Effects of bismuth subsalicylate and encapsulated calcium-ammonium nitrate on enteric methane production, nutrient digestibility, and liver mineral concentration of beef cattle

Abstract

Two randomized block designs were performed to evaluate the effects of bismuth subsalicylate (BSS) and encapsulated calcium-ammonium nitrate (eCAN) on enteric methane production, nutrient digestibility, liver mineral concentration, and performance of beef cattle consuming bahiagrass hay (Paspalum notatum; ad libitum) and sugar cane molasses [1.07 kg/d; dry matter basis]. Experiment 1, used 25 crossbred steers [335 ± 46 kg of initial body weight (BW)] with a 2 × 2 + 1 factorial arrangement of treatments for two 20 d periods. Factors were nonprotein nitrogen (NPN) source (350 mg/kg BW of nitrate or 182 mg/kg BW of urea), BSS (0 or 58.4 mg/kg BW), and a negative control (NCTRL; bahiagrass hay and molasses only). Steers were re-randomized for a second period (n = 10/treatment total). Intake, apparent total tract digestibility and enteric methane were evaluated. Experiment 2 used 75 crossbred heifers in 25 pens (3 heifers/pen; 279 ± 57 kg of initial BW), consuming the same diet and treatments as experiment 1, to determine liver mineral concentration and growth performance over 56 d. Orthogonal contrasts were used to evaluate the effects of NPN (NCTRL vs. others), source of NPN (NS; urea vs. eCAN), BSS, and NS × BSS. For experiment 1, no interactions were observed for any variables, nor were there any effects of NPN on total tract digestibility of nutrients, except for crude protein. Digestibility of all nutrients was reduced (P ≤ 0.021) for steers consuming eCAN compared with urea. There was no effect (P > 0.155) of BSS on digestibility of nutrients; however, BSS reduced (P = 0.003) apparent S retention. Enteric CH₄ emission (g/kg BW^0.75) was decreased (P = 0.051) by 11% with the addition of eCAN compared with urea. For experiment 2, no NS × BSS interactions (P ≥ 0.251) were observed to affect liver mineral concentration; however, the addition of BSS decreased liver concentration of Cu (P = 0.002) while increasing Fe concentration (P = 0.016). There was an NS × BSS interaction (P = 0.048) where heifers consuming eCAN and BSS had lesser final BW compared with heifers consuming urea and BSS. While eCAN may be a viable resource for mitigating enteric CH₄ production of forage-fed cattle, the negative effects on digestibility should be considered. Furthermore, BSS, at the amount provided, appears to have no negative effects on digestibility of nutrients in forage-fed cattle; however, there may be deleterious impacts on performance depending upon what nitrogen source is supplied.

Introduction

There is a growing desire to reduce the environmental impacts, such as emissions of CH₄ via enteric fermentation, of beef production. One strategy that has been evaluated is the addition of nitrate to the diets of cattle in place of traditional urea (van Zijderveld et al., 2011; Newbold et al., 2014; Hegarty et al., 2016). Most of the data indicate that nitrate can reduce enteric CH₄ production by 10% to 30% (Lee and Beauchemin, 2014; Guyader et al., 2015). Enteric CH₄ production can account for 2% to 12% of gross energy losses, depending upon diet type, and it has been theorized that nitrate may increase metabolizable energy supply by reducing the amount of C lost as CH₄; however, most research has focused on the performance of cattle consuming moderate- (Lee et al., 2017a) to high-concentrate (Newbold et al., 2014) diets.

Little is known about the effects of bismuth subsalicylate (BSS) on ruminant animals. For decades, BSS has been reported as a mediator of H₂S in humans, lessening pain in the gastrointestinal tract (Suarez et al., 1998; Levitt et al., 2002; Mitsui et al., 2003). In cattle, in vitro ruminal fermentation has been used to evaluate the possible influence of BSS on in vivo parameters, but in vivo data are needed to truly evaluate the potential impacts on production and performance of cattle (Ruiz-Moreno et al., 2015). Bismuth compounds may have a place in beef production by mitigating the negative effects of S on trace mineral absorption. By binding to S (Suarez et al., 1998), BSS may reduce thiol- compounds which inhibit trace mineral absorption.

To investigate the impacts of nitrate and BSS on beef cattle production and CH₄ emissions, 2 experiments were conducted. In the first experiment, it was hypothesized that the addition of encapsulated calcium-ammonium nitrate (eCAN) to a bahiagrass diet supplemented with molasses will reduce enteric methane production; furthermore, BSS may have antimicrobial effects (Bland et al., 2004), which may lead to a reduction in CH₄ production. The objective of this first experiment was to evaluate the effects of BSS and eCAN, in combination, on apparent total tract digestibility of nutrients, and enteric CH₄ production. For the second experiment, it was hypothesized that BSS would increase trace mineral concentration in beef cattle consuming bahiagrass hay and molasses; furthermore, it was also hypothesized that performance would not be negatively impacted by eCAN or BSS. The objective of this experiment was to evaluate liver mineral concentration and performance of heifers provided eCAN and BSS.

Materials and Methods

All procedures involving animals were approved by the University of Florida Institutional Animal Care and Use Committee (#201609298).

Experiment 1: enteric methane production

Experimental design, animals, and treatments

Twenty-five Bos taurus × Bos indicus [335 ± 46 kg of initial body weight (BW); average BW ± SD] steers were used in a generalized randomized block design with a 2 × 2 + 1 factorial arrangement of treatments. The experiment comprised 2 experimental periods (block) of 20 d each with a 7-d washout period in between where cattle only received bahiagrass (Paspalum notatum) hay and sugar cane molasses (chemical composition presented in Table 1). On day 0 of each period, BW was recorded to determine the amounts of urea, eCAN, and BSS to provide. From days 0 to 13, steers underwent adaptation to treatments and facilities. Within this adaptation period, steers receiving nonprotein nitrogen (NPN) were adapted in the following manner: on days 0 and 1, cattle received 20% of their total supplemental N; on days 2 and 3, cattle received 40% of their total supplemental N; on days 4 and 5, cattle received 60% of their total supplemental N; on days 6 and 7, cattle received 80% of their total supplemental N; and beginning on day 8, cattle were receiving 100% of their total supplemental N as urea or eCAN. From days 7 to 13 steers were equipped with training CH₄ collection canisters. Starting on day 13, hay and molasses samples were collected at time of feeding until day 16. From days 14 to 17 at 0700 and 1500 hours, fecal samples were collected. From days 14 to 18, animals were equipped with CH₄ collection canisters. Beginning with the first period, all steers were stratified by weight and breed characteristics, and randomly assigned to 1 of 5 treatments: (1) no added NPN or BSS (NCTRL), (2) urea supplemented at 182 mg/kg of BW (U), (3) nitrate, in the form of eCAN, supplemented at 350 mg/kg of BW (NIT), (4) urea supplemented at 182 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW (UB), and (5) nitrate, in the form of eCAN, supplemented at 350 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW (NITB). Treatments U, NIT, UB, and NITB were isonitrogenous. The amount of, and adaptation to, eCAN and BSS provided was based on a previous experiment from the same authors (Henry et al., 2020). When these amounts of eCAN and BSS were chosen, it was expected that nitrate would be provided at 2.0% and BSS at 0.33% of the total dry matter (DM) intake (DMI). After evaluating DMI during the experimental periods, steers receiving eCAN and BSS consumed 1.54% of the total DMI of nitrate and 0.25% of the total DMI of BSS. After experimental period 1, the 25 steers were re-randomized for experimental period 2 and received the same treatments and procedures as in experimental period 1.

