β-Alanine decreases plasma taurine but improves nitrogen utilization efficiency in beef steers

Abstract

Recent research has demonstrated that rumen-protected taurine supplementation improves body protein turnover, apparent nitrogen retention (ANR) and nitrogen (N) utilization efficiency (NUE) in beef steers. To further elucidate taurine's role in N metabolism, it is essential to examine whether taurine depletion adversely affects ANR and NUE. Six beef steers (bodyweight 391 ± 10 kg) were allocated in a replicated 3 × 3 Latin square design. Each experimental period was 20 d, including 15 d for adaptation and 5 d for sampling. Three levels of rumen-protected β-alanine (RPβA, a taurine inhibitor)— 0, 17.5, and 35 g/d— were added to the basal diet as dietary treatments. The results showed that RPβA supplementations at 17.5 and 35 g/d linearly decreased the plasma taurine concentrationby 12.54% and 22.54% (P = 0.026), and the urinary taurine excretion by 15.78% and 21.05%(P < 0.001), respectively, while linearly increased ANR (P < 0.001) and NUE (P < 0.001) in steers. Rumen-protected β-alanine supplementation linearly increased the plasma concentrations of methionine (Met, P < 0.001), lysine (Lys, P = 0.018), threonine (Thr, P = 0.011), leucine (Leu, P = 0.042) and histidine (His, P = 0.061), as well as growth hormone (P < 0.001),insulin-like growth factor-1 (P < 0.001), and the total antioxidant capacity (P < 0.001). Rumen-protected β-alanine supplementation tended to decrease the skeletal muscle protein degradation rate (P = 0.055). Specifically, supplementation with 35 g/d RPβA upregulated the plasma amino acid derivatives and oligopeptides, including N-linoleoyl-histidine (P < 0.001), L-Met (P < 0.001), L-4-chlorotryptophan (P = 0.006), L-Thr (P = 0.022), L-Lys (P = 0.026), L-carnitine (P = 0.038), suberic acid (P = 0.036), formyllysine (P = 0.036), N-acetyltyrosine (P = 0.042), histidylglycine (P = 0.045), and N-formyl-L-glutamic acid (P = 0.047). Supplementation with 35 g/d RPβA also altered the muscle cell mRNA expression, upregulated hub genes (GADPH, PFKM, TPII, PGK1, and PKM) and modified arginine-proline metabolism and the AMPK signaling pathway in beef steers. In conclusion, RPβA supplementation effectively reduced the plasma taurine concentrations and improved the ANR and NUE in steers. These effects were mediated by modulation of plasma amino acid profiles and metabolomic pathways, which appear to counteract the negative impacts of taurine depletion on N metabolism.

1. Introduction

The nitrogen utilization efficiency (NUE) of ruminants is lower than monogastric animals (Shurson and Kerr, 2023). About 70% to 95% of the nitrogen (N) intake by ruminants is excreted into the environment (McCoard and Pacheco, 2023), resulting in protein feed loss and environmental pollution (Hristov et al., 2019). Hence, improving NUE is one of the most important objectives in ruminant production.

One effective way to improve steer NUE is supplementation with essential amino acids (AA) such as methionine (Met) and lysine (Lys) to balance intestinal AA profile (Chen et al., 2011; Wang et al., 2022). Taurine is a nonprotein AA that can be synthesized in animal body using Met and cysteine (Cys) as precusors (Kim et al., 2014; Merckx and De Paepe, 2022). The plasma taurine concentration of beef steers (liveweight 250 kg) is 5.67 to 14.29 μmol/L (Sakai and Nagasawa, 1992). A recent study indicated that taurine supplementation improved the apparent nitrogen retention (ANR) and NUE in beef steers (Zhang et al., 2024). To further confirm the impact of taurine on the N metabolism in steers, it is essential to examine whether taurine depletion would negatively affect the N metabolism. Previous studies with cultured rat cardiomyocytes indicated that β-alanine (β-Ala) effectively inhibited taurine absorption and transport (Jong et al., 2010), while in rats administered β-Ala via drinking water (3%, w/v), plasma taurine levels were decreased (Parıldar-Karpuzoğlu et al., 2007). However, it is unclear whether β-Ala supplementation would diminish the taurine level and consequently have adverse impacts on ANR and NUE in steers.

This experiment aimed to investigate whether β-Ala supplementation reduces taurine status in steers, and if consequent taurine depletion negatively affects N metabolism. in steers, and further elucidate the mechanisms through profiling the plasma AA and the metabolomics, and the muscle transcriptomics.

2. Materials and methods

2.1. Animal ethics statement

The procedures and the management of animals were approved by the Laboratory Animal Welfare and Animal Experimental Ethical Inspection of China Agricultural University (AW82803202-1-1).

2.2. Animals and experimental design

Six Simmental steers with initial bodyweight of 391 ± 10 kg were used as experimental animals. The experimental treatments were CON group (basal diet), T1 group (basal diet + 17.5 g/d rumen-protected β-Ala [RPβA]), and T2 group (basal diet + 35 g/d RPβA). The supplemental levels of β-Ala in the present experiment were based on Hu et al. (2024). The animals and treatments were assigned in a replicated 3 × 3 Latin square design. The ingredients and chemical composition of the basal diet are presented in Table 1. To avoid ruminal hydrolysis of β-Ala (Lan et al., 2024), RPβA was used in the experiment. The RPβA product (containing 75% β-Ala and 25% protective coating materials, which were composed of 20% palm oil, 60% corn starch, and 20% sodium carboxymethyl cellulose) was processed by King Techina Feed Co., Ltd. (Hangzhou, China). The in vitro rumen bypass rate and the small intestinal digestibility of RPβA determined using a two-stage digestion technique (Tilley and Terry, 1963) were 77.99% and 85.13%, respectively.

