Impact of Supplementing a Backgrounding Diet with Nonprotein Nitrogen on In Vitro Methane Production, Nutrient Digestibility, and Steer Performance

Abstract

Two experiments were conducted to evaluate the effect of nonprotein nitrogen (NPN) supplementation on in vitro fermentation and animal performance using a backgrounding diet. In experiment 1, incubations were conducted on three separate days (replicates). Treatments were control (CTL, without NPN), urea (U), urea–biuret (UB), and urea–biuret–nitrate (UBN) mixtures. Except for control, treatments were isonitrogenous using 1% U inclusion as a reference. Ruminal fluid was collected from two Angus-crossbred steers fed a backgrounding diet plus 100 g of a UBN mixture for at least 35 d. The concentration of volatile fatty acids (VFA) and ammonia nitrogen (NH₃–N), in vitro organic matter digestibility (IVOMD), and total gas and methane (CH₄) production were determined at 24 h of incubation. In experiment 2, 72 Angus-crossbred yearling steers (303 ± 29 kg of body weight [BW]) were stratified by BW and randomly allocated in nine pens (eight animals/pen and three pens/treatment). Steers consumed a backgrounding diet formulated to match the diet used in the in vitro fermentation experiment. Treatments were U, UB, and UBN and were isonitrogenous using 1% U inclusion as a reference. Steers were adapted to the NPN supplementation for 17 d. Then, digestibility evaluation was performed after 13 d of full NPN supplementation for 4 d using 36 steers (12 steers/treatment). After that, steer performance was evaluated for 56 d (24 steers/treatment). In experiment 1, NPN supplementation increased the concentration of NH₃–N and VFA (P < 0.01) without affecting the IVOMD (P = 0.48), total gas (P = 0.51), and CH₄ production (P = 0.57). Additionally, in vitro fermentation parameters did not differ (P > 0.05) among NPN sources. In experiment 2, NPN supplementation did not change dry matter and nutrient intake (P > 0.05). However, UB and UBN showed lower (P < 0.05) nutrient digestibility than U, except for starch (P = 0.20). Dry matter intake (P = 0.28), average daily gain (P = 0.88), and gain:feed (P = 0.63) did not differ among steers receiving NPN mixtures. In conclusion, tested NPN mixtures have the potential to be included in the backgrounding diets without any apparent negative effects on animal performance and warrant further studies to evaluate other variables to fully assess the response of feeding these novel NPN mixtures.

NPN supplementation increased in vitro fermentation, but the source of NPN did not have differential effects on in vitro fermentation and in vivo animal performance. Novel sources of NPN mixtures can be included in backgrounding diets without affecting animal performance compared to urea.

Introduction

Ruminants can utilize feeds that do not compete with human nutrition, such as high fiber and nonprotein nitrogen (NPN) sources, due to the symbiotic relationship with ruminal microbes (Knapp et al., 2014). Indeed, more than 70% of feeds used in ruminant diets are nonedible foods for human consumption (Mottet et al., 2017). Additionally, protein feed prices have generally increased during the decade, resulting in greater feeding costs and limiting the adequate nutritional balance. In this context, the inclusion of NPN becomes a strategy to supply available nitrogen (N) at a low cost and improve ruminal fermentation and animal productivity, especially in low crude protein (CP) diets (Fonnesbeck et al., 1975; Currier et al., 2004a; Leng, 2008).

Availability of N in the rumen is essential to microbial CP synthesis resulting in greater organic matter (OM) fermentation, volatile fatty acid (VFA) production, and amino acid supply to ruminants fed low-protein diets (Farra and Satter, 1971; Currier et al., 2004b; Leng, 2008). However, fermentation dynamics and microbial CP synthesis are affected by the source of NPN. Urea, biuret, and nitrates have different ruminal fermentation dynamics resulting from dissimilar degrees of hydrolysis, reduction, absorption, and passage rates (Fonnesbeck et al., 1975; Bartle et al., 1998; Nichols et al., 2022). In this regard, the concentration of ammonia nitrogen (NH₃–N), which is the end-product of NPN degradation in the rumen, increased faster when urea was supplemented rather than biuret or nitrate (Bartle et al., 1998; Lee et al., 2017). Also, ruminal degradation of NPN is partly dictated by diet characteristics and microbial adaptation (Schröder and Gilchrist, 1969; Lee and Beauchemin, 2014). In vitro and in vivo responses have been evaluated supplementing urea and biuret or urea and nitrate. However, there is a lack of information on the use of NPN mixtures, precluding recognized possibly synergistic effects of NPN mixtures on rumen fermentation and animal performance. Moreover, NPN does not only provide N; some NPN sources can decrease enteric methane (CH₄) formation. Indeed, nitrate supplementation has been shown to decrease ruminal methanogenesis between 9% and 16%, because nitrate reduction to NH₃ can outcompete the reduction of CO₂ to CH₄ in terms of the use of reducing equivalents (Leng, 2008; Ungerfeld, 2015; Feng et al., 2020). Furthermore, nitrate has inhibitory effects on ruminal methanogenic archaea (van Lingen et al., 2021).