Table 1.

Analyzed¹ chemical composition of bahiagrass hay and sugar cane molasses used in experiment 1

Item, % DM	Bahiagrass hay (average ± SD2)	Sugar cane molasses (average ± SD2)
DM, % as fed	88.8 ± 1.69	79.2 ± 0.71
OM	94.9 ± 0.18	84.2 ± 0.35
CP	10.2 ± 0.72	6.8 ± 0.57
NDF	71.8 ± 2.87	—
ADF	35.2 ± 3.22	—
Sulfur	0.29 ± 0.02	0.78 ± 0.98
Nitrate	-	0.08 ± 0.06

¹Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY).

²Average ± SD calculated from 2 samples; 1 composite sample per period.

To ensure that the additional NPN and BSS were consumed in full, urea, eCAN, and BSS were weighed and mixed into the individual steers’ molasses daily. All steers had ad libitum access to hay and received 1.07 kg/d of sugar cane molasses. Molasses and treatments were consumed within 15 min of presentation. During the experiment, steers were housed in the University of Florida, North Florida Research and Education Center Feed Efficiency Facility in Marianna, FL. Daily DMI of bahiagrass hay was recorded using the GrowSafe feed intake system (GrowSafe Systems Ltd., Airdrie, Alberta, Canada). Five steers were housed in each pen of 108 m² equipped with 2 GrowSafe feed bunks and a single water trough. During the entirety of the experiment, steers were moved from the group pens at 0600 hours, daily, to individual pens to provide molasses + treatments individually.

Sampling procedures

All protocols and procedures used for collecting samples and data from steers were used in an identical manner throughout both experimental periods.

Apparent total tract digestibility of nutrients and apparent S retention

Apparent total tract digestibility of nutrients was determined using indigestible neutral detergent fiber (NDF) as an internal marker. Feed samples were collected daily from days 13 to 16 and fecal samples were collected at 0700 and 1500 hours from days 14 to 17 either by rectal grab or from the ground inside the pen, immediately after the animal defecated. Hay samples were stored at 10 °C, and molasses and fecal samples were stored at −20 °C for further analysis. Hay and fecal samples were dried at 55 °C for 72 hr in a forced-air oven, and ground in a Wiley mill (Arthur H. Thomas Co. Philadelphia, PA) to pass a 2-mm screen. Hay was pooled within pen, and feces was pooled within steer to determine DM, organic matter (OM), crude protein (CP), NDF, acid detergent fiber (ADF), indigestible NDF, and S concentrations.

Methane emissions

Emissions of enteric CH₄ production were measured using the sulfur hexafluoride (SF₆) tracer technique (Johnson et al., 1994) from days 14 to 18 of each period. Brass permeation tubes were dosed via balling gun on day 7 of the first period to each steer. Permeation tubes consisted of brass tube bodies (length = 4.4 cm, outside diameter = 1.43 cm, inside diameter = 0.79 cm, inside depth = 3.8 cm, and volume = 1.86 mL), nylon washers, a Teflon membrane, secured with a porous (2-μm porosity) stainless steel frit and a brass nut. Permeation tubes were filled with ~2.3 g of SF₆ and were kept at 39 °C and weighed 12 times within 38 d. The average SF₆ release rate across steers was 2.46 mg/d. Gas collection canisters were constructed of polyvinyl chloride pipe to have a final volume of 2 L. The samples were collected by evacuating the collection canisters to 68.6 cmHg and connecting the canister to a halter, which was equipped with a crimped capillary tube that was positioned to sample, using a loop design, from both nostrils. The volume of the collection canisters and the crimped capillary tubes were designed to allow half of the vacuum to remain after 24 hr. Three collection canisters and capillary tubes were used to determine environmental CH₄ and SF₆ concentrations. It has been proposed by Haisan et al. (2014) to only include animals with at least 2 d of valid CH₄ measurements in the data set. For the current experiment, only steers with at least 3 successful days of collection and measurement were considered in the final analysis.

Laboratory analyses

All protocols and procedures used for analyzing samples and data from steers were used in an identical manner throughout both experimental periods.

Concentration of DM, OM, CP, NDF, ADF, and S

Dry, hot weight was used to calculate DM of the sample. The sample was then ground to pass through a 2-mm screen in a Wiley mill (Thomas Scientific, Swedesboro, NJ). To determine OM, 0.5 g of ground sample (in duplicate) was weighed into ceramic crucibles and placed in a 105 °C forced air oven for 24 hr to determine sample DM. Dried samples were then placed in a 650 °C muffle furnace for 6 hr before returning to a 105 °C forced air oven. Hot, ashed samples were weighed and used to calculate OM.

Samples of hay and feces were weighed (0.5 g in duplicate) into F57 bags (Ankom Technology Corp., Macedon, NY) and analyzed for NDF, using heat stable α-amylase and sodium sulfite. Subsequent ADF analysis was performed sequentially as described by Van Soest et al. (1991) in an Ankom 200 Fiber Analyzer (Ankom Technology Corp.).

Hay and fecal samples were analyzed for total N and S using a C, H, N, and S analyzer by the Dumas dry combustion method (Vario Micro Cube; Elementar, Hanau, Germany). Crude protein was calculated by multiplying the N concentration of the dry sample by 6.25.

Concentration of indigestible NDF

The concentration of indigestible NDF in hay and feces was determined as described by Gregorini et al. (2008), Cole et al. (2011), and Krizsan and Huhtanen (2013). Briefly, 0.5 g of sample was weighed into Ankom F57 filter bags (Ankom Technology Corp.) and then incubated in the rumen of a cannulated steer grazing a bahiagrass and bermudagrass (Cynodon dactylon) mixed pasture for 288 hr to ensure complete digestion of potentially digestible NDF. After incubation, samples were rinsed twice with tap water followed by 4 rinses with water filtered through a reverse osmosis system. The rinsed samples were then analyzed for NDF as previously described.