Table 1.

Ingredients and nutritional composition of the basal diet (% DM).

Item	Contents
Ingredients
Corn silage	50.00
Corn grain	30.00
Soybean meal	12.00
Wheat bran	6.00
Sodium bicarbonate	1.00
Salt	1.00
Total	100.00
Chemical composition
DM, % fed basis	56.13
OM	95.14
CP	13.16
NDF	37.96
ADF	16.65
GE, MJ/kg	16.27
NEmf,1 MJ/kg	5.58
3-Methylhistidine, μmol/g	1.88

DM = dry matter; OM = organic matter; CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber; GE = gross energy.

¹NE_mf = net energy for maintenance and fattening, calculated based on dietary OM and NDF contents according to the Nutrient Requirements and Feeding Standards of Beef Cattle (Feng, 2000).

The steers were housed in individual pens equipped with feed troughs and had free access to fresh drinking water. Each steer was supplied with 6.98 kg dry matter (DM) of total mixed ration (TMR) containing 3.07 kg corn silage (DM) and 3.91 kg concentrate mixture daily, which met approximately 90% of ad libitum intake. The planned doses of TMR and RPβA were divided into two equal portions and administered to each steer at 07:00 and 17:00. Prior to feeding, RPβA was completely mixed with the TMR. No feed residuals were left from all animals during the experiment. Each experimental period was 20 d, of which 15 d were for adaptation and 5 d for sampling. The liveweight of each beef steer was recorded on the first day and last day of each experimental period before morning feeding. The feed samples were collected weekly during each sampling period and stored at −20 °C.

2.3. Sampling

On the first day of each sampling period, rumen fluid was collected from each steer using a stomach tube through the esophagus tract prior to morning feeding. The first tube of rumen fluid containing approximately 100 mL was discarded to avoid saliva contamination, and the second tube of rumen fluid was retained as a sample. The pH of the rumen fluid sample was immediately measured using a portable pH meter (model 8601, AZ Instrument Corp., Shanghai, China). The rumen fluid samples were then filtered through four layers of surgical gauze and stored in a freezer at −20 °C.

During each 5day sampling period, total feces and urine from each steer were collected daily. The feces were collected using rubber buckets with covers, and the weight of the feces was recorded. After homogenization, 1% of the total feces was sampled and mixed with 20 mL of 10% (v/v) H₂SO₄ to prevent N loss. The urine from each steer was collected using a rubber funnel connected via a polyethylene tube to a 20-L plastic bucket surrounded by ice cubes. Prior to collection, an aliquot of 300 mL 20% (v/v) H₂SO₄ was added to each plastic bucket to prevent N loss. The urine volume from each steer was recorded and homogenized. Then a volume of 100 mL of urine from each steer was taken as a sample. All samples of feces and urine were stored at −20 °C in a freezer for further analysis.

On the last day of each sampling period, before morning feeding, a volume of 20 mL of blood was taken from the jugular vein of each steer using a sterile syringe containing sodium heparin (Shandong Osat Medical Device Co., Ltd., Heze, China). The plasma was obtained after the blood sample was centrifuged at 3000 × g for 15 min at 4 °C. The plasma samples were stored at −20 °C in a freezer.

On the last day of each sampling period, muscle samples were taken from each steer. Briefly, an area of approximately 5 cm × 5 cm between the 12^th and 13^th transverse of the longissimus dorsi muscle was sampled. After the surface hair was removed, the area was disinfected three times with iodophor and 75% alcohol. Local anesthesia was then administered by injecting 7 mL of 5% lidocaine hydrochloride. Five minutes later, the epidermis was incised with a scalpel, and a muscle sample was collected by inserting a sterile puncture device to a depth of 4 to 5 cm. The muscle samples were rinsed with saline, frozen in liquid N, and transferred to a refrigerator at −80 °C.

2.4. Determinations and chemical analyses

The corn silage and feces samples were freeze-dried for 96 h using a freeze dryer (LGJ-12; Beijing Songyuan Huaxing Technology Development Co., Ltd., Beijing, China). The freeze-dried feed samples were milled and the fecal samples were ground using a mortar and a pestle to pass through a 40-mesh sieve. The DM and crude ash contents of feed samples were analyzed according to AOAC (2005) methods 934.01 and 942.05, respectively. The total N of feeds, urine, and feces was determined by the Kjeldahl method using AOAC (2005) method 968.06. The organic matter (OM) of feeds and feces was calculated as DM minus crude ash. The crude protein (CP) of feeds, urine, and feces was calculated as total N × 6.25. The neutral detergent fiber (aNDF) of feeds was analyzed using the procedures of Van Soest et al. (1991) with heat-stable α-amylase and sodium sulfite (Mertens, 2002). The acid detergent fiber (ADF) was analyzed according to AOAC (2005) method 973.18using an ANKOM A200i Fiber Analyzer (ANKOM Technology, Macedon, NY, USA). The gross energy of feeds was analyzed using an oxygen bomb calorimeter (Parr 6300; Parr Instrument Company, Moline, IL, USA). The 3-methylhistidine (3-MeHis) in urine and feeds were analyzed using a microplate reader (DR-200BS; Wuxi Hiwell-Diatek Instruments Co., Ltd., Wuxi, China) using analytical kit (Beijing Sino-UK Institute of Biological Technology, Beijing, China). The ruminal volatile fatty acids (VFA) were analyzed on a gas chromatography (GC-8600; Beijing Beifen Tianpu Instrument Technology Co., Ltd., Beijing, China) using the procedures described by Yang et al. (2017).

