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Impact of dietary supplementation with β-alanine on the rumen microbial crude protein supply, nutrient digestibility and nitrogen retention in beef steers elucidated through sequencing the rumen bacterial community
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Jinming Hu1, Shuo Zhang1, Mengmeng Li, Guangyong Zhao*
Animal Nutrition | 2024, 17(1) : 418 - 427
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Animal Nutrition | 2024, 17(1): 418-427
• Original Research Article •
Impact of dietary supplementation with β-alanine on the rumen microbial crude protein supply, nutrient digestibility and nitrogen retention in beef steers elucidated through sequencing the rumen bacterial community
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Jinming Hu1, Shuo Zhang1, Mengmeng Li, Guangyong Zhao*
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  • State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
doi: 10.1016/j.aninu.2024.02.006
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This study investigated the effects of β-alanine (β-Ala) on rumen fermentation, nutrient digestibility, nitrogen (N) metabolism, plasma biochemical parameters, and rumen bacterial communities in beef steers. Six steers with initial liveweight of 252.8 ± 5.2 kg and 3 treatments of supplementing with 0, 30, or 60 g β-Ala per day to basal diet were allocated in a replicated 3 × 3 Latin square design. Each experimental period was 20 d, of which the first 15 d were for adaptation and the subsequent 5 d were for sampling. The results showed that β-Ala linearly increased the ruminal concentration of microbial crude protein (MCP) (P = 0.005), but it did not affect the ruminal concentrations of ammonia N and total volatile fatty acids (P > 0.10). β-Ala also linearly increased the dry matter (DM) (P = 0.009), organic matter (OM) (P = 0.017) and crude protein (CP) (P = 0.043) digestibility, tended to decrease the acid detergent fiber digestibility (P = 0.077), but it did not affect the neutral detergent fiber digestibility (P = 0.641). β-Ala quadratically increased the relative abundance of ruminal Bacteroidota (P = 0.021) at the phylum level, and increased Prevotella (P = 0.028) and Prevotellaceae_UCG-003 (P = 0.014), and decreased the relative abundance of NK4A214_group (P = 0.009) at the genus level. Feeding steers with β-Ala linearly increased the urinary N (P = 0.006), urea excretions (P = 0.002) and the N retention (P = 0.004), but it did not affect the N utilization efficiency (P = 0.120). β-Ala quadratically increased the plasma concentration of the total antioxidant capacity (P = 0.011) and linearly increased the plasma concentration of insulin-like growth factor-1 (P < 0.001). In summary, dietary supplementation with β-Ala improved the rumen MCP supply and increased the digestibility of DM, OM, CP and the N retention. Further research is necessary to verify the ruminal degradability of β-Ala and to investigate the mechanism of the impact of absorbed β-Ala on the anti-oxidative ability in steers.

β-Alanine  /  Cattle  /  Nitrogen metabolism  /  Rumen bacterial community  /  Rumen fermentation
Jinming Hu, Shuo Zhang, Mengmeng Li, Guangyong Zhao. Impact of dietary supplementation with β-alanine on the rumen microbial crude protein supply, nutrient digestibility and nitrogen retention in beef steers elucidated through sequencing the rumen bacterial community[J]. Animal Nutrition, 2024 , 17 (1) : 418 -427 . DOI: 10.1016/j.aninu.2024.02.006
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1.　Introduction
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β-Alanine (β-Ala) is the only naturally occurring β-amino acid (AA) that exists in many feedstuffs such as whole corn silage (Bai et al., 2021) and Medicago truncatula (Broeckling et al., 2005). β-Ala differs from α-Ala (L-Ala) in that its amino group is located at the beta position of the carboxyl carbon and lacks a chiral center. Although β-Ala and L-Ala differ in the position of only one amino group, the roles they play exhibit significant disparities (Wu, 2009). L-Ala can be directly used for protein synthesis whereas β-Ala serves as a non-proteinogenic AA (Griffith, 1986). In the animal body, however, β-Ala can be used for the synthesis of carnosine, which can improve the activity of glycine-N-methyltransferase in the kidney of mice, and consequently regulate the ratio of S-adenosylmethionine to S-adenosylhomocysteine in the liver (Liu et al., 2020). S-adenosylmethionine acts as a methyl donor for AA and protein synthesis in animals such as mice and dairy cows (Abbasi et al., 2018; Chu et al., 2013). Based on the results of previous studies, it was hypothesized that β-Ala would impact the nitrogen (N) metabolism of animals. To date, however, no reports are available in this aspect.
A previous study showed that supplementing with β-Ala decreased the taurine levels of gastrocnemius muscle and brain in rats (Shaffer and Kocsis, 1981), whereas another study indicated that dietary addition of taurine at 0.2% increased the protein conversion rate, N retention, and N utilization efficiency (NUE) in rats (Liu et al., 1996). The results of the two experiments suggested that supplementing with β-Ala would possibly decrease the N retention and the NUE in rats. In broilers, however, adding β-Ala at 0.5% in diets reduced the feed intake whereas it did not affect the average daily gain (ADG), resulting in an improved feed conversion ratio (Lackner et al., 2021). The results suggested that β-Ala improved the nutrient digestibility and the NUE in broilers. Apparently the results of these experiments on the effects of β-Ala on the nutrient utilization, especially on the N metabolism of animals, are inconsistent. As β-Ala exists in many feeds such as whole corn silage (Bai et al., 2021) and soybeans (Islam et al., 2023) which are the main ingredients of cattle diets, it is necessary to investigate the effects of β-Ala on the nutrient digestibility, NUE, and growth performance in steers and clarify the inconsistency of the results of the previous studies.
