness (Ndegwa et al., 2008).

Environmental variables including soil moisture content, ambient temperature, and humidity, which can influence manure NH3 and GHG emissions, were controlled in the present study. Therefore, this needs to be considered in terms of interpretation of results. For instance, after keeping other factors like temperature constant, Wenzhu et al. (2023) demonstrated an increase in the NH3 emission rate as the soil moisture content decreased from 25% to 5%. To mimic feedlot conditions in summer, dry soil (11% moisture) was used in the present study, which was favorable in terms of NH3 emissions. However, this could have limited N2O emissions since dry conditions are more conducive for nitrification due to increased oxygen availability and limited microbial access to substrates (e.g., NH4+) in soil water films (Meixner and Yang, 2006). Just as in this study, Lee et al. (2011; 2012) also reported low N2O emissions, which was in part attributed to the short duration of measurements (<10 d). Methanogenesis, which is an anaerobic process, could be terminated in soils when desiccated (Conrad, 2020). This was demonstrated by Cardoso et al. (2019), who reported CH4 sequestration rather than emission following soil application of manure (urine and feces) to dry compared to moist soil (22% vs. 34% moisture). Therefore, the aerated and dry soil used possibly accounts for the negative CH4 emission rate in the present study. Emissions of CO2, which besides CH4 is the other product of microbial degradation of fecal OM, can also be limited by a low moisture content (Serrano-Silva et al., 2011). In the present study, the greater CO2 emission rate for CLN-400 than other treatments at the beginning of the incubation period also mirrored NH3 emission data. This is suggestive of the potential contribution of CO2 from hydrolyzed UUN (Serrano-Silva et al., 2011). Besides environmental conditions, other factors like housing and manure management, which could not be accounted for in the current study, also influence NH3 and GHG emissions. Given all these variables, extrapolation of in vitro flux data to on-farm situations is challenging. However, it is important to note that quantifying emissions while controlling for confounding factors like soil moisture and ambient temperature as in the present study, is the best approach to tease out direct effects of different mitigation strategies, including dietary manipulation (Lee et al., 2011, 2012). However, on-farm evaluation of these strategies, where different conditions including climate-related ones are encountered, will be vital in making progress in limiting both reactive N and C emissions.

Besides NH4+, fed CLN also binds diet-derived cations. Ames (1967) ranked the affinity of cations to CLN as follows: K+ > NH4+ > Na+ > Ca2+ > Fe3+ > Mg2+. Therefore, binding of any of the diet-supplied micronutrients could potentially reduce their bioavailability and cause changes in cellular and whole-body metabolism. In the present study, fecal excretion of K and Fe were greater for CLN-fed compared to CON heifers. This is suggestive of binding by fed CLN along the gut, which limited absorption. Different homeostatic pathways maintain the blood concentrations of K and Fe within optimal physiologic ranges. In the case of K, blood concentration was not measured in the present study, but urinary excretion did not differ across diets. However, urinary excretion of NH3 was greatest for CLN-400, intermediate for CLN-30, and lowest for CON heifers. In mammals, hypokalemia can cause an increase in urinary NH3 excretion, which is geared towards maintenance of acid-base homeostasis (Weiner et al., 2015). Therefore, given the observed increase in both fecal K and urinary NH3 excretion, it is possible that the binding of dietary K by fed CLN compromised its absorption in the gut, thereby leading to changes in renal NH3 metabolism. However, increased urinary NH3 excretion could also signal other metabolic disturbances such as liver or kidney damage (Lebeda and Urbankova, 1987). However, to the best of our knowledge, there is no evidence in literature of fed CLN causing liver or kidney damage in ruminants. It is important to note that NH3 excreted in urine possibly contributes to manure NH3 emissions. Serum Ca, P, and Mg did not differ across dietary treatments in the current study. Similarly, feeding CLN had no impact on plasma Ca and P in lambs (2% of diet DM; 500 µm and purity not reported; Pond, 1984) and serum Ca, P, Mg, K, and Na in lactating dairy cows (1.25% and 2.5% of diet DM; < 0.80 mm and 92% purity; Katsoulos et al., 2005). However, urinary Ca excretion was lower for CLN-fed than CON heifers in the current study. This is suggestive of increased renal tubular reabsorption geared towards maintenance of blood Ca concentration within an optimal physiologic range as it is possible that fed CLN bound and reduced intestinal absorption of Ca. However, key regulators of renal reabsorption, intestinal absorption, and bone resorption of Ca like parathyroid hormone and 1,25-dihydroxycholecalciferol (Goff, 2018), were not measured in the present study.