Novel NPN sources can be developed by mixing traditional sources (e.g., urea, biuret, and nitrates) synthesized using technologies that involve controlled chemical reactions. These novel NPN sources may have the potential to modify the ruminal fermentation dynamic and animal performance relative to traditional NPN sources such as urea. However, there is limited information regarding the effect of NPN sources and their mixtures on digestibility, performance, and CH₄ production. Thus, one in vitro and one in vivo experiments were conducted. The objectives of the in vitro and in vivo experiments were to evaluate the effect of two novel mixtures of NPN compared to urea on in vitro fermentation and CH₄ production, and animal performance in backgrounding beef cattle. The experimental hypothesis was that novel NPN mixtures can synchronize ruminal fermentation increasing digestibility, reducing in vitro CH₄ production, and improving animal performance relative to urea in a backgrounding diet.

Materials and Methods

Two experiments were conducted to evaluate the effect of NPN sources on in vitro fermentation parameters and CH₄ production and performance of backgrounding steers. These studies were conducted at the North Florida Research and Education Center in Marianna, FL. All procedures involving animals were approved by the University of Florida Institutional Animal Care and Use Committee (#202111460).

Experiment 1: In vitro fermentation

Animal management and diet adaptation

Two ruminally cannulated Angus-crossbred steers (5 yr of age and 350 ± 15 kg of body weight [BW]) were used as ruminal fluid donors for the in vitro batch culture incubations. The steers were fed a total mixed ratio composed of corn silage, cotton gin byproduct, and a premix of vitamins and minerals [70%, 28%, and 2% on a dry matter (DM) basis, respectively] ad libitum on average 42 d. In addition, each steer received an NPN mixture equivalent to 100 g of urea per day (i.e., 46 g of N daily/steer) composed of an equal amount of N from the different NPN sources to adapt to the rumen microbial community. Ruminal microorganisms require an adaptation period to reduce nitrate and hydrolyze biuret to NH₃–N. Thus, each steer received daily 33 g of urea (U, 46% N, Rumisan, Yara International, Oslo, Norway), 37 g of urea–biuret mixture (UB, 41% N, Yara International), and 43 g urea–biuret–nitrates mixture (UBN, 35% N, Yara International). The UBN source was gradually added into the diet as follows: 30% of the total maximum dose during the first week (13 g/animal/d), 60% of the total maximum dose during the second week (26 g/animal/d), and 100% of the dose since the third week until the end of the experiment (43 g/animal/d).

In vitro incubation procedure and NPN supplementation

In vitro incubations were conducted on three separate days (replicates) using as the incubation substrate the same corn silage and cotton gin byproduct fed to the steers during the adaptation period (70% and 30% on DM basis, respectively, Table 1). Corn silage and cotton gin byproduct were air dried at 55 °C for at least 72 h and ground to pass a 2-mm screen. Treatments were (1) control (i.e., without NPN supplementation), (2) U, (3) UB, and (4) UBN. Diets were designed to be isonitrogenous and equivalent to 1% urea on a DM basis, except for the control treatment. Briefly, a representative sample of rumen digesta was manually collected through the rumen cannula from the ventral, dorsal, cranial, and caudal sacs in the rumen from the two ruminally cannulated steers and strained through four layers of cheesecloth, placed in prewarmed thermos containers, and transported to the laboratory within 30 min of collection. In the laboratory, ruminal fluid was maintained under constant CO₂ flux and was combined in equal proportions of each steer. Combined ruminal fluid was mixed with McDougall’s buffer in a 1:4 relation and used as inoculum.

Table 1.

Chemical composition of the ingredients used as substrate in experiment 1

Chemical analyses1	Ingredient
	Corn silage	Cotton gin byproduct	U2	UB3	UBN4
N	1.3	2.6	46.0	41.0	35
NDF	27.4	61.7	—	—	—
ADF	16.6	58.8	—	—	—
Ash	2.8	10.3	0.2	0.4	12.16
OM	97.2	89.7	99.8	99.5	87.84

¹N, nitrogen; NDF, neutral detergent fiber; ADF, acid detergent fiber; OM, organic matter (OM: 100—Ash (%)).

²Rumisan (Yara International).

³Urea–biuret mixture (Yara International).

⁴Urea–biuret–nitrate mixture (Yara International).

Two bottles of 120 mL per treatment containing 0.7 g of the substrate and 50 mL of inoculum were incubated for 24 h at 39 °C with gentle agitation (60 rpm). Further, two bottles without substrate were incubated as blank bottles. Gas pressure was recorded at 24 h using a manual transducer (Digital Test Gauge, Ashcroft, Ashcroft Inc., Stratford, CT, USA), and a sample of gas was collected using 60-mL syringes and stored in preevacuated 50-mL glass bottles for CH₄ analysis. Previous in vitro batch culture experiments using a corn silage-based substrate and incubated for 24 h have shown an effect of NPN supplementation (Vargas et al., unpublished), thus the choice of 24 h as the fermentation endpoint. At the end of the 24 h of incubation, the final pH of the fermentation fluid was recorded, and fermentation was halted by adding 0.5 mL of a 20% (vol/vol) of H₂SO₄ solution to each bottle. Two 10-mL samples were collected and frozen at −20 °C until analysis of the concentration of VFA and NH₃–N.