2.5. Analyses of plasma biochemical parameters

The plasma albumin (ALB), total protein (TP), urea, triglyceride (TG), total amino acids (T-AA) and total antioxidant capacity (T-AOC) were analyzed using commercial kits (Beijing Sino-UK Institute of Biological Technology Co., Ltd., Beijing, China) on an automatic biochemical analyzer (BS-420; Shenzhen Mindray Biomedical Electronic Co., Ltd., Guangdong, China). The plasma globulin (GLB) was calculated as the difference between TP and ALB. The plasma growth hormone (GH) and insulin-like growth factor-1 (IGF-1) were analyzed using the enzyme-linked immunosorbent assay kits (Beijing Sino-UK Institute of Biological Technology Co., Ltd., Beijing, China) on a semiautomatic biochemical analyzer (7170; Hitachi Ltd., Tokyo, Japan).

The AA profiles of plasma samples were determined using high-performance liquid chromatography (UltiMate 3000, Thermo Fisher Scientific, Waltham, MA, USA) coupled with tandem mass spectrometry (API 3200 Q-TRAP, Sciex, Framingham, MA, USA) (HPLC-MS/MS) following the method described by Shimbo et al. (2010).

2.6. Plasma metabolomics profiling

An aliquot of 100 μL plasma sample of CON group or T2 group was mixed with 400 μL of extraction solvent (acetonitrile:methanol = 1:1) and subjected to low-temperature ultrasonic extraction 30 min (5 °C, 40 kHz). Samples were incubated at −20 °C for 30 min, centrifuged at 13,000 × g and 4 °C for 15 min, and the supernatant was collected and dried by gassing N₂. The residue was reconstituted in 100 μL of acetonitrile:water (1:1, v:v), subjected to 5 min of ultrasonic extraction at 5 °C, centrifuged at 13,000 × g at 4 °C for 10 min, and the final supernatant was transferred to an autosampler vial for analysis. The metabolites were analyzed using a Thermo Scientific UHPLC-Q Exactive system, and the data were processed using Progenesis QI software (Waters Corp., Milford, MA, USA). To ensure analytical stability, a quality control (QC) sample was prepared by mixing all extracted plasma samples at the same proportion. This QC sample was injected at regular intervals (every 5 samples). Plasma metabolites were identified by the Human Metabolome Database (HMDB) (Wishart et al., 2022). The final dataset containing information such as peak number, sample name, and normalized peak area was imported into the SIMCA 16.0.2 software package (Sartorius Stedim Data Analytics AB, Umea, Sweden) for multivariate analysis (Zhang et al., 2023). Significantly changed metabolites (SCMs) were identified based on variable importance (VIP) scores > 1 and P-values < 0.05 from the orthogonal projections to the latent structures-discriminate analysis (OPLS-DA) model (Xia et al., 2022). The metabolic pathway enrichment of SCMs was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) and MetaboAnalyst (http://www.metaboanalyst.ca/), elucidating pathways related to the experimental conditions.

2.7. Muscle transcriptomics profiling

The total RNA was extracted from muscle tissues (CON and T2 groups) TRI-Reagent (T9424, Sigma-Aldrich, St Louis, MO, USA) following the manufacturer's protocol. The concentration and purity of the extracted RNA were measured using a Nanodrop2000 system (Thermo Fisher Scientific, Wilmington, DE, USA), ensuring a ratio of the absorbances at A₂₆₀and A₂₈₀ was within 1.8−2.0. The mRNA was isolated from total RNA via A-T base pairing between polyA and Oligo (dT) - coated magnetic beads and then randomly fragmented into approximately 300 bp fragments. The cDNA was synthesized from these fragments using reverse transcriptase and random primers. The double-stranded cDNA was then ligated with adapters, followed by purification and size selection. The selected fragments were PCR-amplified and further purified to obtain the final library. Different libraries were pooled based on effective concentration and target data volume for sequencing on the Illumina NovaSeq X Plus (San Diego, CA, USA), generating 150 bp paired-end reads with raw data quality checked by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

The clean reads were mapped to the cattle reference genome (ARSUCD 1.4) using Hisat2 (v2.1.0), with gene annotations provided by Ensembl (Release 104) (De los Santos Funes, 2024). The sequence alignment (SAM) files generated from the mapping process were converted to binary alignment (BAM) files using SAMtools (v1.11). Th read counts for each muscle sample in the BAM files were quantified using FeatureCounts (subread package v1.6.3) (Li et al., 2023). DESeq2 (v1.28.1) was used to obtain normalized read counts for gene expression analysis. Differentially expressed genes (DEGs) were identified using the DESeq2 package with a threshold of |log₂(fold change)| > 1 and an adjusted P-value < 0.05. The transcripts per million mapped reads (TPM) were conducted using R (version 4.3.0) dplyr package. The DEGs were analysed using the KEGG pathway to uncover the potential biological functions.

2.8. Calculations and statistical analysis

The ANR and NUE were calculated as the following:

$$\mathrm{A}\text{NR}=\text{N}\text{intake}-\text{Fecal}\mathrm{N}-\text{Urinary}\mathrm{N}$$

$$\text{NUE}\left(\%\right)=\frac{\text{ANR}}{\mathrm{N}\text{Intake}}\times 100$$

The skeletal muscle protein degradation rate was calculated according to Funabiki et al. (1976) and Nishizawa et al. (1979):

$$Y=\frac{0.934\times \left(E-0.8\times F\right)}{P}\text{,}$$

where Y is the skeletal muscle protein degradation rate (%); E is the urinary excretion of 3-MeHis (μmol/d); F is the dietary intake of 3-MeHis (μmol/d); P is the storage of 3-MeHis in steer body (μmol); 0.934 is the ratio of 3-MeHis in skeletal muscle to the body reserve; 0.8 is the recovery rate of ingested 3-MeHis from diets (F).