The experiment aimed to study the effects of dietary supplementation with β-Ala on the rumen fermentation, nutrient digestibility, and N metabolism in beef steers and verify the mechanisms through sequencing the rumen bacterial community.
2.　Materials and methods
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2.1.　 Animal ethics
The procedures of the experiment were approved by China Agricultural University Laboratory Animal Welfare and Animal Experimental Ethical Inspection Committee (AW82803202-2-1).
2.2.　 Animals and dietary treatments
Six Simmental steers with an initial liveweight of 252.8 ± 5.2 kg were used as experimental animals. Three levels of β-Ala (Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China), i.e. 0, 30 and 60 g/d (equivalent to 0%, 0.73% and 1.45% DM, respectively), were supplemented in a basal diet (Table 1), as experimental treatments. Since no references on the suitable supplementation level of β-Ala in steers are available, the supplemental levels of β-Ala in the present experiment referred to that in broilers (0.5%; Lackner et al., 2021) as the basis. Considering β-Ala could possibly be hydrolyzable in the rumen of steers, the actual supplementation levels were higher than that for broilers. The steers and experimental treatments were assigned in a replicated 3 × 3 Latin square design. Each experimental period included a 15-d adaptation phase and a subsequent 5-d sampling phase.
The animals were tethered and housed in individual pens. Each animal was fed with a total mixed ration (TMR), consisting of 2.35 kg whole corn silage and 1.74 kg of concentrated mixture on dry matter (DM) basis daily. The diet could supply about 90% of the ad libitum DM intake of the animals measured in a pre-trial. No feed residuals were left during the whole experiment. The daily amounts of TMR and β-Ala to each steer were equally divided into two portions and supplied to each steer at 07:00 and 17:00, respectively. β-Ala was mixed well with the TMR before feeding. The animals had free access to fresh drinking water. The body weights of the animals were recorded before morning feeding on d 1 and d 20 of each experimental period.
2.3.　 Sampling
During each sampling period, the total feces from each steer were collected and weighed daily. An amount of 200 g feces was sampled from each steer daily after the feces were homogenized. An aliquot of 10 mL H2SO4 (10%, vol/vol) was added to each fecal sample to avoid the N loss. The total urine from each steer was also collected daily using a rubber funnel connected through polyethylene tubing to a plastic bucket in a polystyrene container, surrounded by ice packs. The volume of urine from each steer was recorded daily and well mixed, then 1% of the urine was taken as the sample and added with 10 mL of H2SO4 (10%, vol/vol). Feed samples were collected during each sampling period. All samples were kept in a freezer at –20 °C.
On d 3 of each sampling period, the rumen fluid was collected from each animal using an esophagus tube prior to morning feeding. The first tube of rumen fluid was discarded. Then the second tube of rumen fluid of about 100 mL was taken as the sample. The pH of rumen fluid was immediately measured using a pH meter (PHS-3C; Shanghai Yueping Scientific Instrument Co., Ltd., Shanghai, China). Then the rumen fluid was filtered through four layers of surgical gauze and dispensed into three 15-mL centrifuge tubes and two 5-mL cryopreservation tubes. The rumen fluid samples in 15-mL centrifuge tubes were kept in a freezer at –20 °C for analyzing rumen fermentation parameters, and the rumen fluid samples in 5-mL cryopreservation tubes were stored in liquid N for analyzing the rumen bacterial community.
On d 4 of each sampling period before feeding in the morning, 20 mL blood was taken through the jugular vein of each steer using an anticoagulant tube containing sodium heparin (Greiner Bio-one, Germany). The plasma samples were obtained after the blood samples were centrifuged at 3000 × g at 4 °C for 15 min. The plasma samples were dispensed into 2-mL cryopreservation tubes and kept in liquid N.
2.4.　 Chemical analyses
The corn silage and feces samples were freeze-dried for 72 h in a freeze-dryer (LGJ-12; Beijing Songyuan Huaxing Technology Development Co., Ltd., Beijing, China). The dried fecal samples from each steer during each sampling period were compounded to obtain a fecal sample based on the daily output of feces in proportion. The daily urine samples of each steer were directly mixed to obtain a compound sample. The freeze-dried feces samples were crushed with a pestle and a mortar, and the feed samples were ground to pass through a sieve with a pore size of 1 mm.