Methane and SF₆ analyses

Methane and SF₆ concentrations in collection canisters were analyzed by gas chromatography (Agilent 7820A GC; Agilent Technologies, Palo Alto, CA). A flame ionization detector and electron capture detector were used for CH₄ and SF₆ analysis, respectively, with a capillary column (Plot Fused Silica 25 m × 0.32 mm, Coating Molsieve 5A, Varian CP7536; Varian Inc., Lake Forest, CA). Injector, column, and detector temperatures for CH₄ analysis were 80, 160, and 200 °C, respectively. For SF₆, temperatures were 50, 30, and 300 °C for the injector, column, and detector, respectively. The carrier gas for CH₄ and SF₆ was N₂.

Calculations

Emission of CH₄ produced by steers was determined in relation to the SF₆ tracer gas captured in the collection canisters. The following equation was used to quantify CH₄ production:

$${\displaystyle \begin{array}{l}{\displaystyle {\mathrm{Q}}_{\mathrm{C}\mathrm{H}4}={\mathrm{Q}}_{\mathrm{S}\mathrm{F}6}\times ({\left[\mathrm{C}{\mathrm{H}}_4\right]}_{\gamma }-{\left[\mathrm{C}{\mathrm{H}}_4\right]}_{\beta })÷({\left[\mathrm{S}{\mathrm{F}}_6\right]}_{\gamma }-{\left[\mathrm{S}{\mathrm{F}}_6\right]}_{\beta })}\end{array}}$$

in which Q_CH4 is considered CH₄ emissions per animal (g/d), Q_SF6 is considered SF₆ release rate (mg/d), [CH₄]_γ is considered the concentration of CH₄ in the animals collection canister, [CH₄]_β is considered the concentration of CH₄ in the environmental canisters, [SF₆]_γ is considered the concentration of SF₆ in the animals collection canister, and [SF₆]_β is considered the concentration of SF₆ in the environmental collection canister.

Apparent total tract digestibility of DM, OM, CP, NDF, and ADF, and retention of S was calculated as follows with OM total tract digestibility as an example:

OM digestibility (%) = 100 – 100 × [(indigestible NDF concentration in the feed ÷ 229 indigestible NDF concentration in the feces) × (OM concentration in the feces ÷ OM concentration 230 in the feed)].

Statistical analysis

All data were analyzed as a randomized block design with a 2 × 2 + 1 factorial arrangement of treatments using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC), using steer as the experimental unit. The model included the fixed effects of treatment and the random effects of pen and period. To aid in the interpretation of data, the following contrasts were used: the effect of NPN = NCTRL vs. the mean of U, NIT, UB, and NITB; the effect of NPN source = the mean of U and UB vs. the mean of NIT and NITB; the effect of BSS = the mean of U and NIT vs. the mean of UB and NITB; and NPN source × BSS = the mean of U and NITB vs. the mean of NIT and UB. Significance was declared at P ≤ 0.05.

Experiment 2: liver mineral concentration and performance

Experimental design, animals, and treatments

Seventy-five growing Angus-crossbred heifers [279 ± 57 kg of initial BW] were used in a randomized incomplete block design with a 2 × 2 + 1 factorial arrangement of treatments at the University of Florida, North Florida Research and Education Center Beef Unit in Marianna, FL. The experiment consisted of a 28-d adaptation period followed by a 56-d data collection period in which heifers were weighed every 14 d from days 0 to 56. On days −28 and −27, all heifers were weighed and the average weight of each heifer on those 2 d was considered initial BW. Similarly, the average BW of each heifer on days 55 and 56 was considered final BW. On day −27, heifers were stratified and blocked by BW and allotted to 25 dormant bahiagrass (Paspalum notatum) pastures (3 head/pasture). Pastures (1.34 ha each) were located in 3 different areas of the Beef Unit and were within 0.52 km of each other. The 3 locations were termed North Circle (n = 13; pastures per location), South Circle (n = 6), and R-Pens (n = 6). Pastures were stratified by location and randomly assigned to 1 of 5 treatments: (1) no added NPN or BSS (NCTRL), (2) urea supplemented at 182 mg/kg of BW (U), (3) nitrate, in the form of eCAN, supplemented at 350 mg/kg of BW (NIT), (4) urea supplemented at 182 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW (UB), and (5) nitrate, in the form of eCAN, supplemented at 350 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW (NITB). Treatments U, NIT, UB, and NITB were isonitrogenous.

To reduce any negative effects of nitrate on the health of the heifers, on day −27 cattle began an adaptation to eCAN and urea. From days −27 to −14, cattle received 20% of their total supplemental N; on days −13 and −12, cattle received 40% of their total supplemental N; on day −11 and −10, cattle received 60% of their total supplemental N; on days −9 and −8, cattle received 80% of their total supplemental N; and beginning on day −7, cattle were receiving 100% of their total supplemental NPN as urea or eCAN. One pasture (treatment NITB) was removed from the experiment because the heifers refused to consume the molasses and treatment.

Heifers had ad libitum access to bahiagrass hay (1.2 × 1.5 m round bales) and received 1.07 kg/d (DM basis) of sugar cane molasses (chemical composition is presented in Table 2). This experiment began on February 15, 2017, and prior to initiation, pastures were mob grazed to remove any residual forage; therefore, bahiagrass hay was the only forage available to the heifers. Molasses was weighed and provided daily via plastic feed bunks (2.4 × 0.6 m) and was used as the carrier of treatments. Generally, heifers would consume molasses + treatments within 30 min of feeding.

Table 2.

Analyzed¹ chemical composition of bahiagrass hay and sugar cane molasses used in experiment 2

Item, % DM	Bahiagrass hay	Sugar cane molasses
DM, % as fed	90.0	78.7
OM	93.4	84.4
CP	10.3	7.2
NDF	73.0	-
ADF	45.7	-
TDN	54.0	77
S	0.34	1.47
Nitrate	0.07	0.12

¹Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY).

Sample and data collection

Samples of hay and molasses were collected every 14 d and stored, for further analysis, at 10 °C or −20 °C, respectively. All feed samples were shipped to a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY) for chemical analysis, which are presented in Table 2.

Heifer BW was recorded every 14 d starting on day 0. To calculate average daily gain (ADG), difference in BW was divided by the number of days between BW recordings.