The data were analyzed using the PROC MIXED of SAS 9.4 (SAS Inst. Inc., Cary, NC, USA) with the following model:

$$Y_{ijkl}=\mu +T_i+P_j+S_k+C_l+e_{ijkl}$$

where Y_ijkl is the dependent variable; μ is the overall mean; T_i is the fixed effect of ith treatment (i = 1 to 3); P_j is the mixed effect of jth period (j = 1 to 3); S_k is the random effect of square (k = 1 to 2); C_l is the random effect of the lth steer (l = 1 to 6); e_ijkl is the error residual.

The polynomial contrasts were used to determine the effect (linear and quadratic) of RPβA level. The differences among the treatments were compared using a multiple comparison test following the Tukey method. P ≤ 0.05 indicates a significant difference and 0.05 < P ≤ 0.10 indicates a tendency.

3. Results

3.1. Rumen fermentation

RPβA supplementation did not affect the ruminal pH (P = 0.542), the ruminal concentration of the total VFA (P = 0.332), the molar proportions of acetate (P = 0.240), propionate (P = 0.770), isobutytate (P = 0.271), butyrate (P = 0.882), isovalerate (P = 0.408), valerate (P = 0.536), and the molar ratio of acetate to propionate (P = 0.286) (Table 2).

Table 2.

Effects of RPβA on the rumen fermentation parameters in steers.

Item	RPβA, g/d			SEM	P-value
	0	17.5	35		T	L	Q
pH	6.78	6.75	6.76	0.023	0.542	0.521	0.663
Total VFA, mmol/L	83.50	83.21	82.43	2.921	0.558	0.778	0.311
VFA proportion, mmol/100 mmol
Acetate	55.61	55.59	55.85	0.723	0.240	0.161	0.334
Propionate	26.97	26.93	26.74	0.784	0.770	0.508	0.813
Isobutytate	2.75	2.74	2.80	0.069	0.271	0.207	0.303
Butyrate	11.43	11.52	11.59	0.528	0.882	0.630	0.959
Isovalerate	2.23	2.23	2.27	0.067	0.408	0.292	0.415
Valerate	2.33	2.32	2.17	0.199	0.536	0.334	0.603
Acetate to propionate ratio	2.07	2.08	2.12	0.087	0.286	0.150	0.539

RPβA = rumen-protected β-alanine; SEM = standard error of the mean; T = treatment; L = linear; Q = quadratic; VFA = total volatile fatty acids.

3.2. Nutrient digestibility

RPβA supplementation linearly increased the urinary N excretion (P = 0.004), and it linearly decreased the fecal N excretion (P = 0.026) and the ratio of fecal N to urinary N (P = 0.015), but it did not affect the total N excretion (P = 0.113). RPβA supplementation increased the urinary β-Ala (P < 0.001) and urea excretion (P = 0.011), decreased the urinary taurine excretion (P < 0.001), and increased the ANR (P = 0.025) and NUE (P = 0.036) in a linear manner, but it did not affect the average daily gain (ADG) (P = 0.195) (Table 3).

Table 3.

Effects of RPβA on the N metabolism, urinary excretions of taurine and β-Ala and ADG in steers.

Item	RPβA, g/d			SEM	P-value
	0	17.5	35		T	L	Q
Dry matter intake, kg/d	6.98	6.98	6.98	–	–	–	–
ADG, kg/d	1.05	1.01	0.96	0.038	0.406	0.195	0.874
Feed N, g/d	154.71	154.71	154.71	–	–	–	–
β-Ala N, g/d	0.00	2.34	4.67	–	–	–	–
N intake, g/d	154.71	157.05	159.38	–	–	–	–
Fecal N, g/d	45.95	41.88	37.60	2.197	0.072	0.026	0.970
Urinary N, g/d	73.92b	75.01b	78.42a	0.802	0.013	0.004	0.277
Urinary urea, g/d	55.02b	58.26ab	64.15a	1.937	0.030	0.011	0.595
Urinary β-Ala, mg/d	5.42b	6.87ab	8.21a	0.487	0.002	<0.001	0.471
Urinary taurine, mg/d	193.44a	163.06b	151.48b	5.167	<0.001	<0.001	0.144
Total N excretion, g/d	119.88	116.89	116.03	1.708	0.235	0.113	0.585
Fecal N to urinary N ratio	0.62a	0.55ab	0.47b	0.033	0.045	0.015	0.858
Urinary N/N intake, %	47.78b	48.16ab	50.02a	0.515	0.035	0.016	0.282
Apparent N retention, g/d	34.83	38.85	40.74	1.708	0.065	0.025	0.585
N utilization efficiency, %	22.51	24.94	25.98	1.096	0.089	0.036	0.578

β-Ala = β-alanine; RPβA = rumen-protected β-alanine; ADG = average daily gain; N = nitrogen; SEM = standard error of the mean; T = treatment; L = linear; Q = quadratic.

Within a row, means without a common superscript differ at P ≤ 0.05.

"–", means not analyzed.

3.3. Skeletal muscle protein degradation

RPβA supplementation linearly decreased the urinary excretion of 3-MeHis (P = 0.048) and had a tendency to decrease the skeletal muscle protein degradation rate (P = 0.055) (Table 4).