The DM and crude ash of feeds and feces were analyzed according to the procedures of AOAC (2005) using methods No. 930.15 and 942.05, respectively. The organic matter (OM) content of the feeds and feces was calculated by DM subtracting crude ash content. The total N of feeds, urine, and feces was analyzed using the Kjeldahl method according to AOAC (2005) using method No. 984.13. The neutral detergent fiber (NDF) of feeds and feces were analyzed using the procedures described by Van Soest et al. (1991) with heat-stable α-amylase as well as sodium sulfite and without correction for residual ash as suggested by Mertens et al. (2002). Acid detergent fiber (ADF) was analyzed according to AOAC (2005) using method No. 973.18 on an Ankom A200i fiber analyzer (Ankom Technology, Macedon, NY, USA). The gross energy (GE) of feeds was analyzed on a Parr 6400 automatic oxygen bomb calorimeter (Parr Instrument Company, Moline, IL, USA).
The ammonia nitrogen (NH3–N) concentration of rumen fluid was analyzed using the colorimetric method of Broderick and Kang (1980). The microbial crude protein (MCP) concentration of rumen fluid was analyzed using the colorimetric method of Makkar (1982) on a spectrophotometer (UV-1801; Beijing Rayleigh Analytical Instruments Co., Ltd., Beijing, China). The concentrations of volatile fatty acids (VFA) of rumen fluid were determined on a gas chromatograph (GC-8600; Beijing Beifen Tianpu Instrument Technology Co., Ltd., Beijing, China) using the procedures described by Yang et al. (2017).
The allantoin and uric acid of urine samples were analyzed based on the methods of Chen and Gomes (1992). The urinary hippuric acid, urea, and creatinine of urine samples were analyzed according to China National Hygiene Standard (WS/T 52-1996), the diacetyl monoxime method (Friedman, 1953), and Jaffe's method (Yatzidis, 1974), respectively, on a spectrophotometer (UV-1801; Beijing Rayleigh Analytical Instruments Co., Ltd.).
The total protein, albumin, triglyceride, urea, total antioxidant capacity (T-AOC), growth hormone (GH), and insulin-like growth factor-1 (IGF-1) of plasma samples were analyzed using commercial kits (Beijing Sinouk Institute of Biological Technology, Beijing, China) on an automatic biochemical analyzer (HY-60021; Beijing Sino-UK Institute of Biological Technology, Beijing, China). The globulin of plasma samples was calculated by total protein minus albumin.
2.5.　 β-Ala analysis
The β-Ala of urine and rumen fluid were analyzed using the method of Zhang et al. (2008) with HPLC (LC98-1; Beijing Wenfen Analytical Instrument Development Co., Ltd., Beijing, China) using a HyperClone BDS C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase of acetonitrile-0.05 mol/L NaH2PO4 solution (20:80, vol/vol; pH 6.5) at a flow rate of 1.0 mL/min. The wave length was 360 nm and the column temperature was 30 °C for detection. The samples of urine and rumen fluid were pretreated as follows. A 10-mL brown volumetric flask was used to mix 1 mL urine or rumen fluid sample, 0.5 mL of NaH2PO4 (0.05 M; pH 8.0) and 1 mL 2,4-dinitrofluorobenzene (DNFB) acetonitrile (0.5%, vol/vol). Then, the mixed solution was derivatized for 30 min at 60 °C in a water bath. After the mixture cooled down, ultrapure water was added to the flask to the scale line and mixed well. Then the mixture was centrifuged at 6000 × g for 5 min at 4 °C. The supernatant was collected and passed through a filter membrane with a pore size of 0.45 μm for further analysis.
2.6.　 DNA extraction, PCR amplification, and rumen bacteria sequencing
The Fast DNA SPIN Kit for Soil (MP Biomedicals, Solon, USA) was used to extract the DNA of rumen bacteria. The quality of extracted DNA was assessed using 1% agarose gel. The NanoDrop 2000 UV–Vis spectrophotometer (Thermo Scientific, Wilmington, USA) was applied to determine the concentration and the purity of the extracted DNA.
The hypervariable region V3 to V4 of the bacterial 16S rRNA gene was amplified using the primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR amplification was performed using the ABI GeneAmp 9700 PCR thermocycler (ABI, CA, USA) with TransStart FastPfu DNA Polymerase (AP221-02; TransGen Biotech, Beijing, China), a 20 μL reaction system, 0.8 μmol/L forward and reverse primers, and approximately 10 ng template DNA. The PCR amplification procedures included an initial denaturation at 95 °C for 3 min (1 cycle), then 30 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 45 s, and a final extension at 72 °C for 10 min. Based on the instructions of Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), the purified amplicons were pooled equimolarly and subjected to paired-end sequencing on a NovaSeq PE250 platform (Illumina, San Diego, USA).
The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered using fastp software (version 0.20.0; Chen et al., 2018), and then merged using FLASH software (version 1.2.7; Magoč and Salzberg, 2011) with the following criterias: (1) Reads with an average quality score less than 20 over a 50-bp sliding window were truncated. The reads shorter than 50 bp after truncation or reads containing ambiguous characters were discarded; (2) Overlapping sequences longer than 10 bp were assembled based on their overlaps, with a maximum mismatch ratio of 0.2 in the overlap region. Reads that failed to assemble were discarded; (3) Samples were identified based on their barcodes and primers, and the sequence direction was adjusted accordingly, allowing for exact barcode matching and a 2 nucleotide mismatch in primer matching.