Liver samples were taken on days −28 and −27, and again on days 55 and 56. A random subset of heifers (2 heifers/pen) was selected to collect liver tissue. The same heifers were used for both pre- and posttreatment samples collection. One heifer per pen was randomly selected to have liver sample taken on days −28 and 55, and the second heifer donating liver tissue was collected on days −27 and 56. Biopsies were collected on 2 separate days due to time constraints and with the welfare of cattle in mind. There were no statistical differences (P > 0.05) between cohorts (days −28 and 55; days −27 and 56); therefore, for the rest of the manuscript, liver tissue will be described as initial and final. The techniques described by Arthington and Corah (1995) were used to collect liver tissue. Briefly, a Tru-Cut biopsy needle (CareFusion, 14 gauge by 15 cm; Becton, Dickinson and Comp., Vernon Hills, IL) was inserted between the 11th and 12th intercostal space until Glisson’s capsule was penetrated. Three core tissue samples (25 mg each) were collected from each animal at each collection. All tissue samples were stored in Zn-free microcentrifuge tubes (Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) at −20 °C until further analysis. Liver tissue mineral concentration was determined using ICP-MS (Animal Health Diagnostic Laboratory, Lansing, MI).

On days −28, −27, 55, and 56, carcass measurements were taken using ultrasonography (3.5-mHz linear array transducer, Aloka 500V; Corimetrics Medical Systems Inc., Wallingford, CT). Two-thirds of the heifers were measured on days −28 and 55 (all animals not donating liver tissue plus the first cohort of liver tissue donors) and one-third was measured on days −27 and 56 (the second cohort of liver tissue donors). No differences were found between days −28 and −27, nor days 55 and 56; therefore, for the remainder of the manuscript, ultrasonography measurements will be described as initial and final. Ultrasonography measurements were made between the 12th and 13th intercostal space. Images were used to assess longissimus muscle (LM) area and back fat thickness.

Statistical analysis

Data were analyzed as a randomized incomplete block design with a 2 × 2 + 1 factorial arrangement of treatments using pasture as the experimental unit. The model included the fixed effect of treatment and the random effects of block and location (North Circle, South Circle, and R-Pens). Pens were blocked (n = 5) by heifer BW at day −28. Initial liver mineral concentration and ultrasound measurements were used as covariates for final liver mineral concentration and ultrasound measurements, respectively. Denominator degrees of freedom were adjusted using the Kenward–Rogers adjustment. The following contrasts were used to aid in the interpretation of data: the effect of NPN = NCTRL vs. the mean of U, NIT, UB, and NITB; the effect of NPN source = the mean of U and UB vs. the mean of NIT and NITB; the effect of BSS = the mean of U and NIT vs. the mean of UB and NITB; and NPN source × BSS = the mean of U and NITB vs. the mean of NIT and UB. Significance was declared at P ≤ 0.05.

Results and Discussion

Experiment 1

Intake and apparent total tract nutrient digestibility data are presented in Table 3. There was no interaction (P ≥ 0.887) or effect (P ≥ 0.288) of NPN, NPN source, or BSS on intake of DM, OM, NDF, or ADF during the 4-d digestibility period. This experiment was not designed to test effects on intake; therefore, it cannot be assumed that eCAN and BSS have no impact on DMI. It appears from published literature that the effect of eCAN on intake is conditional, depending upon diet type and the quality of the diet. Data reported by Lee et al. (2015b) indicated that heifers consuming a diet of 55% forage (50% and 5% of barley silage and grass hay, respectively) reduced DMI by 6% when nitrate was used as a source of NPN rather than urea. Lee et al. (2017b) observed that when growing steers were placed on a backgrounding diet with a greater concentration of forage (65% corn silage), DMI was not affected by the inclusion of eCAN at 1.25% or 2.5% of the diet DM. The difference between these 2 studies may be related to the source of nitrate used: Lee et al. (2015b) provided heifers with an unencapsulated form of calcium-ammonium nitrate, whereas Lee et al. (2017b) used an encapsulated form of the same nitrate. Encapsulation of nitrate has been observed to abate the release of nitrate in vitro, compared with unencapsulated calcium-ammonium nitrate, without affecting NH₃-N or total volatile fatty acid (VFA) concentration (Lee et al., 2017b). Likewise, encapsulation of calcium-ammonium nitrate essentially eliminated nitrite accumulation in vitro where the unencapsulated form allowed accumulation to occur. It is possible that the accumulation of nitrate and nitrite in the rumen may have negative effects on intake of ruminants due to the inhibitory actions on fiber-degrading bacteria (Zhou et al., 2012). Others have reported reductions in DMI of animals consuming 78% concentrate (2.57% calcium-ammonium nitrate; Velazco et al., 2014) and 90% concentrate when provided 2.5% eCAN, but not 1.25% (Lee et al., 2017c). As expected, CP intake during the 4-d digestibility period was increased with the addition of NPN (P = 0.0021).

Table 3.

Experiment 1: effect of BSS and eCAN¹ on nutrient intake and apparent total tract digestibility, and sulfur intake and retention

	Treatment2						P-value3
Item	NCTRL	U	NIT	UB	NITB	SEM	NPN	NS	B	B × N
4-d intake, kg/d
DM	8.31	8.17	7.79	8.43	7.92	0.442	0.636	0.321	0.666	0.887
OM	7.76	7.63	7.26	7.87	7.39	0.418	0.632	0.320	0.666	0.888
CP	0.81	0.98	0.94	1.00	0.96	0.043	0.002	0.353	0.714	0.953
NDF	5.11	5.00	4.71	5.18	4.80	0.323	0.609	0.306	0.674	0.907
ADF	2.52	2.46	2.30	2.54	2.35	0.162	0.576	0.288	0.690	0.931
S	0.039	0.038	0.037	0.039	0.037	0.001	0.627	0.290	0.727	0.963
Digestibility, %
DM	52.58	52.99	51.65	52.66	50.66	0.658	0.424	0.015	0.323	0.620
OM	54.09	54.87	53.59	54.28	52.69	0.601	0.727	0.021	0.222	0.795
CP	34.66	46.82	43.67	46.14	41.89	1.444	<0.001	0.014	0.401	0.707
NDF	52.09	52.22	50.81	52.40	50.98	0.536	0.419	0.012	0.744	0.990
ADF	49.36	50.16	47.25	50.25	49.30	0.737	0.881	0.013	0.155	0.193
S retention4, %	61.94	62.41	61.31	57.49	56.01	1.642	0.158	0.437	0.003	0.908

¹eCAN = encapsulated calcium-ammonium nitrate (5Ca(NO₃)₂–NH₄NO₃) – 65.1% nitrate DM basis.

²NCTRL, treatment 2.7 g/kg BW molasses; U, NCTRL + 182 mg/kg BW urea; NIT, NCTRL + 538 mg/kg BW eCAN; UB, treatment U + 58.4 mg/kg BW BSS; NITB, treatment NIT + 58.4 mg/kg BW BSS. All groups contained 10 experimental units. Means and largest SEM presented.