To measure the amount of undigested OM, two 100-mL polypropylene tubes per treatment and two blanks (without substrate) were incubated for 24 h at 39 °C with gentle agitation (60 rpm) on each replicate day. These tubes contained 0.7 g of the substrate and 50 mL of inoculum was added. After a 24-h of incubation, 6 mL of 20% HCl and 2 mL of a 5% (wt/vol) pepsin (1:3,000; Amresco Inc., Solon, OH) solution were added to each tube, and incubated for another 48 h before filtering, drying at 105 °C in a forced-air oven for 24 h, and ashing at 550 °C for 6 h.

Experiment 2: Animal performance

Animals, experimental design, and treatments

A total of 72 crossbred yearling steers (approximately 10 mo of age and 303 ± 29 kg initial BW) were stratified by BW and randomly allocated to three treatments. All steers were offered a basal diet composed of corn silage, cotton gin byproduct, and a premix of vitamins and minerals (77%, 20%, and 2% on DM basis, respectively, Table 2). Animals were housed in 9 concrete-floored pens (8 steers/pen, 3 pens/treatment) of 111 m² each. The basal diet was supplemented with the different NPN sources, the same products used in experiment 1, on an isonitrogenous basis, using as a benchmark the N provided by urea when included at 1% on a DM basis. Thus, treatments were (1) U included at 1.00% of the diet DM; (2) UB, included at 1.12% of the diet DM; and (3) UBN, included at 1.31% of the diet DM. All treatment diets contained the same amount of N coming from the test NPN source.

Table 2.

Ingredients, chemical composition, and particle size of the experimental diets used in experiment 2

Variable	Treatment1
	U	UB	UBN
Ingredient, % of DM
Corn silage	77.00	76.88	76.69
Cotton gin byproduct	20.00	20.00	20.00
Mineral	2.00	2.00	2.00
NPN source	1.00	1.12	1.31
Composition2, % of DM
DM, % as Fed	37.13	36.17	36.97
CP	13.20	12.70	12.90
Sol CP, % of CP	49.00	53.00	55.00
aNDF	41.40	38.30	39.00
ADF	30.10	29.40	31.00
Starch	27.70	29.80	27.20
Ash	5.91	5.78	6.34
Particle size, %
>19 mm	12.96	13.94	14.41
8–19 mm	60.34	60.16	58.77
4–8 mm	13.70	13.41	13.59
<4 mm	13.00	12.49	13.23

¹Nonprotein nitrogen supplements, U, urea; UB, urea–biuret mixtures; UBN, urea–biuret–nitrates mixtures.

²DM, dry matter; CP, crude protein; Sol CP, soluble crude protein; aNDF, amylase neutral detergent fiber; ADF, acid detergent fiber.

Animals were adapted to NPN supplementation during 17 d using a stepwise approach consisting of feeding 20% of the total supplemental NPN on days 1 to 2, 40% on days 3 to 5, 60% on days 6 and 9, 80% on days 10 and 14, and 100% on days 15 onwards. On days 30 to 34 following the adaptation period, digestibility evaluation was performed using 36 randomly selected animals (4 animals/pen). From days 35 to 91, animal performance was assessed using 72 animals. Steers were weighed on days 34 and 35 to obtain the initial BW and on days 90 and 91 to record the final BW. In addition to the initial and final BW, unshrunk weights and blood samples were obtained every 14 d during the performance evaluation. Individual feed intake was recorded daily using the GrowSafe intake monitoring system (GrowSafe Systems Ltd., Airdrie, AB, Canada).

Apparent total tract digestibility of nutrients

A subset of 32 steers (12 animals/treatment, 4 animals/pen) was sampled to determine the apparent total tract digestibility of nutrients using indigestible neutral detergent fiber (iNDF) as an internal marker. Feed samples were collected daily from days 30 to 33, and fecal samples were collected twice daily (0800 and 1600 hours) from days 31 to 34 by rectal grab. Feed and fecal samples were immediately dried at 55 °C for 72 h in a forced-air oven and stored for further analyses. Subsequently, samples were ground in a Willey mill (Arthur H. Thomas Co., Philadelphia, PA) to pass a 2-mm sieve and pooled on an equal weight basis per steer to determine nutrient and marker concentration.

Blood measurements

Blood samples were collected every 14 d during the performance evaluation via jugular venipuncture into 10-mL evacuated tubes containing Na heparin (BD Vacutainer; Franklin Lakes, NJ), placed on ice, and centrifuged at 1,500 × g for 15 min at 4 °C. Plasma was transferred to polypropylene tubes (12 × 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and stored at −20 °C for further analysis.