UPARSE version 7.1 (Edgar, 2013; Stackebrandt and Goebel, 1994) was used to generate the operational taxonomic units (OTU) with a 97% similarity cutoff and the chimeric sequences were removed. The taxonomy of each representative sequence of OTU was determined using RDP Classifier version 2.2 (Wang et al., 2007) against the 16S rRNA database with a confidence threshold of 0.7.
2.7.　 Calculations and statistical analysis
The rumen microbial N (MN) supply was estimated using the daily urinary excretions of total urinary purine derivatives (PD) according to the methods of Chen and Gomes (1992).
where X (mmol/d) represents the excretion of absorbed PD; BW0.75 (kg) represents the metabolizable live weight of the steer; 0.85 represents the recovery of absorbed purine as PD in urine; 0.385 (mmol/kg BW0.75 per day) represents the endogenous excretion of PD.
where X (mmol/d) represents the PD absorption; 70 (mg N/mmol) represents the purine N content; 0.116 represents the ratio of purine N to total mixed rumen microbial N; 0.83 represents the rumen microbial purine digestibility.
The data were statistically analyzed using the mixed linear model in SAS 9.4 (SAS Inst. Inc., Cary, NC, USA) as follows:
where Yijk is the dependent variable; μ is the overall mean; Ti is the fixed treatment effect; Pj is the period effect; Sk is the steer effect; eijk is random error.
The linear and quadratic effects were evaluated using the CONTRAST procedure in SAS 9.4. The Duncan method was used for multiple comparisons of the mean values among treatments. P ≤ 0.05 was declared as significant difference and 0.05 < P ≤ 0.10 as a tendency.
The correlations between the distance-corrected dissimilarity of the domain genera and the nutrient digestibility were evaluated using the Mantel test using the linkET package in R (version 3.5.1).
3.　Results
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3.1.　 Rumen fermentation parameters
Table 2 indicates that supplementing with β-Ala increased the ruminal concentrations of MCP (P = 0.005) and β-Ala (P = 0.003). No significant differences were found in the ruminal pH, the concentrations of total VFA and individual VFA, the ratio of acetate to propionate, and the NH3–N concentration (P > 0.10).
3.2.　 Apparent nutrient digestibility
Table 3 shows that supplementing with β-Ala linearly increased the DM (P = 0.009), OM (P = 0.017), and CP (P = 0.043) digestibility, tended to decrease the ADF digestibility (P = 0.077), but it did not affect the NDF digestibility (P = 0.641).
3.3.　 N balance, urine nitrogenous compounds and ADG
Table 4 indicates that supplementing with β-Ala linearly increased the urinary urea excretion (P = 0.006), the ratios of urinary urea N to N intake (P = 0.015) and urinary urea N to total N excretion (P = 0.002). β-Ala did not affect the urinary excretions of uric acid (P = 0.656), allantoin (P = 0.453), and hippuric acid (P = 0.839), but linearly decreased the ratios of uric acid N (P = 0.016), allantoin N (P = 0.049), and hippuric acid N (P = 0.007) to total urinary N and tended to decrease the ratio of creatinine N to urinary N (P = 0.077). β-Ala also linearly increased the urinary excretion of the PD (P = 0.001) and consequently linearly increased the estimated rumen MN supply (P = 0.048). β-Ala was undetectable in the urine samples.
Table 5 shows that supplementing with β-Ala linearly decreased the fecal N excretion (P = 0.019) and increased the urinary N excretion (P = 0.006) and the total N excretion (P = 0.019). It also decreased the ratio of fecal N to urinary N (P = 0.007) and increased the N retention (P = 0.004) in a linear manner. However, β-Ala did not affect the NUE (P = 0.277) and the ADG (P = 0.430).
3.4.　 Plasma biochemical parameters
Supplementing with β-Ala did not affect the total protein (P = 0.446), globulin (P = 0.567), and triglyceride (P = 0.408) concentrations in plasma, but linearly increased the urea (P = 0.018) and IGF-1 (P < 0.001) concentrations and linearly tended to increase the GH (P = 0.084) and quadratically tended to increase the albumin (P = 0.058) and T-AOC concentrations (P = 0.011) in plasma (Table 6).
3.5.　 Richness and diversity of the rumen bacterial community
A total of 1,315,676 clean reads were detected with over 73,093 for each sample. The Chao1 and Shannon indices were used to generate the rarefaction curves to assess the adequacy of the sampling depth. The rarefaction curves indicated a diminishing rate of clean reads identification as the number of reads per sample increased. With all rarefaction curves of clean reads tending to plateau at 45,000 tags, the sequencing depth and the amount were saturated (Figs. S1A and B). Similarly, the Good's coverage of all samples exceeded 98%, indicating the high accuracy and reproducibility of the sequencing. The indices of Chao1 and Shannon were used to present the richness and the diversity of bacteria (Table S1). The nonmetric multidimensional scaling analysis (NMDS) plots were performed based on the Bray Curtis distance. The clearly clustered bacterial communities indicated distinct structures among treatments (Fig. S2).