Mumpton and Fishman (1977) suggested that fed CLN could improve ruminal N use as the binding and slow release of NH3 would limit its spillage into blood while increasing the probability of its capture and use for MPS. Since it supplies up to 80% of the N required, it has been suggested that optimal MPS occurs when ruminal NH3 concentration is at least 5.0 to 11.8 mg/dL (Satter and Slyter,1974; Reynal and Broderick, 2005). In the present study, ruminal NH3 concentration, which was lower for CLN-30 than CON and CLN-400 heifers, was also below the 5 mg/dL threshold at all sampling times. However, this possibly did not compromise MPS since estimated microbial N supply did not differ across diets. Since ruminal NH3 concentration was only measured at set intervals over the first 12 h post-feeding, we cannot fully account for all the changes that could have occurred during a full 24-h feeding cycle. However, as in this study, Urias-Estrada et al. (2017) also did not observe changes in microbial N supply in finishing steers fed 1% to 3% CLN (DM basis). Ruminal fermentable energy supply is a major determinant of MPS (Hristov and Jouany, 2005). Although it was not measured in the present study, both dietary OM and starch content were 2 percentage units lower for the CLN than CON diet. However, since MPS did not differ across diets, this difference might not have been substantial enough to compromise microbial growth. Given the lack of a dietary treatment effect on microbial N flow and other indicators of nutrient supply like intake and apparent total tract digestibility, and ruminal SCFA profile, it is not surprising that apparent N retention also did not differ across dietary treatments. It is important to note that spot urine and grab fecal samples were used to measure digestibility, N utilization, and manure GHG emissions. Although deemed adequate for making treatment comparisons, potential errors related to accuracy of obtained estimates cannot be discounted (Morris et al., 2018; Lee et al., 2019). However, total urine and feces collection, which is the alternative method, also has limitations. For instance, waiting for 24 h before collection of subsamples for analysis from total excreted urine, even when an acid is used to lower pH, increases the likelihood of substantial losses of volatile N, leading to underestimation N excretion. In the present study, steps like the collection of a minimum of 6 evenly spaced samples in a 24-h feeding cycle and the snap freezing of all samples immediately after collection, were followed. However, even with these measures, there is still a need to use caution when interpreting results.

Conclusion

Feeding supplemental CLN (90% purity; 2.5% of diet DM) with a particle size of 30- but not 400-µm led to a decrease in ruminal NH3 concentration and UUN excretion in finishing cattle; however, NH3 emission rate and cumulative emissions were lower for manure from heifers fed 400-µm but not 30-µm CLN. Although reasons for this are not clear, it is possible that changes in physiological parameters of digesta like pH and ionic concentration as it moved through different gut segments could have led to divergent changes in cation adsorption and resorption properties of fed CLN that ended up excreted. Increased excretion of K and Fe in feces and NH3 in urine, and decreased excretion of Ca in urine by CLN-fed heifers, suggests binding of other cations along the gut that could have also led to changes in whole-body metabolism. Unlike when fed, surface application of CLN was not effective in reducing NH3 emissions. However, further research, especially measurement of manure N and C emissions under practical settings when CLN is fed and/or applied to the pen surface is warranted.

Acknowledgments

This work was supported by the USDA National Institute of Food and Agriculture (Hatch project IDA01570) and Idaho Beef Council (AG5509). The authors thank Ida-Ore Zeolite for supplying clinoptilolite for this study. The authors also thank the staff of the Beef Unit at the Palouse Research, Extension and Education Center (University of Idaho) and students in the Ruminant Nutrition Laboratory (Kylee Elmore, Elizabeth Worley, Clair Feldmann, Harleigh Johnson, and Ani Husaby) for excellent animal care and assisting with sample collection. The authors want to thank the USDA-ARS Research Station in Kimberly, ID for the use of the static flux chambers.

Abbreviations

ADF acid detergent fiber

BW body weight

C carbon

Ca calcium

CHO carbohydrate

CLN clinoptilolite

CO2 carbon dioxide

CP crude protein

DM dry matter

DMI dry matter intake

Fe iron

FTIR Fourier transform infrared

H2PO4 metaphosphoric acid

H2SO4 sulfuric acid

iNDF indigestible neutral detergent fiber

K potassium

Mg magnesium

Mo molybdenum

MPS microbial protein synthesis

N nitrogen

Na sodium

NDF neutral detergent fiber

N2O nitrous oxide

NH3–N ammonia-nitrogen

NH4+ ammonium

NHx-N total ammoniacal-nitrogen

NOx nitrous oxides

NO2− nitrites

NO3− nitrates

OM organic matter

P phosphorus

PD purine derivatives

PUN plasma urea-nitrogen

RH relative humidity

SCFA short-chain fatty acid

TMR total mixed ration

UUN urine urea-nitrogen

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.
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