Laboratory analyses

The concentration of CH₄ in the gas subsamples was determined by gas chromatography (Agilent 7820A GC, Agilent Technologies, Palo Alto, CA) using flame ionization and a capillary column (Plot Fused Silica 25 m × 0.32 mm, Coating Molsieve 5A, Varian CP7536; Varian Inc., Lake Forest, CA). The injector, column, and detector temperatures were 80, 160, and 200 °C, respectively, and N₂ was the carrier gas flowing at 3.3 mL/min. The split ratio for the injected CH₄ sample was 100:1.

The concentration of VFA was determined in a liquid–liquid solvent extraction using ethyl acetate methodology (Ruiz-Moreno et al., 2015). Briefly, incubation fluid samples were thawed and centrifuged for 15 min at 10,000 × g. Ruminal fluid supernatant was mixed with a meta-phosphoric acid (25% wt/vol): crotonic acid (2 g/L, internal standard) solution at a 5:1 ratio, and samples were frozen overnight, thawed, and centrifuged for 10 min at 10,000 × g. The supernatant was transferred into glass tubes (12 mm × 75 mm; Fisherbrand; Thermo Fisher Scientific Inc.) and mixed with ethyl acetate in a 2:1 ratio of ethyl acetate to the supernatant. After shaking tubes vigorously and allowing the fractions to separate, the ethyl acetate fraction (top layer) was transferred to vials (9 mm; Fisherbrand; Thermo Fisher Scientific Inc). Samples were analyzed by gas chromatography (Agilent 7820A GC, Agilent Technologies) using a flame ionization detector and a capillary column (CP-WAX 58 FFAP 25 m × 0.53 mm, Varian CP7767, Varian Inc). The column temperature was maintained at 110 °C, and injector and detector temperatures were 200 and 220 °C, respectively.

Concentration of NH₃–N in the incubation fluid was measured after thawing and centrifuging at 10,000 × g for 15 min at 4 °C (Avanti J-E, Beckman Coulter Inc., Palo Alto, CA) following the methodology described by Broderick and Kang (1980). Briefly, 1 mL of a phenol reagent was pipetted into 12 × 75 mm borosilicate disposable culture tubes (Fisherbrand; Thermo Fisher Scientific Inc). A 20-μL aliquot of the supernatant from the centrifuged sample was transferred to the phenol-containing culture tubes. After vortexing, 0.8 mL of a hypochlorite solution was added to the mixture and vortexed again. The culture tubes were covered with glass marbles and placed in a water bath at 95 °C for 5 min. Absorbance was read on 200 µL samples at OD620 in flat-bottom 96-well plates (Corning Costar 3361, Thermo Fisher Scientific Inc.) using a plate reader (Fisherbrand UV/VIS AccuSkan GO Spectrophotometer, Thermo Fisher Scientific Inc).

Samples of feed and feces previously dried at 55 °C for 72 h, were weighed in duplicate (approximately 0.5 g of sample), dried in a forced-air oven at 100 °C for 24 h, and ashed at 550 °C for 6 h to determine DM and OM. For determination of the fibrous component, samples were weighed in duplicate into F57 bags (Ankom Technology Corp., Macedon, NY) and analyzed for neutral detergent fiber (NDF), using heat-stable α-amylase and sodium sulfite, and subsequently for acid detergent fiber (ADF) as described by Van Soest et al. (1991) in an Ankom 200 Fiber Analyzer (Ankom Technology Corp). Samples of 0.5 g were weighed in duplicate into F57 bags (Ankom Technology Corp.) and were incubated in the rumen of one ruminally cannulated steers for 288 h (Cole et al., 2011; Krizsan and Huhtanen, 2013) to determine the iNDF concentration in feed and feces. The residue was analyzed for NDF concentration as previously described. The concentration of N was analyzed through the Dumas dry combustion method using a Vario Micro Cube (Elementar, Manchester, UK) after samples were ball milled using a Mixer Mill MM400 (Retsch) at 25 Hz for 9 min. Feed and fecal samples were analyzed for starch concentration through the enzymatic–colorimetric method of Hall (2015) with the following modifications: glucose was analyzed using a quantitative colorimetric kit (G7521-1L; Pointe Scientific Inc., Canton, MI), absorbance was read on 200-µL samples at OD520 in flat-bottom 96-well plates (Corning Costar 3361, Thermo Fisher Scientific Inc) using a plate reader (Fisherbrand UV/VIS AccuSkan GO Spectrophotometer, Thermo Fisher Scientific Inc). Plasma was analyzed for urea N (PUN) using a quantitative colorimetric kit (B7551-120; Pointe Scientific Inc).