3.6.　 Composition of the rumen bacterial community
The taxonomic analysis of the reads revealed that there were more than 9 bacterial phyla in the rumen fluid. The dominant phyla were Bacteroidota and Firmicutes, accounting for 55.03% and 38.75% of total reads, respectively (Fig. 1A). Among these phyla, supplementing with β-Ala quadratically increased the relative abundance of Bacteroidota (P = 0.021) (Table 7). At the genus level, 9 genera with high relative abundance were identified in the rumen fluid. The top genera were Prevotella (18.85%), Rikenellaceae_RC9_gut_group (15.16%), norank_f__F082 (5.66%), and Ruminococcus (5.29%) (Fig. 1B). Table 7 shows that β-Ala exerted a pronounced impact on the dominant bacteria at the genus level, leading to a quadratic change in the relative abundance of Prevotella (P = 0.028), NK4A214_group (P = 0.009), and Prevotellaceae_UCG-003 (P = 0.014).
3.7.　 Biomarker taxa of the rumen bacterial community
Linear discriminant analysis (LDA) effect size (LEfSe) was used to integrate the rank sum tests with taxonomic data, allowing for the identification of biomarker taxa (with a logarithmic LDA score ≥ 3.0) that significantly influenced the structure of the rumen bacterial community. The LDA histogram (Fig. 2) indicates the presence of a total of 20 biomarkers from the phylum level to the genus level, of which 5, 1 and 14 biomarkers were detected for the groups supplementing with 0, 30 and 60 g β-Ala/d, respectively, as follows: o__Lachnospirales, f__Lachnospiraceae, g__Acetitomaculum, g__Defluviitaleaceae_UCG-011, f__Defluviitaleaceae; g__Prevotellaceae_UCG-003; f__Oscillospiraceae, f__Bacteroidales_RF16_group, g__norank_f__Bacteroidales_RF16_group, c__Coriobacteriia, o__Coriobacteriales, g__Sediminispirochaeta, g__GCA-900066575, g__hoa5-07d05_gut_group, g__norank_f__Coriobacteriales_Incertae_Sedis, f__Coriobacteriales_Incertae_Sedis, g__Lachnoclostridium, f__Atopobiaceae, g__norank_f__Atopobiaceae, g__Atopobium, where prefix c represents the Class, o represents the Order, f represents the Family, and g represents the Genus.
3.8.　 Correlations between the bacterial genera and the nutrient digestibility
The Mantel test shows that positive correlations were found between the relative abundance of Prevotellaceae UCG-003 and the DM digestibility (P < 0.05, 0.1 < r < 0.3), the OM digestibility (P < 0.05, 0.1 < r < 0.3), and the CP digestibility (P < 0.05, r > 0.3). A positive correlation was also found between the relative abundance of Prevotella and the CP digestibility (P < 0.05, r > 0.3) (Fig. 3).
4.　Discussion
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In ruminants, dietary CP can be extensively degraded into peptides, AA, and NH3 by ruminal microorganisms. Ruminal NH3–N can be used for MCP synthesis, absorbed into blood and moved into the lower digestive tract with digesta. As a free AA, L-Ala is highly hydrolyzable in rumen fermentation (Sok et al., 2017). Since β-Ala and L-Ala are only slightly different in molecular structure, it could be speculated that β-Ala could be highly hydrolyzed by ruminal microorganisms to NH3 and CO2. However, the results of the present experiment showed that supplementing with β-Ala did not affect the ruminal concentration of NH3–N. One reason for the results could be that the rumen fluid was taken before morning feeding while another reason could be that part of the NH3–N from β-Ala was utilized for ruminal MCP synthesis. The results of the present experiment also showed that supplementing with β-Ala increased the ruminal MCP concentration and MN supply predicted based on the urinary excretion of PD. Since β-Ala is a non-proteinogenic AA, the increased ruminal MCP should be synthesized through utilizing the NH3–N released from β-Ala.
In ruminants, the absorbed NH3 from the rumen into the blood can be used for urea synthesis in the liver (Reynolds and Kristensen, 2008). Part of the urea in the blood can go back into the rumen through saliva secretion and across the rumen epithelium, while part of the urea can be excreted into urine (Zhao, 2019). Urinary urea excretion is positively correlated with plasma urea concentration (Calsamiglia et al., 2010) and urinary urea accounts for a major part of the urinary nitrogenous compounds in cattle (Gao et al., 2021). The results of the present experiment showed that supplementing with β-Ala increased the plasma urea concentration and urinary excretion of urea, suggesting that part of the NH3 degraded from β-Ala was absorbed into the blood and utilized for urea synthesis.
The results of the present experiment also showed that dietary supplementation with β-Ala linearly decreased the fecal N excretion and increased the urinary N excretion. The decreased fecal N excretion could be resulted from the increased rumen MCP synthesis because rumen MCP has a more balanced AA composition and can be more easily digestible and utilizable than feed CP (Storm et al., 1983b; Storm and Ørskov, 1983a) while the increased urinary N excretion could be due to the increased urinary excretion of urea.