Table 4.

Effects of RPβA on the urinary 3-MeHis excretion and skeletal muscle protein degradation rate in steers.

Item	RPβA, g/d			SEM	P-value
	0	17.5	35		T	L	Q
Urinary 3-MeHis excretion, mmol/d	2.83a	2.53ab	2.45b	4.584	0.041	0.048	0.069
3-MeHis reserve in steer body, mmol	90.79	90.79	90.55	0.898	0.218	0.134	0.364
Skeletal muscle protein degradation rate, %	2.80a	2.50ab	2.40b	0.083	0.042	0.055	0.064

RPβA = rumen-protected β-alanine; 3-MeHis = 3-methylhistidine; SEM = standard error of the mean; T = treatment; L = linear; Q = quadratic.

Within a row, means without a common superscript differ at P ≤ 0.05.

3.4. Plasma biochemical parameters

RPβA supplementation did not affect the plasma concentrations of TP (P = 0.579), ALB (P = 0.462), GLB (P = 0.199), TG (P = 0.208), and urea (P = 0.242), but it linearly increased the plasma concentrations of GH (P < 0.001), IGF-1 (P < 0.001), and T-AOC (P < 0.001) (Table 5).

Table 5.

Effects of RPβA on the plasma biochemical indexes in steers.

Item	RPβA, g/d			SEM	P-value
	0	17.5	35		T	L	Q
Nutrients
Total protein, g/L	70.42	71.58	70.67	2.234	0.579	0.832	0.320
Albumin, g/L	31.94	31.37	31.25	0.621	0.462	0.257	0.656
Globulin, g/L	38.48	40.21	39.42	2.278	0.199	0.312	0.132
Triglyceride, mmol/L	0.20	0.23	0.17	0.023	0.208	0.283	0.150
Urea, mmol/L	4.13	4.32	4.46	0.203	0.242	0.103	0.885
Hormones
GH, ng/mL	3.37c	4.83b	6.13a	0.286	<0.001	<0.001	0.780
IGF-1, ng/mL	249.05b	279.34b	343.27a	7.967	<0.001	<0.001	0.117
Antioxidant
T-AOC, U/mL	8.33b	11.66a	13.03a	0.324	<0.001	<0.001	0.046

RPβA = rumen-protected β-alanine; SEM = standard error of the mean; T = treatment; L = linear; Q = quadratic; GH = growth hormone; IGF-1 = insulin-like growth factor; T-AOC = total antioxidant capacity.

Within a row, means without a common superscript differ at P ≤ 0.05.

3.5. Plasma taurine and other AA

RPβA supplementation linearly increased the plasma β-Ala concentration (P < 0.001) and decreased the taurine concentration (P = 0.026). It also linearly increased the plasma concentrations of Met (P < 0.001), Lys (P = 0.018), threonine (Thr, P = 0.011), leucine (Leu, P = 0.042) and histidine (His, P = 0.061), and tended to increase the plasma concentration of total essential amino acids (EAA) (P = 0.085) (Table 6).

Table 6.

Effects of RPβA on the plasma amino acids in steers (µmol/L).

Item	RPβA, g/d			SEM	P-value
	0	17.5	35		T	L	Q
β-Ala	1.30b	1.61a	1.85a	0.099	0.001	<0.001	0.703
Taurine	11.40a	9.97b	8.83b	1.492	0.072	0.026	0.866
EAA
Histidine	59.46	63.53	67.48	2.567	0.155	0.061	0.985
Isoleucine	73.35	74.76	69.70	4.408	0.521	0.430	0.419
Leucine	98.98	100.83	108.68	4.072	0.092	0.042	0.415
Lysine	82.55b	97.43ab	101.55a	4.941	0.043	0.018	0.364
Methionine	16.78b	19.63a	21.51a	0.798	<0.001	<0.001	0.492
Phenylalanine	34.80	36.85	34.00	2.707	0.442	0.724	0.231
Tryptophan	29.06	31.03	28.35	1.808	0.455	0.742	0.238
Threonine	51.66b	58.28ab	67.38a	3.151	0.031	0.011	0.771
Tyrosine	27.21	27.22	27.28	2.859	0.799	0.984	0.994
Valine	158.67	158.67	158.17	6.661	0.996	0.941	0.966
Total EAA	640.73	670.60	678.38	22.132	0.180	0.085	0.525
NEAA
Anserine	0.25b	0.30ab	0.32a	0.011	0.009	0.003	0.426
Arginine	73.43b	84.85ab	93.85a	3.465	0.009	0.002	0.780
Asparagine	61.33	64.63	58.75	4.746	0.707	0.719	0.466
Aspartic acid	3.10	2.64	2.58	0.228	0.310	0.170	0.520
Carnosine	7.14b	7.72ab	8.27a	0.761	0.044	0.019	0.955
Cysteine	0.19b	0.24b	0.25a	0.012	0.016	0.007	0.230
Glutamine	220.83b	226.67b	236.33a	2.121	0.003	0.001	0.493
Glutamic acid	52.65	50.03	47.26	3.335	0.177	0.341	0.106
Glycine	263.50	249.67	225.67	17.586	0.327	0.151	0.811
Proline	69.40	74.61	68.55	3.078	0.474	0.872	0.240
Serine	74.25	79.66	74.85	3.970	0.627	0.922	0.351
Total NEAA	826.60	853.28	830.64	28.92	0.803	0.927	0.525
EAA + NEAA	1467.33	1523.88	1509.02	41.747	0.644	0.512	0.517

β-Ala = β-alanine; RPβA = rumen-protected β-alanine; SEM = standard error of the mean; T = treatment; L = linear; Q = quadratic; EAA = essential amino acids; NEAA = non-essential amino acids; TAA = total amino acids.