Calculations

The in vitro OM digestibility (IVOMD) was calculated according to the following formula:

$${\displaystyle \begin{array}{l}{\displaystyle 100\times \left(\frac{\mathrm{I}\mathrm{n}\mathrm{c}\mathrm{u}\mathrm{b}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{O}\mathrm{M}-\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{d}\mathrm{u}\mathrm{a}\mathrm{l}\mathrm{O}\mathrm{M}}{\mathrm{I}\mathrm{n}\mathrm{c}\mathrm{u}\mathrm{b}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{O}\mathrm{M}}\right)}\end{array}}$$

Initial BW was calculated as the average unshrunk BW of the steers on days 34 and 35, while final BW was the average unshrunk BW measured on days 90 and 91. The average daily gain (ADG) was determined by the difference between the final and initial BW divided by the number of days of the performance evaluation (i.e., 56 d). The gain-to-feed ratio (G:F) was computed as the ratio of ADG to daily dry matter intake (DMI), whereas the feed-to-gain ratio (F:G) was the reciprocal of the G:F. The net energy of gain (NEg) for each diet was estimated from the performance data using the equation proposed by Zinn and Shen (1998) based on individual animal DMI and ADG.

The apparent total tract digestibility of DM, OM, NDF, ADF, CP, and starch was calculated using the following formula:

$${\displaystyle \begin{array}{l}{\displaystyle 100-100\times [\left(\frac{\mathrm{m}\mathrm{a}\mathrm{r}\mathrm{k}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{f}\mathrm{e}\mathrm{e}\mathrm{d}}{\mathrm{m}\mathrm{a}\mathrm{r}\mathrm{k}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{s}}\right)\times \left(\frac{\mathrm{n}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{s}}{\mathrm{n}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{e}\mathrm{d}}\right)]}\end{array}}$$

Statistical analysis

Data from experiment 1 were analyzed as a randomized complete block design using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). The experimental unit was considered the average of two bottles or tubes within an incubation day. The model included the fixed effect of treatment and the random effect of incubation day (replicate). Significance was considered at P < 0.05, and tendencies were considered when 0.10 > P ≥ 0.05.

Data from experiment 2 were analyzed as a randomized block design using the MIXED procedure of SAS (SAS Institute Inc.). Animals were considered the experimental unit, and the model included the fixed effects of NPN supplementation and initial BW as covariates, as well as the random effect of animals within treatment. Significance was declared at P < 0.05, and tendencies were considered when 0.10 > P ≥ 0.05.

Results

In vitro fermentation and methane production when supplementing backgrounding diets with NPN

The inclusion of NPN increased the concentration of total VFA and propionate (P = 0.01) in vitro (Table 3). However, the proportion of the individual VFA, final pH, IVOMD, total gas, and CH₄ production did not change (P > 0.05) when NPN sources were included. In contrast, the concentration of acetate, butyrate, and NH₃–N was greater (P = 0.03, 0.04, and 0.01, respectively) in UB and UBN than in the control treatment. The concentration of the total VFA and the proportion of the individual VFA, final pH, IVOMD, total gas, and CH₄ production did not differ (P > 0.05) among NPN sources after 24 h of incubation.

Table 3.

Effect of NPN supplementation on in vitro fermentation and methane production. Experiment 1

Variable1	Treatment2				SEM3	P value 4
	Control	U	UB	UBN
pH	6.50	6.49	6.49	6.56	0.029	0.201
Total VFA, mM	88.72b	99.10a	105.78a	101.69a	2.996	0.006
Acetate, mM	55.57b	61.06ab	65.41a	63.04a	2.254	0.025
Propionate, mM	18.19b	21.80a	22.12a	21.46a	0.920	0.007
Butyrate, mM	10.79b	11.85ab	13.21a	12.46a	0.609	0.037
Acetate, mol/100 mol	62.64	61.56	61.76	61.95	0.477	0.371
Propionate, mol/100 mol	20.52	22.01	20.89	21.13	0.470	0.133
Butyrate, mol/100 mol	12.15	12.02	12.57	12.28	0.785	0.858
Acetic:propionic ratio	3.05	2.80	2.97	2.95	0.060	0.086
NH3–N, mM	3.25b	4.67ab	6.96a	6.71a	0.876	0.011
IVOMD, %	54.40	49.27	54.96	58.33	6.128	0.484
Gas, mL/g OMD	300.26	333.84	301.37	270.52	45.265	0.510
Methane, mM/g OMD	33.73	34.20	32.74	26.66	5.665	0.568

¹VFA, volatile fatty acid; IVOMD, in vitro organic matter digestibility; OMD, organic matter digested.

²Treatments. Control: Without NPN supplementation; U, urea; UB, urea–biuret mixture; UBN, urea–biuret–nitrate mixture.

³Standard error of the mean, n = 3/treatment.

⁴Observed significance for the main effects of treatment.

^a,bWithin a row, means with different superscripts differ, P < 0.05.

Nutrient digestibility and animal performance when supplementing backgrounding diets with NPN

During the digestibility period, DM and nutrient intake were not different (P > 0.05) among NPN supplements (Table 4). However, DM, OM, CP, NDF, and ADF digestibility were 10%, 9%, 15%, 13%, and 17% lesser (P < 0.01) in UB and UBN relative to U treatment, respectively. Only starch digestibility did not differ (P = 0.20) among the different sources of NPN supplementation (Table 4).