Qi et al. (2018) reported that dietary supplementation with 1000 mg/kg β-Ala for broilers increased the ADG and elevated the β-Ala content in breast muscle. Wang et al. (2022) reported that dietary addition of β-Ala at 600 mg/kg increased the lean meat percentage and decreased the backfat thickness in finishing pigs. Although the results of the two studies suggested that β-Ala had beneficial effects on the growth performance of broilers and pigs, no direct evidence was available on the impacts of β-Ala on the N retention of broilers and pigs. The results of the present experiment showed that adding β-Ala at 30 and 60 g/d increased the N intakes of the two treatments by 4.92% and 9.84%, respectively, whereas it increased the N retention by 9.97% and 19.94%, respectively. Apparently, the increased N retention rates were higher than the increased N intake rates. The results suggested that β-Ala not only could be used as an N source but also as a potential promoter for N utilization. It should be noted, however, that supplementing with β-Ala did not affect the NUE in steers. The reason for the results could be that a considerable part of β-Ala was hydrolyzed in rumen fermentation. Feeding experiments with higher level of β-Ala or rumen-protected β-Ala need to be conducted to verify the effects.
The present experiment showed that supplementing with β-Ala did not affect the richness and the diversity (Chao1 and Shannon index) of the rumen bacterial community in steers. At the phylum level, the dominant ruminal bacteria were found to be Bacteroidota and Firmicutes. These results were consistent with Zeineldin et al. (2018) who indicated that the predominant phyla present in the rumen are Firmicutes and Bacteroidetes. Supplementing with β-Ala quadratically increased the relative abundance of rumen Bacteroidota and linearly increased the CP digestibility. Since the phylum Bacteroidota were able to improve feed protein degradation by producing aminopeptidases in the rumen (Hernandez et al., 2022), the linearly increased nutrient digestibility of DM, OM, and CP could be partly attributed to the increased relative abundance of Bacteroidota.
At the genus level, Prevotella are the most abundant ruminal bacteria in the Bacteroidetes group. The results of the present experiment showed that the main ruminal bacteria were Prevotella and Rikenellaceae_RC9_gut_group for all groups and supplementing with β-Ala quadratically increased the relative abundances of Prevotella and Prevotellaceae_UCG-003 and reduced the relative abundance of NK4A214_group. Since Prevotella are able to improve the feed protein degradation in rumen fermentation (Liu et al., 2019), the increased CP digestibility should be partly due to the increased relative abundance of Prevotella and Prevotellaceae_UCG-003 by the impact of β-Ala. The speculation was confirmed by the Mantel test analysis which showed that the digestibility of DM, OM or CP was positively correlated with the relative abundance of Prevotella and Prevotellaceae_UCG-003 in the rumen.
In pigs, it was reported that Ruminococcaceae_NK4A214_group are the predominant genera of gut microbiota to improve the apparent total tract digestibility of ADF (Niu et al., 2022). The results of the present experiment showed that supplementing with β-Ala decreased the relative abundance of ruminal NK4A214_group and tended to decrease the ADF digestibility. The impact of β-Ala on decreasing the relative abundance of NK4A214_group could be one of the reasons for the tendency to decrease the ADF digestibility.
In the present experiment, supplementing with β-Ala increased the plasma concentration of IGF-1. The results are consistent with Pence et al. (2016) who reported that feeding 17-month-old mice with 3.43 mg/kg β-Ala for 41 d increased the plasma concentrations of IGF-1. However, the mechanism by which β-Ala stimulates the production of IGF-1 remains unclear.
β-Ala can be used for carnosine synthesis in many species of animals such as poultry, pigs and rats (Boldyrev et al., 2013). One of the important functions of carnosine is the antioxidative effect (Wu, 2020). It was reported that supplementing with β-Ala at 600 mg/kg to weaned piglets increased the superoxide dismutase (SOD) activity in the jejunum (Chen et al., 2023), and feeding 1200 mg/kg of β-Ala to 1-d-old pullets for 42 d increased the T-AOC content in the breast muscle (Qi et al., 2018). The present experiment showed that supplementing steers with β-Ala quadratically increased the plasma concentration of T-AOC, suggesting that part of the absorbed β-Ala was used for carnosine synthesis, resulting in the increased plasma T-AOC concentration.
5.　Conclusion
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Dietary supplementation with β-Ala linearly increased the DM, OM and CP digestibility but tended to decrease the ADF digestibility. Supplementing with β-Ala also linearly increased MN supply and improved the N retention but did not affect the NUE in beef steers. β-Ala could be highly hydrolyzable in rumen fermentation, but further research is needed to verify this speculation.
References
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Abbasi I, Abbasi F, Wang L, Abd EHM, Swelum AA, Hao R, Yao J, Cao Y. Folate promotes s-adenosyl methionine reactions and the microbial methylation cycle and boosts ruminants production and reproduction. AMB Express 2018;8(1):65. https://doi.org/10.1186/s13568-018-0592-5.
AOAC. Official methods of analysis. 18th ed. Washington, DC: AOAC International; 2005.
Bai C, Wang C, Sun L, Xu H, Jiang Y, Na N, Yin G, Liu S, Xue Y. Dynamics of bacterial and fungal communities and metabolites during aerobic exposure in wholeplant corn silages with two different moisture levels. Front Microbiol 2021;12:663895. https://doi.org/10.3389/fmicb.2021.663895.