³Observed significance levels for: NPN, effect of NPN, NCTRL vs. the mean of U + NIT + UB + NITB; NS, effect of NPN source (excludes NCTRL); B, effect of BSS (excludes NCTRL); B × N, interaction of BSS and eCAN.

⁴S absorption was calculated as follows: 100 – (100 × (([diet iNDF] ÷ [fecal iNDF]) × ([fecal S] ÷ [diet S]))).

The addition of NPN had no effect (P ≥ 0.419) on apparent total tract digestibility of DM, OM, NDF, and ADF. It was hypothesized that nutrient digestibility would have been enhanced with the addition of an NPN source; however, this effect was not observed. In the current experiment, the DMI of the NCTRL steers was composed of nearly 9.8% CP, which is a drastic difference from the 4% CP oaten chaff that was provided to Merino ewes by Nguyen et al. (2016). Nguyen et al. (2016) reported that the addition of NPN in the form of calcium-ammonium nitrate increased DM digestibility by 12% along with an increase in DMI of more than 200 g/d. The authors attributed this change in digestibility to an increase in ruminal NH₃-N. In an experiment utilizing 10 cannulated beef cattle (Henry et al., 2020), identical treatments were provided to investigate ruminal metabolism. Cattle from that experiment, as those in experiment 1, were provided more CP than what is recommended (calculated using NASEM, 2016). Data indicated that, over 24 hr, regardless of the peak at hour 2 (6.35 mM), average ruminal concentration of NH₃–N was 20% less (2.85 mM) than what has been recommended in the literature (3.57 mM; Satter and Slyter, 1974) for NPN treatments (Henry et al., 2020). It is imperative that constant distribution of NH₃ occur throughout the day to support maximal ruminal fermentation and digestion; therefore, it is likely that cattle in experiment 1 did not have sufficient distribution of NH₃-N even when additional NPN sources were provided.

There was a negative effect of eCAN on DM (P = 0.015), OM (P = 0.021), NDF (P = 0.012), and ADF (P = 0.013) total tract digestibility. In the literature, there is no consensus on the impact of nitrate on nutrient digestibility. Researchers have reported increases in DM digestibility when nitrate was included in a 35% forage diet (Li et al., 2013), while others reported no change in nutrient digestibility of diets ranging from 50% (Olijhoek et al., 2016) to 70% forage (Klop et al., 2017; Sun et al., 2017); furthermore, linear reductions in DM digestibility have been reported for diets containing 55% forage (Lee et al., 2015b). More research is needed to evaluate the effects of nitrate on nutrient digestibility of cattle consuming a range of diet compositions fed ad libitum and restrictively. To speculate, the reductions in nutrient digestibility observed in the current experiment may be partially caused by an accumulation of nitrate/nitrite in the rumen and/or an increase in the concentration of H₂ in the rumen. It is evident that in the current experiment, the reductions in DM and OM total tract digestibility were largely accounted for by the reduction in digestibility of NDF and ADF. As discussed previously, nitrate and nitrite have toxic effects on certain populations of microbial species in the rumen. Several research groups have observed reductions in the relative abundance of cellulolytic microbial populations in the rumen when nitrate was provided (Marais, 1988; Zhou et al., 2012; Asanuma et al., 2015). These inhibitory effects on fiber-degrading microorganisms may explain the reductions in nutrient digestibility in the rumen. If it was not a direct effect of nitrate/nitrite on microbial populations, it is plausible that the increase in H₂ occasionally observed when nitrate is introduced to a ruminal environment, would constrain the activity of certain cellulolytic bacteria (Janssen, 2010; Guyader et al., 2015). It is probable that the reduction in fiber digestion, leading to reduction in DM and OM, was a result of both the toxic effects of nitrate/nitrite on ruminal microorganisms and an increase in H₂ concentration in the rumen.

Total tract digestibility of CP was reduced by 8% when eCAN was provided as an NPN source compared with urea (P = 0.014). It is possible that this is an actual difference and eCAN does reduce the digestibility of CP in the total tract; however, an observation was made when collecting feces from the steers during the digestibility portion of this experiment: eCAN prills were found, intact, in the feces. By observing eCAN prills in the feces of these animals, it was considered that the amount of nitrogen analyzed in the feces would be greater, thereby causing a reduction in the digestibility of CP in the total tract. Other researchers have reported that the percentage of intake nitrogen retention is linearly increased as eCAN is provided up to 3% of DMI (Lee et al., 2015b); however, the heifers used were provided a basal diet that had a greater inclusion of concentrate (45% concentrate) compared with the current experiment (~14% molasses). It is possible that the encapsulation of the nitrate used in the current experiment was over-protected, allowing some nitrate to pass through the total tract unmetabolized.

Apparent total tract digestibility of nutrients was not affected by BSS (P ≥ 0.155). In a companion in vitro study (D. D. Henry, unpublished data), a linear reduction in in vitro OM digestibility (IVOMD) was observed when BSS was included in the substrate DM up to 1.0%; however, when BSS was included at 0.00%, 0.05%, 0.10%, or 0.33% of the substrate DM, no effect of BSS on IVOMD was observed. Another research group reported that BSS, included at 1.0% of a high-concentrate substrate, did not negatively affect fermentation (Ruiz-Moreno et al., 2015). The impact of BSS on fermentation is likely diet dependent in regards to S content of a diet and ruminal pH (concentrate- vs. forage-based diets). Furthermore, it can be speculated that when provided with a high-forage diet or substrate, inclusion rates of BSS should be lesser than when provided with a high-concentrate diet or substrate. The data from the current experiment indicates that at ~0.25% of DMI of a forage-based diet, BSS does not impact total tract digestibility of nutrients.

There was no interaction or effect (P ≥ 0.290) of NPN, NPN source, or BSS on the intake of S during the 4-d digestibility portion of this experiment; however, S retention was reduced (P = 0.003) by 8% with the inclusion of BSS. It has been proposed that BSS in humans reacts in the following manner:

$${\displaystyle \begin{array}{l}{\displaystyle 2\mathrm{B}{\mathrm{i}}^{3+}+3{\mathrm{H}}_2\mathrm{S}\leftrightarrow \mathrm{B}{\mathrm{i}}_2{\mathrm{S}}_3+6{\mathrm{H}}^{+}.}\end{array}}$$

Bismuth sulfide is an insoluble, black salt that, theoretically, is not absorbed through the intestinal wall (Suarez et al., 1998). In the rumen of cattle that are consuming diets which would promote the production of H₂S (high-S, high-concentrate diets; lesser ruminal pH), it may be that BSS acts in a similar manner (Ruiz-Moreno et al., 2015); however, it is not evident what reaction is occurring when H₂S is not present, or is present in lesser concentrations, such as that observed in the companion metabolism experiment (Henry et al., 2020). Whatever the mode of action, the data indicate that BSS is shifting a portion of the intake S that would apparently be retained to pass though the gastrointestinal tract and be excreted in the feces of the animal.