Table 4.

Effect of the NPN supplementation on nutrient intake and apparent total tract digestibility of steers. Experiment 2

Variable1	Treatment2			SEM3	P value4
	U	UB	UBN
Intake, kg/d
DM	5.96	4.99	5.45	0.432	0.333
OM	5.64	4.73	5.14	0.409	0.338
CP	0.79	0.63	0.70	0.056	0.202
NDF	2.47	1.91	2.13	0.172	0.108
ADF	1.79	1.47	1.69	0.130	0.235
Starch	4.31	3.50	3.97	0.310	0.227
Digestibility, %
DM	64.51a	59.07b	57.13b	0.648	<0.001
OM	66.67a	61.43b	60.03b	0.633	<0.001
CP	55.73a	48.59b	46.56b	1.471	0.001
NDF	45.80a	41.00b	38.56b	0.938	<0.001
ADF	42.50a	35.18b	35.38b	0.886	<0.001
Starch	98.27	97.79	98.14	0.181	0.196

¹DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber.

²Nonprotein nitrogen supplementation. U, urea; UB, urea–biuret mixture; UBN, urea–biuret–nitrate mixture.

³Standard error of the mean, n = 24 steers/treatment.

⁴Observed significance for the main effects of NPN supplementation.

^a,bWithin a row, means with different superscripts differ, P < 0.05.

Initial and final BW did not differ (P = 0.61) among treatments, resulting in no difference (P > 0.05) ADG after 56 d of performance evaluation (Table 5). However, UBN showed a greater (P = 0.05) ADG than UB during the first 28 d of performance evaluation, while U showed an intermediate value (data not shown). Feed intake did not differ (P = 0.28) among steers receiving different NPN sources, but DMI expressed per unit of BW tended to be greater (P = 0.06) when steers were supplemented with UB than U; while UBN showed an intermediate value (Table 5).

Table 5.

Effect of the NPN supplementation on growth performance and PUN concentration of steers. Experiment 2

Item1	Treatment2			SEM3	P value4
	U	UB	UBN
Initial BW, kg	288.69	283.18	289.92	5.057	0.612
Final BW, kg	343.69	342.11	349.48	5.781	0.643
ADG, kg	1.01	1.05	1.05	0.063	0.881
DMI, kg/d	7.29	7.98	7.52	0.294	0.275
DMI, % of BW	2.31y	2.54x	2.34xy	0.079	0.056
G:F, kg/kg	0.14	0.13	0.14	0.005	0.628
NEg, Mcal/kg of DM	0.55	0.52	0.55	0.017	0.379
PUN, mg/dL	8.20	8.60	8.01	1.122	0.994

¹ADG, average daily gain; DMI, dry matter intake; G:F, gain to feed ratio; F:G, feed to gain ratio; NEg, net energy of gain calculated from performance; PUN, plasma urea nitrogen.

²Nonprotein nitrogen supplementation. U, urea; UB, urea–biuret mixture; UBN, urea–biuret–nitrate mixture.

³Standard error of the mean, n = 24 steers/treatment.

⁴Observed significance for the main effects of NPN supplementation.

^x,yWithin a row, means with different superscripts tended to differ, 0.10 > P ≥ 0.05.

Efficiency expressed as G:F was not different (P = 0.63) among sources of NPN during the performance evaluation (Table 5). G:F was greater (P = 0.02) for UBN than UB during the first 28 d, while U showed an intermediate value (data not shown). Diets did not differ (P = 0.38) on the values of NEg calculated according to the animal performance. Finally, PUN concentration was similar (P = 0.99) among animals receiving different NPN supplements during the performance period (Table 5).

Discussion

In vitro fermentation and methane production when supplementing backgrounding diets with NPN

The inclusion of NPN in a hay-based substrate increased the OM digestibility and NH₃–N concentration in vitro without affecting the concentration of VFA (Henry et al., 2021). Differently, in this study, supplementing a corn silage-based substrate with NPN increased the concentration of the total VFA and propionate but did not change the proportion of individual VFA, IVOMD, and total gas and CH₄ production after 24 h of incubation. The fermentation dynamics between the studies by Henry et al. (2021) and this one may explain these differences in OM digestibility in vitro.

Increased concentration of NH₃–N in response to NPN supplementation is associated with enhanced fermentation in low CP diets (Oltjen et al., 1969). In turn, improved fermentation is usually associated with greater synthesis of VFA, digestibility, and gas production (Pell and Scofield, 1993). In this experiment, the greater availability of NH₃–N due to NPN supplementation could have promoted greater microbial activity, especially of fibrolytic bacteria, resulting in greater VFA production.