Within a row, means without a common superscript differ at P ≤ 0.05.

RPβA supplementation linearly increased the plasma concentrations of the NEAA including carnosine (P = 0.019), anserine (P = 0.003), Cys (P = 0.007), glutamine (Gln) (P = 0.001), and arginine (Arg) (P = 0.002), but it did not affect the plasma concentration of total NEAA (P = 0.803).

3.6. Plasma metabolome

A total of 1662 metabolites were detected from the CON and T2 groups. The OPLS-DA analysis showed clear separations between the two groups, and the intragroup variation of the CON group was higher than that of the T2 group (Fig. 1A). A total of 189 SCMs between the CON and the T2 groups were identified, of which 74 displayed higher abundances in the T2 group, while 114 were enriched in the control group (Fig. 1B). The upregulated SCMs in the T2 group were classed into six metabolic superclasses according to HMDB, among which 18 metabolites (24.19 %) belonged to organic acids and derivatives (Fig. 1C). The KEGG enrichment analysis based on the SCMs revealed that adding RPβA at 35 g/d (T2 group) significantly altered the pathways of glutathione metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, histidine metabolism, protein digestion and absorption, and D-amino acid metabolism (Fig. 1D). The key SCMs belonged to organic acids and derivatives were extracted for further analysis. Dietary supplementation with RPβA at 35 g/d (T2 group) upregulated the relative abundances of N-linoleoyl_histidine, Met, L-4-chlorotryptophan, L-Thr, L-Lys, L-carnitine, suberic acid, formyllysine, N-acetyltyrosine, histidylglycine, and N-formyl-L-glutamic acid (Fig. 1E).

Fig. 1.

Comparison of the plasma metabolite signatures in steers between the control group (CON) fed 0 g/d rumen-protected β-alanine (RPβA) and the group fed 35 g/d RPβA (T2). (A) The orthogonal partial least squares discriminant analysis (OPLS-DA) of plasma metabolites. (B) The changes in plasma metabolites of T2 group compared to CON group. The below panel refers to the change of metabolite variable importance (VIP). The above panel refers to the density of the VIP of the metabolites and thepanel on the right refers to density of the adjusted P-value. (C) The classification of significantly changed metabolites (SCMs) at the superclass level in CON and T2 groups. (D) The significantly enriched metabolic pathways of SCMs (P < 0.05). (E) The abundances of the key SCMs in CON and T2 groups.

3.7. Muscle transcriptome

After stringent quality filtering of the raw reads, 29,122,451 clean reads on average were obtained per sample, and more than 95% of the reads had a quality score of Q30. A total of 1139 genes were found differentially expressed between the CON and T2 groups. Compared with the CON group, the T2 group upregulated 666 genes and downregulated 473 genes (Fig. 2A). The KEGG enrichment analysis based on the DEGs indicated that the top 10 enriched pathways included starch and sucrose metabolism, Arg and proline metabolism, biosynthesis of amino acids, pancreatic secretion, gonadotropin hormone-releasing hormone (GnRH) signaling, carbon metabolism, adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling, cyclic adenosine monophosphate (cAMP) signaling, cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling, and calcium signaling (Fig. 2B). Among these pathways, 10 genes were found to be related to amino acid synthesis, 7 genes to Arg and proline metabolism, 15 genes to AMPK signaling and 24 genes to cAMP signaling (Fig. 2C).

Fig. 2.

Comparison of the muscle transcriptome in steers between the control group (CON) fed 0 g/d rumen-protected β-alanine (RPβA) and the group fed 35 g/d RPβA (T2). (A) The upregulated (n = 666, red) and downregulated differentially expressed genes (DEGs) (n = 473, blue) in the T2 group compared with the CON group. (B) The significantly enriched metabolic pathways of DEGs (P < 0.05). (C) The amino acid metabolic pathways and their associated genes. (D) The hub genes selected using cytoHubba plugin with the maximal clique centrality (MCC) method. (E) The expression of hub genes. The red asterisks represent significant differences (P < 0.05). (F) The correlations between the hub genes and the plasma amino acids. ∗ Refers to significant correlation (P < 0.05) and ∗∗ refers to extremely significant correlation (P < 0.01), and the color scale bar indicates the correlation coefficient.

The maximal clique centrality (MCC) method of the cytoHubba plugin was used to select the hub genes related to the pathway of amino acid synthesis (Fig. 2D). The 5 top genes ranked by the clustering coefficient method as hub genes, including GADPH, PFKM, TPII, PGK1, and PKM. The full names of the genes are listed in Table S1 and were all significantly upregulated in the T2 group (Fig. 2E). Correlation analysis between the hub genes with the plasma AA indicated that the expressions of GADPH, PFKM, TPII and PKM were positively correlated with the plasma concentration of Met (Fig. 2F) and the expressions of PGK1 or PKM were positively correlated with the plasma concentration of β-Ala. The results also showed that the expression of PGK1 was positively correlated with the plasma concentration of Cys, and GADPH was positively correlated with the plasma concentration of Thr.

4. Discussion

The present experiment showed that RPβA supplementation did not affect the ruminal pH and the ruminal concentration of total VFA of beef steers, suggesting that β-Ala was successfully protected from rumen microbial degradation. RPβA supplementation increased the plasma concentration of β-Ala and the urinary excretion of β-Ala, indicating that RPβA was well digested and β-Ala was absorbed in the hindgut.