Boldyrev AA, Aldini G, Derave W. Physiology and pathophysiology of carnosine. Physiol Rev 2013;93(4):1803-45. https://doi.org/10.1152/physrev.00039.2012.
Broderick GA, Kang JH. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J Dairy Sci 1980;63(1):64-75. https://doi.org/10.3168/jds.S0022-0302(80)82888-8.
Broeckling CD, Huhman DV, Farag MA, Smith JT, May GD, Mendes P, Dixon RA, Sumner LW. Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism. J Exp Bot 2005;56(410):323-36. https://doi.org/10.1093/jxb/eri058.
Calsamiglia S, Ferret A, Reynolds CK, Kristensen NB, van Vuuren AM. Strategies for optimizing nitrogen use by ruminants. Animal 2010;4(7):1184-96. https://doi.org/10.1017/S1751731110000911.
Chen XB, Gomes MJ. Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivatives - an overview of Technical details. Occasional Publication. International Feed Resources Unit, Rowett Research Institute; 1992.
Chen S, Zhou Y, Chen Y, Gu J. FASTP: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018;34(17):i884-90. https://doi.org/10.1093/bioinformatics/bty560.
Chen L, Zhong Y, Ouyang X, Wang C, Yin L, Huang J, Li Y, Wang Q, Xie J, Huang P, Yang H, Yin Y. Effects of β-alanine on intestinal development and immune performance of weaned piglets. Anim Nutr 2023;12:398-408. https://doi.org/10.1016/j.aninu.2022.10.008.
China National Hygiene Standard. Urine-determination of hippuric acid spectrophotometric method (WS/T 52-1996). Beijing: China Standards Press; 1996.
Chu J, Qian J, Zhuang Y, Zhang S, Li Y. Progress in the research of s-adenosyl-lmethionine production. Appl Microbiol Biotechnol 2013;97(1):41-9. https://doi.org/10.1007/s00253-012-4536-8.
Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 2013;10(10):996-8. https://doi.org/10.1038/nmeth.2604.
Feng Y. The Nutrient Requirements and Feeding Standards of Beef Cattle. Beijing: China Agricultural University Press; 2000.
Friedman HS. Modification of determination of urea by diacetyl monoxime method. Anal Chem 1953;25(4):662-4. https://doi.org/10.1021/ac60076a040.
Gao J, Cheng B, Liu Y, Li MM, Zhao G. Dietary supplementation with red cabbage extract rich in anthocyanins increases urinary hippuric acid excretion and consequently decreases nitrous oxide emissions in beef bulls. Anim Feed Sci Technol 2021;281:115075. https://doi.org/10.1016/j.anifeedsci.2021.115075.
Griffith OW. Beta-amino acids: mammalian metabolism and utility as alpha-amino acid analogues. Annu Rev Biochem 1986;55:855-78. https://doi.org/10.1146/annurev.bi.55.070186.004231.
Hernandez R, De Mares MC, Jimenez H, Reyes A, Caro-Quintero A. Functional and phylogenetic characterization of bacteria in bovine rumen using fractionation of ruminal fluid. Front Microbiol 2022;13:813002. https://doi.org/10.3389/fmicb.2022.813002.
Islam N, Krishnan HB, Slovin J, Natarajan S. Metabolic profiling of a fast neutron soybean mutant reveals an increased abundance of isoflavones. J Agric Food Chem 2023;71(26):9994-10003. https://doi.org/10.1021/acs.jafc.3c01493.
Lackner J, Albrecht A, Mittler M, Marx A, Sauerwein H. Effect of feeding histidine and β-alanine on carnosine concentration, growth performance and meat quality of broiler chickens. Poult Sci 2021;100:101393. https://doi.org/10.1016/j.psj.2021.101393.
Liu XJ, Li YS, Shen LF. Effect of taurine on protein utilization, growth and cognitive ability in rats. Acta Nutr Sin 1996;18(2):149-54 [in Chinese].
Liu H, Xu T, Xu S, Ma L, Han X, Wang X, Zhang X, Hu L, Zhao N, Chen Y, Pi L, Zhao X. Effect of dietary concentrate to forage ratio on growth performance, rumen fermentation and bacterial diversity of Tibetan sheep under barn feeding on the Qinghai-Tibetan plateau. PeerJ 2019;7:e7462. https://doi.org/10.7717/peerj.7462.
Liu XQ, Jiang L, Lei L, Nie ZY, Zhu W, Wang S, Zeng HX, Zhang SQ, Zhang Q, Yard B, Wu YG. Carnosine alleviates diabetic nephropathy by targeting GNMT, a key enzyme mediating renal inflammation and fibrosis. Clin Sci 2020;134(23):3175-93. https://doi.org/10.1042/CS20201207.
Magoč T, Salzberg SL. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011;27(21):2957-63. https://doi.org/10.1093/bioinformatics/btr507.
Makkar HP, Sharma OP, Dawra RK, Negi SS. Simple determination of microbial protein in rumen liquor. J Dairy Sci 1982;65(11):2170-3. https://doi.org/10.3168/jds.S0022-0302(82)82477-6.
Mertens DR. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: collaborative study. J AOAC Int 2002;85(6):1217-40. http://refhub.elsevier.com/S2405-6545(24)0004-1/sref25.