Methane production parameters are presented in Table 4. It has been widely accepted that nitrate reduces enteric methane emissions of ruminants (Hulshof et al., 2012; Newbold et al., 2014; Lee et al., 2015b). This reduction in CH₄ production is thought to be related to 2 factors: nitrate acting as an H₂ sink (Leng, 2008), and the inhibitory effects of nitrate, more specifically nitrite, on methanogens (Duin et al., 2016). In the current experiment, there were no interactions observed for methane production. A reduction (P = 0.039) in CH₄ (g/d) was observed when eCAN was provided as a NPN source rather than urea. When evaluating grams of CH₄ produced per kilogram of OM intake and OM digested, there was no effect of NPN source (P ≥ 0.649). When metabolic BW was taken into account, eCAN reduced (P = 0.051) CH₄ production (g/kg of BW^0.75) by 11% compared with urea. There was no effect (P ≥ 0.264) of NPN or BSS on any of the CH₄ variables evaluated in this experiment.

Table 4.

Experiment 1: effect of BSS and eCAN¹ on enteric CH₄ production of beef steers

	Treatment2						P-value3
Item4	NCTRL	U	NIT	UB	NITB	SEM	NPN	NS	B	B × N
CH4 emissions, g/d	121.16	117.27	115.57	134.09	100.04	10.986	0.673	0.039	0.951	0.121
CH4 emissions, g/kg OMI	15.35	15.42	16.44	15.59	13.67	1.197	0.954	0.689	0.264	0.209
CH4 emissions, g/kg OMD	28.72	28.63	30.33	29.36	25.70	2.246	0.921	0.649	0.372	0.222
CH4 emissions, g/kg MBW	1.51	1.39	1.44	1.55	1.18	0.126	0.341	0.051	0.689	0.063

²eCAN = encapsulated calcium-ammonium nitrate (5Ca(NO₃)₂-NH₄NO₃) – 65.1% nitrate DM basis.

²NCTRL, treatment 2.7 g/kg BW molasses; U, NCTRL + 182 mg/kg BW urea; NIT, NCTRL + 538 mg/kg BW eCAN; UB, treatment U + 58.4 mg/kg BW BSS; NITB, treatment NIT + 58.4 mg/kg BW BSS. Groups contained 10 (NCTRL), 8 (U, UB) and 7 (NIT, NITB) experimental units. Largest SEM is provided.

³Observed significance levels for: NPN, effect of NPN, NCTRL vs. the mean of U + NIT + UB + NITB; NS, effect of NPN source (excludes NCTRL); B, effect of BSS (excludes NCTRL); B×N, interaction of BSS and eCAN.

⁴CH₄ was determined from the average of at least 3 out of 5 24 hr periods of breath sample collection. OM, OM intake; OMD, OM digested; MBW, BW^0.75.

Experiment 2

Growth performance data are presented in Table 5. At the beginning of experiment 2, heifers began with similar BW (P ≥ 0.349). By the end of the experiment, BW was not affected by NPN (P = 0.470), NPN source (P = 0.384), or BSS (P = 0.600); however, there was an interaction (P = 0.048) between BSS inclusion and source of NPN. After adjusting with Tukey’s, no treatment mean differences were observed for final BW (treatment mean differences are not shown); however, numerically, BSS increased ADG in urea fed cattle, but decreased ADG in eCAN fed cattle. There was no effect of NPN supplementation, NPN source, BSS, or an interaction for ADG measured within any time points (P ≥ 0.125). The lack of NPN effect is likely due to the kinetics of urea and eCAN degradation in the rumen. As previously discussed, a metabolism experiment conducted using identical diets and treatments provided data that indicate a rapid conversion of both NPN sources to NH₃ (within 2 hr of feeding), followed by ruminal concentrations below recommendations for optimal ruminal fermentation (Satter and Slyter, 1974; Henry et al., 2020). The authors attribute the lack of NPN effect on ADG to a potential lack of distribution of NH₃ throughout the day, which inhibited benefits of the NPN sources.

Table 5.

Experiment 2: effect of BSS and eCAN¹ on growth performance of beef heifers

	Treatment2						P-value3
Item	NCTRL	U	NIT	UB	NITB	SEM	NPN	NS	B	B × N
Initial BW4, kg	274	275	276	279	277	3.1	0.349	0.930	0.435	0.615
Final BW5, kg	335	335	342	352	332	6.7	0.470	0.384	0.600	0.048
ADG, kg
Days -28 to 0	0.87	0.80	0.97	1.07	0.80	0.139	0.794	0.729	0.731	0.125
Days 0 to 14	0.67	0.69	0.83	1.01	0.80	0.141	0.286	0.772	0.307	0.218
Days 0 to 28	0.70	0.75	0.75	0.86	0.83	0.110	0.401	0.898	0.388	0.898
Days 0 to 42	0.78	0.76	0.74	0.90	0.76	0.092	0.891	0.398	0.347	0.508
Days 0 to 56	0.65	0.69	0.68	0.74	0.55	0.061	0.865	0.125	0.545	0.139

¹eCAN = encapsulated calcium-ammonium nitrate (5Ca(NO₃)₂-NH₄NO₃) – 65.1% nitrate DM basis.

²NCTRL, treatment 2.7 g/kg BW molasses; U, NCTRL + 182 mg/kg BW urea; NIT, NCTRL + 538 mg/kg BW eCAN; UB, treatment U + 58.4 mg/kg BW BSS; NITB, treatment NIT + 58.4 mg/kg BW BSS. Groups contained 5 (NCTRL, U, NIT, UB) and 4 (NITB) experimental units. Largest SEM is provided.

³Observed significance levels for: NPN, effect of NPN, NCTRL vs. the mean of U + NIT + UB + NITB; NS, effect of NPN source (excludes NCTRL); B, effect of BSS (excludes NCTRL); B × N, interaction of BSS and NPN source (excludes NCTRL).

⁴Initial BW was the average of BW recorded on days −28 and −27.

⁵Final BW was the average of BW recorded on days 55 and 56.