Evaluations of different sources and mixtures of NPN on in vitro fermentation and CH₄ emissions are scarce. In experiment 1, fermentation products were not different among NPN sources after 24 h incubation. NPN supplements are utilized more efficiently in high-energy and low-protein diets and after an appropriate adaptation period (Bauriedel et al.,1971; Fonnesberk et al., 1975; Leng 2008). In this regard, fluid donor animals were fed a corn silage-based diet along with the different NPN sources on average for 42 d resulting in a similar fermentation profile among NPN treatments and suggesting an appropriate microbial adaptation. Additionally, the inclusion of nitrates can reduce CH₄ emissions both in vitro and in vivo because nitrate reduction to NH₃–N competes with methanogenesis, and some nitrate reduction intermediate compounds are toxic to methanogenic microorganisms (i.e., archaea, Ungerfeld, 2015; Yang et al., 2016; Granja-Salcedo et al., 2019; van Lingen et al., 2021). In experiment 1, the nitrate concentration was 0.28% in DM basis for the UBN treatment. The low concentration of nitrates relative to other experiments (1.67%, Feng et al., 2020) might have limited the possibility of reducing CH₄ emissions under the in vitro conditions used herein.

Nutrient digestibility and animal performance when supplementing backgrounding diets with NPN

Supplementation of NPN increases intake when CP concentration is low in ruminant diets (Löest et al., 2001; Currier et al., 2004a, b; Nguyen et al., 2016). However, DM and nutrient intake were similar among animals receiving different NPN sources in experiment 2 of the current study. Generally, NPN supplementation is associated with increasing ruminal fermentation, microbial population, fiber particle reduction, and passage rate resulting in greater intake capacity (Oltjen et al., 1969). In experiment 2, diets were formulated to be isonitrogenous meeting the nutrient requirements of backgrounding steers (NASEM, 2016). In this regard, Fonnesbeck et al. (1975) suggested that NPN supplementation does not increase intake when the diet provides adequate CP levels. Thus, differences in nutrient intake from the feeding of NPN were not expected because the supply of CP was sufficient by design.

Nutrient and DM intake have been compared in animals consuming urea and biuret or urea and nitrate; however, there is a scarcity of information about the use of NPN mixtures. Biuret and urea resulted in similar DMI when the sources of NPN were either mixed in a complete diet (Carver and Pfander 1974; Currier et al., 2004a) or supplied using a lick block (Löest et al., 2001). Although nitrates were frequently found to have no depressing effect on DMI when included at less than 2.0% of DM basis (van Zijderveld et al., 2011; Olijhoek et al., 2016; Henry et al., 2020a, b), some experiments report DMI reduction with even lower inclusion rates in the diet (Lee et al., 2015; Rebelo et al., 2019). In experiment 2, nitrate concentration was 0.28% in the UBN treatment, which could have accounted for the lack of negative effects on DM and nutrient intake.

Supplementation of NPN in low CP diets is associated with improved ruminal fermentation and DMI (Oltjen et al., 1969). In this study, UB supplementation resulted in lower DM and nutrient digestibility than U. Biuret supplementation has shown contradictory results on nutrient digestibility. Johnson and McClure (1964) and Schaadt et al. (1966) did not find differences in nutrient digestibility when urea or biuret was supplemented; however, in other studies, feeding urea increased the concentration of VFA and NDF digestibility relative to biuret (Oltjen et al., 1968; Löest et al., 2001). Greater digestibility in animals receiving urea rather than biuret is associated with increased N (i.e., NH₃–N ) concentration in the rumen (Chicco et al., 1971; Currier et al., 2004a, c) resulting from the faster hydrolysis rate of urea (Fonnesbeck et al., 1975; Bartle et al., 1998). Biuret hydrolysis is driven by biuretases, which are bacterial enzymes that depending on the diet characteristics, require from 14 to 40 d to be synthesized (Oltjen et al., 1968; Fonnesbeck et al., 1975). In this regard, shorter adaptation periods are expected when diets have low CP and high starch concentration (Schröder and Gilchrist, 1969). In experiment 2, an assessment of digestibility was conducted after 30 d of NPN exposure. Considering that treatments resulted in similar PUN, it seems reasonable to expect that feeding U and UB generated similar NH₃–N concentrations and N availability in the rumen. Thus, ruminal microbes were likely adapted to the different NPN sources under the experimental conditions reported herein, ensuring sufficient N supply to not limit DM and nutrient digestibility. Alternatively, alterations in nutrient synchronization or changes in ruminal microbial communities could have explained the reduction of nutrient digestibility when UB was included in the diet (Belasco, 1954; Leng and Nolan, 1984). However, future work is necessary to test these hypotheses.