Niu Q, Pu G, Fan L, Gao C, Lan T, Liu C, Du T, Kim SW, Niu P, Zhang Z, Li P, Huang R. Identification of gut microbiota affecting fiber digestibility in pigs. Curr Issues Mol Biol 2022;44(10):4557-69. https://doi.org/10.3390/cimb44100312.
Pence BD, Gibbons TE, Bhattacharya TK, Mach H, Ossyra JM, Petr G, Martin SA, Wang L, Rubakhin SS, Sweedler JV, McCusker RH, Kelley KW, Rhodes JS, Johnson RW, Woods JA. Effects of exercise and dietary epigallocatechin gallate and β-alanine on skeletal muscle in aged mice. Appl Physiol Nutr Metab 2016;41(2):181-90. https://doi.org/10.1139/apnm-2015-0372.
Qi B, Wang J, Ma YB, Wu SG, Qi GH, Zhang HJ. Effect of dietary β-alanine supplementation on growth performance, meat quality, carnosine content, and gene expression of carnosine-related enzymes in broilers. Poult Sci 2018;97(4):1220-8. https://doi.org/10.3382/ps/pex410.
Reynolds CK, Kristensen NB. Nitrogen recycling through the gut and the nitrogen economy of ruminants: an asynchronous symbiosis. J Anim Sci 2008;86(14 Suppl.):E293-305. https://doi.org/10.2527/jas.2007-0475.
Shaffer JE, Kocsis JJ. Taurine mobilizing effects of beta alanine and other inhibitors of taurine transport. Life Sci 1981;28(24):2727-36. https://doi.org/10.1016/0024-3205(81)90173-9.
Storm E, Ørskov ER, Smart R. The nutritive value of rumen micro-organisms in ruminants. 2. The apparent digestibility and net utilization of microbial N for growing lambs. Br J Nutr 1983b;50(2):471-8. https://doi.org/10.1079/bjn19830115.
Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 1991;74(10):3583-97. https://doi.org/10.3168/jds.S0022-0302(91)78551-2.
Sok M, Ouellet DR, Firkins JL, Pellerin D, Lapierre H. Amino acid composition of rumen bacteria and protozoa in cattle. J Dairy Sci 2017;100(7):5241-9. https://doi.org/10.3168/jds.2016-12447.
Stackebrandt E, Goebel BM. A place for DNA-DNA reassociation and 16s ribosomal-RNA sequence-analysis in the present species definition in bacteriology. Int J Syst Bacteriol 1994;44(4):846-9. https://doi.org/10.1099/00207713-44-4-846.
Storm E, Ørskov ER. The nutritive value of rumen micro-organisms in ruminants. 1. Large-scale isolation and chemical composition of rumen micro-organisms. Br J Nutr 1983a;50(2):463-70. https://doi.org/10.1079/bjn19830114.
Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 2007;73(16):5261-7. https://doi.org/10.1128/AEM.00062-07.
Wang F, Yin Y, Wang Q, Xie J, Fu C, Guo H, Chen J, Yin Y. Effects of dietary β-alanine supplementation on growth performance, meat quality, carnosine content, amino acid composition and muscular antioxidant capacity in Chinese indigenous Ningxiang pig. J Anim Physiol Anim Nutr 2022;107(3):878-86. https://doi.org/10.1111/jpn.13797.
Wu G. Amino acids: metabolism, functions, and nutrition. Amino Acids 2009;37(1):1-17. https://doi.org/10.1007/s00726-009-0269-0.
Wu G. Important roles of dietary taurine, creatine, carnosine, anserine and 4-hydroxyproline in human nutrition and health. Amino Acids 2020;52(3):329-60. https://doi.org/10.1007/s00726-020-02823-6.
Yang K, Wei C, Zhao GY, Xu ZW, Lin SX. Effects of dietary supplementing tannic acid in the ration of beef cattle on rumen fermentation, methane emission, microbial flora and nutrient digestibility. J Anim Physiol Anim Nutr 2017;101(2):302-10. https://doi.org/10.1111/jpn.12531.
Yatzidis H. New method for direct determination of “True” creatinine. Clin Chem 1974;20(9):1131-4. https://doi.org/10.1093/clinchem/20.9.1131.
Zeineldin M, Barakat R, Elolimy A, Salem A, Elghandour M, Monroy JC. Synergetic action between the rumen microbiota and bovine health. Microb Pathog 2018;124:106-15. https://doi.org/10.1016/j.micpath.2018.08.038.
Zhang ZB, Gao LJ, Qiu JP. Determination of β-alanine in biocatalysis process. Bul Sci Technol 2008;6(24):779-784+791 [in Chinese].
Zhao GY. Improving feed protein utilization rate in cattle through nutritional approaches. Curr Protein Pept Sci 2019;20(2):164-71. https://doi.org/10.2174/1389203719666180514153236.
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doi: 10.1016/j.aninu.2024.02.006
  • Receive Date：2023-09-22
  • Online Date：2026-01-28
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  • Received：2023-09-22
  • Revised：2024-02-17
  • Accepted：2024-02-29
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    State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

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Corresponding author. E-mail address: (G. Zhao).
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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