When considering the proposed mode of action of the reduction of CH₄ production with the addition of nitrate, it may be that performance of the ruminant being provided nitrate would increase. When nitrate is reduced in the rumen to NH₃, H₂ that would be used to reduce CO₂ to CH₄ would theoretically no longer be available; therefore, a shift in the VFA profile to produce more propionate could possibly occur, leading to increases in performance. Data from the current experiment do not support this theory nor do a majority of the literature on nitrate report such findings (Newbold et al., 2014; Lee et al., 2017a, 2017c). It has been hypothesized that the production of propionate is not increased with the addition of nitrate because of the thermodynamics of VFA production (Ungerfeld and Kohn, 2006; Janssen, 2010; Ungerfeld, 2015). In a review by Janssen (2010), evidence was presented that indicated a shift in VFA production to H₂ producing pathways (i.e., 2 acetate + 4 H₂) was more thermodynamically favorable when the concentration of H₂ was decreased; therefore, increasing the amount of C used for acetate production at the expense of more energetically favorable VFA, such as propionate. However, in a companion experiment, molar proportions of acetate, propionate, and butyrate were not altered by NPN source (urea or eCAN; Henry et al., 2020).

To the best of the authors’ knowledge, this is the first data set describing the performance of ruminants provided BSS. A plethora of data has been reported indicating the negative effects of S on beef cattle performance (Gould, 1998; Felix et al., 2012; Drewnoski et al., 2014). It was hypothesized that BSS may be able to thwart these negative effects by binding to ruminal S and transporting it, as Bi₂S₃, out of the gastrointestinal tract via feces. In the current experiment, the diet consisted of ~0.51% S (DM basis), which is marginally greater than what is considered safe for grazing beef cattle (NASEM, 2016). Early research has indicated that growing cattle consuming forage-based diets and high-S water (~0.55% S in DMI) experience reductions in DMI and BW gain (Weeth and Hunter, 1971; Weeth and Capps, 1972). In the current experiment, heifers not receiving BSS did not perform less favorably compared with those which were provided BSS, indicating that heifers were not affected by the S concentration of the diet. In experiment 1, data regarding the impact of BSS on S retention indicated that BSS reduced S retention; therefore, it is likely that the dietary concentration of S in the current experiment, in combination with a higher ruminal pH due to a forage-based diet, was not sufficient to have deleterious effects on performance. Data from a ruminal metabolism experiment (Henry et al., 2020) indicated that BSS had minimal effects on the concentration of H₂S in the gas cap of ruminally cannulated cattle consuming bahiagrass hay and molasses (0.40% S DM basis). Previous work has indicated that the activity of BSS is impacted by pH, with more acidic environments favoring the formation of H₂S (Sox and Olson, 1989; Drewnoski et al., 2014). It is possible that the rumen environment was not as conducive for BSS activity in the current experiment compared with a ruminant fed a high-grain diet. Researchers published data from an in vitro batch culture experiment using a 90% concentrate substrate with 1% BSS (DM basis), in which fermentation was not negatively impacted by BSS, while H₂S was reduced by 34%, indicating that BSS was binding to S (Ruiz-Moreno et al., 2015); however, in a separate in vitro continuous culture experiment, the same inclusion rate of BSS reduced H₂S by nearly 99%. When evaluating the pH of each in vitro experiment, the batch culture had a final pH of ~6.3, whereas the mean pH of the continuous culture was ~5.4 (Ruiz-Moreno et al., 2015). An in vitro batch culture (D. D. Henry, unpublished data) was used to evaluate BSS at up to 1.0% of the DM of a bahiagrass hay and molasses substrate. At 1.0%, H₂S was reduced by 100%; however, IVOMD was decreased by 29% compared with control. When included in the substrate at 0.33%, BSS reduced H₂S by 61% without negatively affecting in vitro fermentation. Currently, there is not enough data to conclude exactly what is occurring in the rumen when BSS is provided, but the data from the current experiment does not support the hypothesis that BSS will improve the performance of cattle consuming high-S, forage-based diets.

Carcass ultrasound results are presented in Table 6. There was no effect of NPN supplementation, NPN source, BSS, or interaction on LM area (P ≥ 0.431). As previously discussed, there was an expectation for NPN to improve performance, thereby, possibly increasing LM area; however, this was not observed in the current experiment. The lack of change in LM area when eCAN was provided in place of urea, is in agreement with much of the literature (Hegarty et al., 2016; Lee et al., 2017c). In general, the benefits of nitrate on performance are observed in increases in G:F (Newbold et al., 2014; Lee et al., 2017c). Surprisingly, a BSS × NPN source interaction (P < 0.001) was observed for 12^th–rib fat thickness on day 56 and change in fat thickness from days −28 to 56. The data from this experiment indicate that BSS may increase fat deposition when urea is provided as an NPN source; however, potentially, a decrease in fat thickness may occur when BSS is provided with eCAN. Furthermore, by providing NPN, fat thickness at the 12th rib was increased (P = 0.042) over time.

Table 6.

Experiment 2: effect of BSS and eCAN¹ on carcass ultrasound measurements

	Treatment2						P-value3
Item4	NCTRL	U	NIT	UB	NITB	SEM	NPN	NS	B	B × N
LM area, cm2	54.36	56.54	54.41	58.07	56.79	2.677	0.441	0.493	0.431	0.871
LM area change7, cm2	10.26	12.33	11.38	15.12	12.32	2.221	0.298	0.395	0.397	0.671
12th-–rib fat thickness, cm	0.50	0.42	0.47	0.56	0.40	0.025	0.204	0.012	0.080	<0.001
12th-rib fat thickness change8, cm	0.063	-0.025	0.025	0.117	-0.045	0.019	0.042	0.009	0.069	<0.001

¹eCAN = encapsulated calcium-ammonium nitrate (5Ca(NO₃)₂–NH₄NO₃) – 65.1% nitrate DM basis.

²NCTRL, treatment 2.7 g/kg BW molasses; U, NCTRL + 182 mg/kg BW urea; NIT, NCTRL + 538 mg/kg BW eCAN; UB, treatment U + 58.4 mg/kg BW BSS; NITB, treatment NIT + 58.4 mg/kg BW BSS. Groups contained 5 (NCTRL, U, NIT, UB) and 4 (NITB) experimental units. Largest SEM is provided.

³Observed significance levels for: NPN, effect of NPN, NCTRL vs. the mean of U + NIT + UB + NITB; NS, effect of NPN source (excludes NCTRL); B, effect of BSS (excludes NCTRL); B × N, interaction of BSS and NPN source (excludes NCTRL).

⁴LM area and 12th-rib fat thickness was measured on day 56 using day −28 as a covariate. Difference in LM area and 12th-rib fat thickness measured between days −28 and 56.