The effects of nitrate supplementation have been inconsistent. In several studies, nitrate supplementation reduced nutrient digestibility (Marais et al., 1988; Nolan et al., 2010; Henry et al., 2020a), especially the digestibility of the fiber fraction (van Zijderveld et al., 2010; de Raphélis-Soissan et al., 2016). However, in other reports nutrient digestibility was not affected (Olijhoek et al., 2016; Henry et al., 2020b; Almeida et al., 2022a, b) or even increased (Patra and Yu, 2013; Adejoro et al., 2020) when nitrates were included in ruminant diets. The depressing effect of nitrates on digestibility is related to the inhibitory effect of some intermediate compounds produced during nitrate reduction (i.e., nitrites or nitrous oxide) on ruminal bacteria and archaea, which ultimately affects nutrient fermentability (Lin et al., 2013; Yang et al., 2016; Granja et al., 2019; Ortiz-Chura et al., 2021). In experiment 2, digestibility of fiber was affected to a greater extent than that of OM (16% vs. 10% respectively), suggesting a deleterious influence of nitrates on fibrolytic microorganisms. However, it was not expected that UBN could result in reduced nutrient digestibility compared with U because the concentration of nitrates was low, and animals received UBN supplementation at least 30 d before the digestibility evaluation. In this regard, Leng (2008) suggested that nitrate adaptation requires at least 3 wk, thereby improving the rate of nitrate reduction and nutrient fermentability (van Zijderveld et al., 2010; Lee and Beauchemin, 2014). Additionally, nitrate reduction to nitrite has an optimum pH of 6.5; whereas the reduction of nitrite to NH₃–N has an optimum ruminal pH of 5.5, implying that diet characteristics determine the potential accumulation of antimicrobial compounds (Latham et al., 2016). In this regard, it is likely that corn silage-based diets as used herein would favor the occurrence of low rumen pH promoting the accumulation of nitrites and thereby depressing ruminal fermentation. However, this hypothesis should be explored in further experiments.

In experiment 2, animals receiving the different mixtures of NPN showed similar intake and performance but different digestibility. These findings suggest that animals that consumed UB and UBN were more efficient in the uptake and/or utilization of nutrients than the U-fed counterparts. Possibly, biuret supplementation might have increased bacterial efficiency by facilitating a more constant availability of NH₃–N during the fermentation process (Currier et al., 2004b) which ultimately enhanced microbial protein synthesis relative to urea. Additionally, biuret might have formed complexes with true protein in the rumen increasing the outflow of undegradable protein and avoiding energy losses associated with ruminal fermentation (Fonnesbeck et al., 1975). Other authors observed that biuret supplementation reduced daily gain compared with urea, but these results were likely associated with poor adaptation and deficient NH₃–N availability in the rumen (Berry et al., 1956; Farlin et al., 1968). In addition to modifying rumen microbiota, nitrates can directly affect animal metabolism by increasing red blood cell synthesis (El-Zaiat et al., 2014; Latham et al., 2016). Additionally, nitrates can be reduced to nitric oxide, reducing platelet aggregation, promoting fat mobilization, and regulating blood flow (Marais et al., 1988; Nnate and Achi, 2016; Ma et al., 2018). In the gastrointestinal tract, nitric oxide is an antimicrobial compound that acts upon microbial population reducing pathogenic bacteria (Lee and Beauchemin, 2014; Nnate and Achi, 2016). Furthermore, nitrate supplementation can reduce heat production, resulting in greater net energy available for maintenance or production (van Zijderveld et al., 2010). Several mechanisms therefore exist for mediating the animal responses to UB and UBN. Yet, a definitive explanation for the findings reported herein remains elusive.

The evaluation of NPN mixtures using in vitro and in vivo models allows for a better comprehension of a feeding intervention. Supplementation with different NPN mixtures did not modify in vitro fermentation (experiment 1) or animal performance (experiment 2) relative to the inclusion of urea. Further research should include the evaluation of other response variables when NPN mixtures are supplemented. For example, fermentation changes due to supplementation with NPN mixtures might result in a different ruminal microbiome, potentially modifying methane emissions. In addition, the optimal inclusion level of NPN mixtures should be determined in further studies to optimize ruminal fermentation and animal performance.

Conclusions

In experiment 1, supplementation of NPN increased the concentration of VFA without affecting the IVOMD and total gas and CH₄ production. In addition, in vitro fermentation was similar among NPN sources. In experiment 2, the performance of animals supplemented with NPN mixtures did not differ from those receiving U, despite having decreased nutrient digestibility. These findings suggest that NPN mixtures have the potential to be included in backgrounding diets without compromising animal performance and warrant further studies to evaluate potential responses on other outcome variables (e.g., methane production, VFA concentration, and microbial protein supply) to fully assess the potential benefits of feeding NPN mixtures.

Glossary

Abbreviations

ADF

acid detergent fiber

ADG

average daily gain

BW

body weight

CP

crude protein

DM

dry matter

DMI

dry matter intake

F:G

feed to gain ratio

G:F

gain:feed ratio

iNDF

indigestible neutral detergent fiber

IVOMD

in vitro organic matter digestibility

NEg

net energy of gain

NDF

neutral detergent fiber

NH₃–N

ammonia nitrogen

NPN

nonprotein nitrogen

OM

organic matter

PUN

plasma urea nitrogen

VFA

volatile fatty acids

Conflict of interest statement. Isabel Ruiz is an employee of the R&D department of YARA Industrial Solutions. The other authors declare no real or perceived conflicts of interest.