The present experiment showed that the plasma concentration of taurine ranged from 8.83 to 11.40 μmol/L in steers, which was within the range reported by Sakai and Nagasawa (1992) that the plasma concentration of taurine in beef steers (liveweight 250 kg) was from 5.67 to 14.29 μmol/L. The present experiment also showed that compared with the CON group, the plasma taurine concentrations of T1 and T2 groups were linearly decreased from 11.40 to 9.97 and to 8.83 μmol/L, by 12.54% and 22.54%, respectively, and the urinary excretions of taurine were linearly decreased from 193.44 to 163.06 and to 151.48 mg/d, by 15.78% and 21.05%, respectively. In cultured rat cardiomyocytes, Jong et al. (2010) found that β-Ala inhibited taurine absorption and transport, and in rats, Parıldar-Karpuzoğlu et al. (2007) reported that administering β-Ala in drinking water (3%, w/v) decreased plasma taurine. The present experiment with steers demonstrated that β-Ala supplementation effectively inhibited the plasam taurine concentration and the urinary taurine excretion. It is worth noting that RPβA supplementation decreased the plasma taurine concentration and the urinary taurine excretion at similar rates, suggesting that the steers were capable to equilibrate the taurine status in their bodies (El Idrissi, 2019).

Recent studies showed that supplementing with rumen-protected taurine improved the ANR and the NUE in steers (Zhang et al., 2024). In the present experiment, although RPβA supplementation decreased the plasma taurine concentration and the urinary taurine excretion, the ANR and the NUE were increased rather than decreased. One possible reason for the results could be that the decreased plasma concentration of taurine by 12.54% and 22.54% were not high enough to negatively affect the ANR and the NUE, respectively. Another reason could be that β-Ala counteracted the adverse impact of plasma taurine depletion and therefore improved the ANR and the NUE in steers. Hu et al. (2024) reported that dietary supplementation with unprotected β-Ala at 30 or 60 g/d linearly improved the ANR but did not affect the NUE in beef steers. The reason for the difference in the effect of β-Ala on the N metabolism in steers between the two experiments could be that in the experiment of Hu et al. (2024), β-Ala was extensively hydrolyzable in rumen fermentation (Lan et al., 2024) and the amount of β-Ala absorbed was not high enough to improve the NUE in beef steers. However, in the present experiment, β-Ala was supplied to the steers in the form of RPβA. Most part of the β-Ala in RPβA should have been absorbed, therefore resulting in improving the ANR as well as the NUE in beef steers. The results also indicated that the impact of β-Ala on the ANR and the NUE was dose-dependent. The present experiment showed that RPβA supplementation did not affect the ADG of the steers. The results indicated that β-Ala had no negative impact on the performance and the health of the steers. However, whether higher level of β-Ala supplementation is safe or not is unclear. Further research is necessary to investigate the possible negative impacts of β-Ala on the performance and health of steers.

Essential amino acids are crucial for body protein synthesis in animals. Met and Lys are limiting AA for ruminants (Park et al., 2020). Many studies showed that supplementation with Met and Lys increased the ANR and the NUE in beef cattle (Li et al., 2019; Liu et al., 2021; Zou et al., 2023). Non-essential AA, such as Cys, Gln, and Arg, also play crucial roles in protein homeostasis and N metabolism in animals (Muthuraman et al., 2021; Requejo et al., 2010; Rubio-Aliaga and Wagner, 2016). Lee and Kim (2007) reported that supplementation with 3% β-Ala in drinking water improved the glutathione synthesis and increased the liver Cys concentration in mice. Wang et al. (2023) reported that dietary supplementation with β-Ala at 600 mg/kg increased the concentrations of Arg and Gln in the longissimus dorsi muscle of pigs. The present experiment showed that supplementing with RPβA linearly increased the plasma concentrations of EAA, including Met, Lys, Thr, Leu, and His. The results were consistent with the KEGG enrichment analysis which indicated that β-Ala enriched the pathways of D-amino acid metabolism and protein digestion and absorption. The present experiment also showed that supplementing with RPβA linearly increased the plasma concentrations of NEAA including Cys, Gln, and Arg in beef steers, which were in consistent with Lee and Kim (2007) and Wang et al. (2023). Therefore, the improved ANR and the NUE by RPβA could be mainly attributed to the effects of β-Ala on increasing the plasma concentrations of some EAA and NEAA in beef steers.

In swine, it was reported that carnitine supplementation improved protein accretion (Owen et al., 2001). The untargeted metabolomic profiling in the present experiment showed that supplementing with RPβA increased the plasma concentration of carnitine, which could be another factor for the improvement of the ANR and the NUE in beef steers. The untargeted metabolomic profiling also indicated that RPβA supplementation increased the plasma concentrations of some plasma AA derivatives and oligopeptide, including N-linoleoyl-histidine, N-palmitoyl-methionine, L-carnitine, formyllysine, N-acetyltyrosine, histidylglycine, and N-formyl-L-glutamic acid. The increased plasma AA derivatives and oligopeptide should be resulted from the elevated plasma AA levels by RPβA supplementation.

The metabolic pathway analysis indicated that RPβA supplementation altered the pathways of phenylalanine, tyrosine and tryptophan biosynthesis, aminoacyl-tRNA biosynthesis, histidine metabolism, protein digestion and absorption, and D-amino acid metabolism, which are related with AA metabolism and consequently the ANR and the NUE in beef steers.