The concentration of trace minerals in the liver from cattle in the current experiment can be found in Table 7. Concentration of minerals in the liver was not affected by NPN source (P ≥ 0.116). To the best of the authors’ knowledge, this is the first data representing the effects of nitrate supplementation on liver mineral concentration. There were no interactions between BSS and source of NPN (P ≥ 0.251) affecting liver mineral concentration. The addition of NPN increased liver concentrations of Fe (P = 0.013) and Mn (P = 0.006) by 37% and 15%, respectively. The reason for this change in Fe and Mn is unknown to the authors.

Table 7.

Experiment 2: effect of BSS and eCAN¹ on liver mineral concentration

	Treatment2						P-value3
Item	NCTRL	U	NIT	UB	NITB	SEM	NPN	NS	B	B × N
Final liver mineral4, mg/kg DM
Cu	54.98	55.69	53.93	11.35	21.66	9.771	0.088	0.666	0.002	0.533
Fe	189.33	213.63	235.73	268.00	318.15	24.274	0.013	0.116	0.016	0.527
Zn	123.56	128.83	134.93	137.20	131.60	4.983	0.087	0.958	0.604	0.251
Mn	11.14	12.56	12.34	13.00	13.45	0.487	0.006	0.815	0.134	0.475
Se	0.33	0.38	0.37	0.31	0.33	0.035	0.613	0.921	0.125	0.637

¹eCAN = encapsulated calcium-ammonium nitrate (5Ca(NO₃)₂–NH₄NO₃) – 65.1% nitrate DM basis.

²NCTRL, treatment 2.7 g/kg BW molasses; U, NCTRL + 182 mg/kg BW urea; NIT, NCTRL + 538 mg/kg BW eCAN; UB, treatment U + 58.4 mg/kg BW BSS; NITB, treatment NIT + 58.4 mg/kg BW BSS. Groups contained 5 (NCTRL, U, NIT, UB) and 4 (NITB) experimental units. Largest SEM is provided.

³Observed significance levels for: NPN, effect of NPN, NCTRL vs. the mean of U + NIT + UB + NITB; NS, effect of NPN source (excludes NCTRL); B, effect of BSS (excludes NCTRL); B×N, interaction of BSS and NPN source (excludes NCTRL).

⁴Liver mineral analyzed on day 56 using day −28 as a covariate.

It was hypothesized that BSS may increase the liver concentration of trace minerals when BSS was provided with a high S diet. The effects of diets with large concentrations of S on trace mineral absorption has been reported in both grazing cattle (Arthington et al., 2002) and grain-fed cattle (Pogge and Hansen, 2013; Pogge et al., 2014). In the rumen, S, along with Mo, can act as an antagonist to trace mineral (i.e., Cu) absorption. The S binds with Mo to form thiomolybdates that bind to minerals, leaving them unabsorbable (Drewnoski et al., 2014). It was speculated that BSS may reduce the amount of available S in the rumen by forming Bi₂S₃. Without having available S to bind to trace minerals, the minerals, theoretically, would have a greater chance of absorption. The data from this experiment do not support this hypothesis. Liver mineral concentrations of Cu were reduced (P = 0.002) by 70% when BSS was provided to heifers consuming bahiagrass hay and molasses. Unfortunately, very little data are available evaluating the effects of bismuth on the mineral status of humans and rats. One experiment injected BiCl₃ subcutaneously into rats to evaluate the effects of bismuth on Cu concentration of differing rat organs (Szymanska and Zelazowski, 1979). The researchers found an increase in total body Cu content with no change in liver concentrations of Cu; however, the concentration of Cu observed in the kidneys was increased with the injection of bismuth. Assuming that bismuth causes a shift in Cu transportation to the kidneys, it may be possible that the heifers receiving BSS in the current experiment eliminated Cu via urine. Another possibility is that formations of Bi-Cu-S compounds bound Cu, and excreted the Cu via feces. Researchers have discussed the synthesis of Cu₃BiS₃ nanocrystals in the laboratory (Yan et al., 2013); however, there is no evidence that this can occur naturally in the rumen.

The inclusion of BSS in the diets of heifers was also associated with a 30% increase (P = 0.016) in liver concentration of Fe. There has been some evidence that bismuth may act as an antimicrobial by inhibiting bacterial uptake of Fe (Domenico et al., 1996). More recently, other research groups have contested this theory claiming that the effects of bismuth on bacteria (i.e., Helicobacter pylori) is not similar to intracellular Fe deprivation (Bland et al., 2004). Further research needs to focus on the mode of action of bismuth as an antimicrobial. If there is a link between bismuth and the uptake of Fe by bacteria, it is possible that more Fe is available for absorption in the duodenum; however, this is unlikely. It is more probable that the increase in liver Fe concentration is related to the depletion of Cu in the liver (Suttle, 2010). Copper is vital for the activity of ferroxidase, an enzyme which mobilizes Fe out of the liver; therefore, if Cu is limiting in the liver, Fe is likely to accumulate in the hepatic tissue (Mills et al., 1976; Hansen et al., 2010; Suttle, 2010).

In conclusion, the addition of BSS does not negatively affect apparent total tract digestibility of nutrients, nor does BSS appear to mitigate enteric CH₄ emissions. Further research is needed to evaluate the effects of BSS on differing dietary compositions in regards to nutrient digestibility. Alone, BSS did not hinder growth of heifers consuming bahiagrass hay and molasses. The diet in the current experiment contained ~0.5% S (DM), which may not have been great enough to observe the potential benefits of BSS on performance. Regardless of performance, heifers consuming BSS exhibited decreased liver Cu and increased liver Fe accumulation, which may have detrimental effects in the long term. More research is required to determine the effects of BSS on cattle consuming diets of differing composition and S content. It may be plausible in the future to provide BSS to finishing cattle consuming by-products, such as distiller’s grains, to mitigate the negative effects associated with high-S diets. The inclusion of eCAN in the diets of ruminants forage may reduce daily emissions of CH₄ (g/d and g/kg of BW^0.75); however, the effect of nitrate on digestibility should be considered. Future research should focus on the effects of nitrate on grazing cattle and cattle consuming different forage types.

Glossary

Abbreviations

ADF

acid detergent fiber

ADG

average daily gain

BSS

bismuth subsalicylate

BUN

blood urea nitrogen

BW

body weight

CP

crude protein

DM

dry matter

DMI

DM intake

eCAN

encapsulated calcium-ammonium nitrate

IVOMD

in vitro OM digestibility

NDF

neutral detergent fiber

NPN

nonprotein nitrogen

NS

source of NPN

OM

organic matter

VFA

Volatile fatty acid

Conflict of interest statement

Rafael C. Araujo reports that he is the R&D manager for GRASP Ind. & Com. LTDA, the company which donated calcium-ammonium nitrate. The other authors declare no real or perceived conflicts of interest.