Previous studies showed that the plasma concentration of total EAA was positively correlated with the levels of GH and IGF-1 in the hepatic of rats (Xu et al., 2021). Growth hormone stimulated the IRS1/Akt and the mitogen-activated protein kinase (MAPK) pathways to enhance protein synthesis (Consitt et al., 2017) and IGF-1 activated the Akt-mTORC1 pathway to improve protein synthesis (Schiaffino and Mammucari, 2011). The results of the present experiment indicated that RPβA supplementation linearly increased the plasma concentrations of GH and IGF-1. The results were consistent with Pence et al. (2016) who reported that feeding 17-month-old mice with β-Ala at 3.43 mg/kg feed for 41 d increased the plasma concentration of IGF-1. The results suggested that the impact of β-Ala on improving the ANR and the NUE of beef steers in the present experiment can be partly attributed to the increased levels of plasma GH and IGF-1. It should be noted that no differences were found in ADG among different groups. The reason for the results is that each period of the present experiment was only 20 d which were short for measuring ADG and the steers experienced intensive treatments. Therefore, the observed effects of taurine on steer ADG should be interpreted as indicative rather than conclusive.

Skeletal muscle is the primary site for AA and protein storage and metabolism in animals (Kamei et al., 2020). 3-MeHis is produced in skeletal muscle after His is methylated and it is released in muscle proteolysis. However, the released 3-MeHis can not be used for body protein synthesis but is excreted in urine (Young et al., 1972). Hence, 3-MeHis can serve as a suitable biomarker for identifying the skeletal muscle protein degradation in animals (Kochlik et al., 2018). In the present experiment, dietary supplementation with RPβA linearly decreased the urinary excretion of 3-MeHis and tended to decrease the skeletal muscle protein degradation rate. The results were consistent with the improved ANR and the NUE in beef steers. The degradation of skeletal muscle protein is primarily influenced by the metabolism of AA in animal body (Kamei et al., 2020; Lundholm et al., 1981). It was reported that carnosine can inhibit protein degradation by suppressing the calpain activity (Liao et al., 2014) and Leu also has an impact on inhibiting muscle protein degradation (Rehman et al., 2023). The present experiment showed that supplementing with RPβA increased the plasma concentration of carnosine and Leu, which could be another important reason for the decreased skeletal muscle protein degradation by β-Ala. It should be noted that dietary supplementation with RPβA linearly increased the urinary N and urea excretion in steers in the present experiment. This could be due to the increased N intake from RPβA supplementation.

In the present experiment, five hub genes, including GADPH, PFKM, TPII, PGK1, and PKM were selected in the longissimus dorsi muscle of beef steers using the MCC method of the cytoHubba. Correlation analysis indicated that the expressions of GADPH, PFKM, TPII, and PKM were positively correlated with the plasma Met concentration. The pathway enrichment analysis also showed that these five genes were all associated with the body AA synthesis in steers. However, previous studies indicated that the five genes were closely associated with the glycolysis and energy metabolism in animals (Barber et al., 2005) but no direct evidence was available for the specific link of these five genes to the AA and N metabolism in animals (Ludwig et al., 2001; Oparina et al., 2013). Therefore, β-Ala might regulate the AA and N metabolism through regulating the energy metabolism in animals.

Carnosine and anserine play vital roles in antioxidation and enhancing muscle health in mammals (Solana-Manrique et al., 2022; Wang et al., 2024). β-Ala is a key precursor for carnosine and anserine synthesis (Indriani et al., 2024). Previous studies showed that oral intake of β-Ala at 4 to 6 g/d for 4 to 10 weeks increased the carnosine and anserine concentrations in skeletal muscle by 40% to 80% in humans (Blancquaert et al., 2016) and supplementation with 10.5 g β-Ala and 24.7 g L-His to 1 kg basal diet in a water tank increased the intramuscular concentration of carnosine in yellowtails (Ogata, 2002). The present experiment indicated that RPβA supplementation increased the plasma concentrations of carnosine and anserine, which could be one important reason for the effect of β-Ala on increasing the plasma T-AOC in beef steers.

The present experiment also indicated that RPβA supplementation linearly decreased the fecal N excretion in beef steers. This could be another reason for increasing the ANR and NUE in beef steers. It is speculated that β-Ala released from RPβA decreased the fecal N excretion through modifying the lower gut bacterial community or improved the N absorption rate. However, this hypothesis needs to be clarified in further research.

5. Conclusion

This experiment demonstrated that dietary supplementation with 17.5 and 35 g/d RPβA linearly decreased plasma concentration and urinary excretion of taurine and confirmed the inhibitory impact of β-Ala on taurine in beef steers. This experiment also indicated that decreased plasma taurine level by RPβA supplmentation did not adversely affect the ANR and NUE in beef steers. Instead, the ANR and NUE in beef steers were improved through increasing plasma concentrations of some EAA, NEAA and AA derivatives, as well as enrichment of D-amino acid metabolic pathway. The results can be attributed to β-Ala's positive influence on N metabolism, which potentially counteracted the adverse effects of taurine depletion. Future research should explore the effects of reducing plasma taurine level by administering other taurine inhibitors than β-Ala on the N metabolism in beef steers to verify whether lowered taurine level would impair the N metabolism in beef steers.

Credit Author Statement

Shuo Zhang: Methodology, Investigation, Formal analysis, Data curation, Writing-original draft. Yufeng Liu: Investigation, Formal analysis. Jinming Hu: Formal analysis. Cheng Liu: Formal analysis. Mengmeng Li: Writing – review & editing. Guangyong Zhao: Conceptualiztion, Funding acquisition, Methodology, Writing – review & editing.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work.

Appendix A. Supplementary data

The following is the Supplementary data to this article: