Introduction
The rapid expansion of the poultry industry in China is increasingly constrained by the shortage and rising cost of soybean meal, the primary protein source in feed
[1,2]. This situation has prompted significant research into reducing dietary CP levels and identifying alternative strategies that can sustain growth performance and animal health while decreasing soybean meal usage and nitrogen emissions
[3,4]. However, a low-protein diet may impair growth performance in broilers even when supplemented with crystalline amino acids
[5], primarily due to deficiencies in essential amino acids and increased metabolic stress
[6,7]. Furthermore, an imbalance between AME and CP in low-protein formulations may result in fatty liver and abdominal fat accumulation
[8]. Consequently, there is an urgent need for functional feed additives that can mitigate the physiological challenges associated with low-protein diets.
Betaine (trimethylglycine) is a well-established methyl donor that participates in the methionine cycle, contributing labile methyl groups for the synthesis of metabolically active substances such as creatine and carnitine
[9]. Betaine serves as an organic osmolyte, stabilizing cellular hydration and protein conformation under osmotic stress, which protects intestinal epithelial cells and maintains mucosal barrier integrity
[10]. Previous studies have shown that dietary betaine supplementation can improve growth performance
[11], enhance meat quality
[12], and modulate intestinal microbiota in broilers
[13], particularly under heat stress conditions. Moreover, betaine is involved in the synthesis of proteins and the metabolism of fats in animals, while there is a scarcity of research on its utilization under low-protein conditions
[14]. Compared with conventional betaine, betaine phosphate (BETP) may provide both methyl-donor capacity and available phosphorus, suggesting a potential dual role in methyl metabolism and nutrient utilization. However, its biological effects under low-energy and low-protein conditions remain poorly understood. The dose-dependent remodeling of BETP on the ileal microbiota and its functional implications under protein restriction remain uncharacterized.
In our study, dietary energy was reduced alongside crude protein (CP) to maintain a balanced energy-to-protein ratio and prevent excessive lipid deposition, which can occur when low-protein diets are formulated with relatively high energy levels. It was hypothesized that supplementing low-energy and low-protein diets with BETP would improve feed efficiency and carcass performance by modulating host energy metabolism and altering the ileal microbial community. The present study aimed to determine the optimal supplemental level of BETP under low-energy and low-protein conditions and to assess changes in feed efficiency, carcass performance, energy metabolism, and ileal microbiota. The findings from this research will provide reference data and a theoretical basis for the application of dietary BETP under low-energy and low-protein conditions in poultry production.
Materials and methods
Ethics statement
This experiment was carried out with the approval of the China Agricultural University Laboratory Animal Welfare and Experimental Ethical Committee (Approval number AW50206202-1-05) at the experimental farm of China Agricultural University, Hebei Zhuozhou Experimental Base.
Animals, housing, and experimental design
The BETP used in this study was provided by Sunwin Biotech Shandong Co., Ltd., (Weifang, China) and added by replacing the rice husk powder in the basal diet.
A total of 480 healthy male Arbor Acres chicks (initial body weight 42.02 ± 0.53g) were randomly divided into 5 treatment groups, with 8 replicates and 12 birds each. The control group (CON) was fed basal diet (starter, 1-21d, 23.00% CP; grower, 22-42d, 21.50% CP), and experimental groups were fed a low-energy (lower by 0.21 MJ/kg than the control group which is 12.35 MJ/kg AME at starter and 13.19 MJ/kg AME at grower) and low-protein (starter, 21.00% CP; grower, 19.50% CP) (LL) diet and supplemented with 0, 1000, 1500, 2000 mg/kg BETP (LL, BETP1000, BETP1500, and BETP2000), respectively. The entire experimental period was 42 days.
All the broilers were fed in a fully enclosed chicken house with 3-tier battery cages (200cm [length] × 80cm [wide] × 60cm [high]). Feed and water were provided ad libitum throughout the experimental period. The house temperature was initially maintained at 34 ℃ for the first week and was subsequently reduced by 2℃ to 3℃ per week until a final temperature of 23℃ was reached. The lighting program consisted of 23 hours of light and 1 hour of darkness for the first 5 days, followed by 20 hours of light and 4 hours of darkness from day 6 until the end of the trial. The basal diets were pelleted according to the Feeding Standard of Chicken
[15] to meet the nutritional requirements (
Table 1).
Growth performance
The body weight and feed intake of the chicks were recorded after 12 hours of starvation on day1, day10, day21, and day42. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated for the phases of 0-10d, 11-21d, 22-42d, and 1-42d. Mortality was recorded daily, and the European production efficiency factor (EPEF) was calculated at the end of the trial.
Sample collection
At d 14 and d 42, one chick close to the average body weight was selected from each replicate. Blood was collected from the wing vein and centrifuged at 1500 × g for 15 min at 4 ℃ to obtain serum, which was then stored at -20 ℃. Liver and ileum mucosa samples were collected into 2 mL cryotubes, immediately snap-frozen in liquid nitrogen, and stored at -80 ℃. Pectoral muscle samples of approximately 2 cm × 2 cm were excised, fixed, and embedded in optimal cutting temperature compound, and stored at -20 ℃. Ileal digesta samples were collected into 2mL cryotubes, snap-frozen in liquid nitrogen, and stored at -80 ℃.
Carcass performance
At 42 days of age, one chick close to the average body weight was selected and euthanised from each replicate. The carcass cutting operation was carried out according to “Chicken carcass segmentation, GB/T 24864-2010”
[16]. Dressed weight, half-eviscerated weight, eviscerated weight, leg muscle weight, and pectoral muscle weight were measured according to “Performance terminology and measurements for poultry, NY/T 823-2020”
[17], and the corresponding yields were calculated.
Hepatic FGF21 content
The liver samples were accurately weighed and diluted with normal saline at a 1: 9 (w/v) ratio to make a 10% tissue homogenate. The contents of FGF21 (ml941205) were determined by assay kits purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China).
Serum antioxidant enzyme and gut barrier
The contents of GSH-Px (A005-1-2), SOD (A001-1-2), MDA (A003-1-2), and T-AOC (A015-1-2) were determined by assay kits purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, Jiangsu, China). The contents of DLA (MC827L) and DAO (ml036981) were determined by ELISA kits purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China).
RNA isolation and real-time polymerase chain reaction
Total RNA was extracted from the ileum and liver tissues using AIPzol reagent (RE205-02, I-presci Scientific Co., Ltd., Beijing, China) according to the manufacturer’s instructions at the end of the trial, and RNA purity and concentration were determined using a spectrophotometer (ND5000, Thermo Fisher Scientific Co., Ltd., Portland, OR, USA). Reverse transcription was performed using UltraScript First-Strand cDNA Synthesis RT-PCR Kit (R317-02, I-presci Co., Ltd., Beijing, China). Real-time quantitative PCR was carried out with an Applied Biosystems 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA) and AipMix 2× SYBR Green qPCR SuperMix (FP308-02, I-presci Scientific Co., Ltd., Beijing, China). The results were normalized to
β-actin, and the relative gene expression levels were calculated via the 2
-ΔΔCT method. The primer sequences for target genes are given in
Table 2.
Histomorphology
The fixed breast muscles were cut at 8 μm along the vertical direction of the muscle fibers, and subsequently mounted on slides. The tissue slides were stained with H＆E after fixing in a 4% paraformaldehyde solution. For each sample, three non-overlapping fields of view were randomly captured at 200 × magnification using a light microscope. ImageJ software 1.51h (National Institutes of Health, Bethesda, MD, USA) was employed to quantify the areas and numbers of pectoral muscle fibers. The number of muscle fibers was quantified by counting all intact fibers within each field and then expressed as fiber density, and the cross-sectional area was calculated by the software after tracing the fiber boundaries. The average values from the three fields per sample were used for statistical analysis.
16S rRNA sequencing of microorganisms in the ileum
DNA extraction and gene amplification
Total community genomic DNA was extracted from ileal digesta samples using the E.Z.N.A DNA Kit (Omega, USA). DNA concentration and quality were assessed using a Qubit 2.0 Fluorometer (Life Technologies, USA). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using KAPA HiFi Hot Start Ready Mix (2×) (TaKaRa Bio Inc., Japan) and universal primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC). Each 30 μL PCR reaction contained 2 μL of microbial DNA (10 ng/μL), 1 μL of each primer (10 μM), and 15 μL of 2× KAPA HiFi mix. PCR was performed on an Applied Biosystems 9700 thermal cycler (USA) under the following conditions: 95℃ for 3 min; 5 cycles of 95℃ for 30 s, 45℃ for 30 s, 72℃ for 30 s; 20 cycles of 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s; and final extension at 72℃ for 5 min. The PCR products were checked using electrophoresis in 1 % (w/v) agarose gels in TBE buffer (Tris, boric acid, EDTA) stained with ethidium bromide (EB) and visualized under UV light.
Library construction, quantification, and sequencing processing
Amplicons were purified with AMPure XP beads to remove primer dimers. Libraries were constructed by Sangon BioTech (Shanghai, China) using Illumina adapters and indices. DNA concentration was quantified using Qubit 2.0, and fragment size distribution was assessed with an Agilent 2100 Bioanalyzer (USA). Equimolar pooling of libraries was performed, and paired-end sequencing (2 × 300 bp) was conducted on an Illumina MiSeq platform (Illumina, USA) according to standard protocols. Raw sequencing data were processed as follows: (1) paired-end reads were merged using PEAR (v0.9.6); (2) sequences containing ambiguous bases, lengths > 480 bp or < 200 bp, or homopolymers > 6 bp were removed; (3) identical sequences were merged; (4) sequences were aligned against a customized reference database; (5) adapters and indices were trimmed; (6) noise reduction and chimera removal were performed using Pre.cluster and UCHIME within the mothur pipeline (v.1.44.3). Taxonomic classification was conducted using the RDP Classifier against the SILVA database. Alpha diversity indices were calculated in mothur.
Statistical analysis
All the data were analyzed using one-way analysis of variance (ANOVA), and the means were compared using Duncan’s multiple range test in SAS 9.4 (SAS Institute Inc. Cary, NC, USA). Linear and quadratic effects of dietary BETP concentrations (0, 1000, 1500, and 2000 mg/kg) were analyzed by orthogonal comparisons among the LL and BETP-supplemented groups. The CON group was excluded from the dose-response analysis because it differed from the LL-based treatments in dietary energy and crude protein concentrations. Differences were considered statistically significant at P< 0.05. The data are presented as the mean and pooled SEM.
Results
Growth performance
The growth performance is shown in
Table 3. The LL group had a significantly higher FCR during d10-21 than the CON group (
P< 0.05). D10-21 FCR decreased linearly with increasing BETP supplementation (
P< 0.05). Compared with the LL group, supplementation of the low-energy and low-protein diet with 1000 and 2000 mg/kg BETP decreased d1-42 ADFI, d10-21, d22-42, and d1-42 FCR significantly (
P< 0.05). There was no significant difference among all treatments in mortality and EPEF (
P> 0.05).
Carcass performance
Carcass performance of broilers is presented in
Table 4. Compared with the CON and LL groups, dietary low-energy and low-protein supplementation with 1500 and 2000 mg/kg BETP significantly increased the semi-eviscerated yield percentage (
P< 0.05). All BETP supplementation groups increased the eviscerated yield percentage significantly (
P< 0.05).
Hepatic energy metabolism
The results showed that a low-energy and low-protein diet with BETP supplementation decreased liver FGF21 content compared with the CON and LL groups at d 42 (
P< 0.001). FGF21 content decreased linearly and quadratically with increasing BETP supplementation at d 42 (
P< 0.001) (
Table 5).
Fig. 1 showed that dietary low-energy and low-protein with 2000 mg/kg BETP supplementation upregulated d 14
PPARα relative mRNA expression significantly compared with other groups (
P< 0.05), and downregulated d 14
AMPK relative mRNA expression compared with that of the 1000 mg/kg BETP group (
P< 0.05). Compared with the CON group, dietary low-energy and low-protein with 1500 and 2000 mg/kg BETP supplementation upregulated d 42
CPT1 relative mRNA expression (
P< 0.05) while downregulated d 42
AMPK relative mRNA expression significantly (
P< 0.05). With the supplementation of BETP, d 14
PPARα relative mRNA expression showed a linear and quadratic increase (
P< 0.01), d 14 and d 42
AMPK relative mRNA expression showed a linear decrease (
P< 0.05), and d 42
CPT1 relative mRNA expression increased linearly (
P< 0.05).
Serum antioxidant
As shown in
Table 6, a low-energy and low-protein diet with 2000 mg/kg BETP supplementation showed higher MDA content among all groups at d 42 (
P< 0.05). With the increment of BETP supplementation, MDA content showed a quadratic increase (
P< 0.05), GSH-Px activity showed a linear decrease (
P< 0.05), although the ANOVA was not significant at d 42 (
P> 0.05).
Intestinal barriers
As presented in
Table 7, compared with the control group, dietary low-energy and low-protein increased d 14 serum DAO content significantly (
P< 0.05), compared with the LL group, low-energy and low-protein with BETP supplementation increased d 42 serum DAO content significantly (
P< 0.05). With the increment of BETP supplementation, d 42 DAO content showed a linear and quadratic increase significantly (
P< 0.05), and d 42 DLA content showed a quadratic increase significantly (
P< 0.001).
Fig. 2 showed that dietary low-energy and low-protein and supplementation with 2000 mg/kg BETP downregulated d 14
OCLN relative mRNA expression compared with the control group (
P< 0.05), and low-energy and low-protein with 2000 mg/kg BETP supplementation downregulated d 42
OCLN relative mRNA expression compared with the 1000 mg/kg group (
P< 0.05).
As for ileal immune factors (
Fig. 3), the low-energy and low-protein diet with 2000 mg/kg BETP supplementation showed higher relative mRNA expression of
IFN-γ on d 14 and
TNF-α on d 42 compared with other groups (
P< 0.05). All of those groups showed linear and quadratic increase with BETP supplementation (
P< 0.01).
Histomorphology
As shown in
Fig. 4, there was no significant difference in pectoralis major muscle histomorphology among the treatments (
P> 0.05).
16S rRNA sequencing of microorganisms
As illustrated in
Fig. 5A and
B, rarefaction curves plateaued at approximately 40000 sequences, confirming that sequencing depth was sufficient to cover the vast majority of microbial species in the samples at d 14 and d 42.
Our experimental treatment had a significant effect on the alpha diversity of ileal microbiota at d 14 (
Fig. 5C,
E-H). The Simpson index showed a significant reduction compared with the LL group (
P< 0.05).
As for the ileal microbiota at d 42 (
Fig. 5D,
I-L), the Sobs, Chao1, and ACE index in the BETP1000 and 1500 groups and the Shannon index in the BETP1000 group were higher than those of the CON, LL, and BETP2000 groups (
P< 0.05), while the Simpson index in the BETP2000 group was higher than that of the other groups. With the increment of dietary BETP supplementation, the Chao1, ACE, and Shannon index showed a linear increase (
P< 0.01), while the Simpson index showed a linear and quadratic increase (
P< 0.05).
A total of 400 (CON), 423 (LL), 438 (BETP1000), 423 (BETP1500), and 437 (BETP2000) OTUs were detected in the ileal samples, and 257 OTUs common to all subgroups at d 14 (
Fig. 6A). Beta-diversity analysis based on Bray-Curtis distances revealed non-significant separation among groups in both principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) plots (PERMANOVA, R
2=0.16151,
P=0.082; Stress=0.06821;
Fig. 6B and
C).
A total of 435 (CON), 503 (LL), 756 (BETP1000), 755 (BETP1500), and 397 (BETP2000) OTUs were detected in the ileal samples, and 246 OTUs common to all subgroups at d 42 (
Fig. 7A). Beta-diversity analysis demonstrated significant separation among groups in both PCoA and NMDS (PERMANOVA, R
2=0.21833,
P=0.001; Stress=0.03672;
Fig. 7B and
C).
At the phylum level, the top 5 phyla in relative abundance of the microorganisms in the ileal chyme at d 14 were
Firmicutes,
Proteobacteria,
Cyanobacteria,
Actinobacteriota, and
Bacteroidota, and there was no significant difference between the relative abundance and the ratio between
Firmicutes and
Bacteroidota (
Fig. 8A and
C). As shown in
Fig. 8B and
C, the d 42 top 5 phyla in terms of relative abundance in ileal chyme were
Firmicutes,
Cyanobacteria,
Bacteroidota,
Proteobacteria, and
Verrucomicrobiota, and compared with the control group, the relative abundance of
Cyanobacteria in the low-energy and low-protein groups is numerically lower, while compared with the low-energy and low-protein group, low-energy and low-protein with 1000 and 1500 mg/kg BETP supplementation increased it significantly (
P< 0.05), and compared with the control and low-energy and low-protein group, the ratio of F/B was numerically lower in the BETP1000 and BETP1500 group while higher in the BETP2000 group (
P> 0.05). At the genus level, low-energy and low-protein group increased the relative abundance of
Ligilactobacillus numerically compared with the control group (
P> 0.05), the low-energy and low-protein with 1000 mg/kg BETP supplementation reversed it and showed the highest relative abundance of
Lactobacillus among all groups at d 14 (
P< 0.05) (
Fig. 8D). Compared with the control group, the low-energy and low-protein group increased the relative abundance of
Lactobacillus significantly (
P< 0.05) (
Fig. 8E).
LEfSe analysis (LDA> 4.0) at d 14 and d 42 was shown in
Fig. 6F-G and
Fig. 7F-G, respectively. Only g_
Ligilactobacillus showed significant changes at d 14. At d 42, the control group was characterized by enrichment of
Proteobacteria, the low-energy and low-protein showed a significant enrichment in
Lactobacillus and
Bacilli, while a low-energy and low-protein diet with 1000 mg/kg BETP enriched fiber-degrading taxa (
Bacteroidota,
Oscillospirales,
Ruminococcaceae, and
Lachnospiraceae), the 1500 mg/kg dose shifted toward protein-fermenting bacteria (
Romboutsia,
Peptostreptococcaceae, and
Clostridiaceae). No significant discriminant taxa were observed for the BETP2000 group at the LDA> 4.0 threshold.
Discussion
Low-protein diets have emerged as a critical approach to reducing nitrogen emissions and feed costs as animal husbandry evolves toward sustainability and efficiency
[18]. However, the reduction of protein levels is frequently accompanied by a variety of consequences, such as impaired intestinal barrier function
[19], disturbed amino acid metabolism
[20], and decreased growth performance
[21]. These issues necessitate immediate improvement through nutritional regulation. This study showed that a low-energy and low-protein diet with BETP supplementation could effectively enhance feed efficiency, improve intestinal health and modulate the microbiota, and had no adverse effect on the meat quality and nutritional utilization efficiency of broilers. The recommended supplemental dosage in low-energy and low-protein diets was 1000 mg/kg in this study.
Precision feeding, which involves the use of a low-protein diet fortified with essential amino acids, has gained commercial acceptance as a means of enhancing protein utilization and reducing environmental impact
[22]. Nevertheless, previous studies have demonstrated that broiler growth performance may be diminished by a low-protein diet
[23], which is consistent with our results that a low-energy and low-protein diet increases the feed conversion ratio. BETP supplementation decreased ADFI and FCR without affecting ADG, indicating that BETP supplementation could improve nutrient utilization. Notably, the BETP1000 group consistently exhibited the lowest ADFI over the entire 42 d period while maintaining ADG comparable to other groups, suggesting that the improved FCR was driven by both enhanced nutrient utilization and a sustained reduction in voluntary feed intake, without compromising growth. Economic viability of the broiler industry is contingent upon slaughter performance, which is a critical metric for assessing meat yield in livestock
[24]. The results showed that BETP supplementation not only enhanced semi-eviscerated and eviscerated yields but also increased the breast muscle index by approximately 1.5%. Although the latter finding did not achieve statistical significance, it provides preliminary support for the commercial potential of BETP supplementation. Besides, our research found that BETP supplementation downregulated the relative mRNA expression of
AMPK while upregulated the relative mRNA expression of
CPT1, which diverged from the canonical
AMPK-
CPT1 activation axis, and the concurrent upregulation of d 14
PPARα indicated that
CPT1 upregulation may be more closely associated with
PPARα activation than with
AMPK signaling in the present study. Enhanced fatty acid oxidation driven by this pathway may supply energy for protein synthesis in skeletal muscles (increased slaughter performance and breast muscle yield), which was in accordance with a previous study that reducing energy levels in the low-protein diet to adjust the energy-to-protein ratio may improve fat deposition
[25]. Furthermore, the observed
AMPK downregulation may represent a negative feedback response to improve cellular energy status rather than a direct regulatory cause. The marked reduction in hepatic FGF21 content following BETP supplementation suggests an alleviation of the metabolic stress induced by the low-energy and low-protein diet, as FGF21 is known to be upregulated under conditions of protein and energy restriction
[26]. This reduction, together with the upregulation of
PPARα and
CPT1, indicates that BETP supplementation effectively improved hepatic energy homeostasis. All in all,
PPARα-
CPT1 activation combined with reduced FGF21 suggests that BETP supplementation may improve hepatic energy metabolism, which in turn supports increased feed efficiency and meat yield.
The intestinal mucosa contains an extensive enzyme and non-enzyme antioxidant system
[27], while previous research has demonstrated that dietary betaine has the potential to enhance the non-enzymatic antioxidant effects by restoring S-adenosyl methionine
[28]. This restoration could subsequently increase the synthesis of GSH
[29], stabilize the intracellular environment as an osmoprotectant
[30], and activate endogenous antioxidant defense pathways
[31]. These expected changes were not observed in our experimental results. Our results showed that high BETP concentration (2000 mg/kg) in a low-energy and low-protein diet increased MDA content. An insufficient availability of methyl acceptors may lead to hepatic S-adenosylhomocysteine accumulation, which acts as a potent competitive inhibitor of methyltransferase
[32], finally leading to a decline in antioxidant capacity. In addition, the alteration of intestinal inflammatory factors was in accordance with the fact that an excessive BETP concentration may induce endoplasmic reticulum stress, which in turn activates inflammatory pathways. Thus, a low-energy and low-protein diet with 1000 and 1500 mg/kg BETP supplementation supported antioxidant homeostasis in birds.
Serum DAO and DLA content are the biomarkers indicative of leaky gut syndrome
[33], which indirectly reflect permeability and barrier function of intestinal mucosa and the integrity of intestinal epithelial cells
[34]. The elevated DAO level in the BETP1000 group may indicate mild changes in intestinal permeability or epithelial turnover. However, because DLA was not increased and OCLN expression was not reduced, this response may not necessarily reflect severe barrier disruption. This was additionally corroborated by the observed decrement in DLA content. On the other hand, the 2000 mg/kg group showed higher DAO and DLA but lower
OCLN, and the mRNA expression of
IFN-γ and
TNF-α was upregulated.
TNF-α could enhance intestinal permeability
[35] while suppressing the transcription of
OCLN and other tight junction proteins
[36]. Consequently, the moderate dosage may trigger a regulated epithelial remodeling process that ultimately reinforced barrier integrity, while high dosage (2000 mg/kg) BETP not only inflicted epithelial damage but also fundamentally impaired the mucosal intrinsic repair capacity through inflammatory signaling, which is consistent with the antioxidant capacity. In addition, the downregulated
AMPK signaling pathway is supposed to promote the synthesis of inflammatory factors, but
CPT1-mediated enhanced energy metabolism reduces lipotoxicity and endoplasmic reticulum stress, thereby decreasing the expression of inflammatory factors. On the whole, a low-energy and low-protein diet with an appropriate level of BETP (1000 mg/kg) could improve the intestinal health of broilers.
The intestinal microbiota serves as a critical mediator between dietary interventions and host physiological responses, and plays a crucial role in regulating growth performance by maintaining intestinal health
[37,38]. Our findings indicate that BETP induces a dose-dependent remodeling of the ileal microbiota. Community separation, although not fully resolved at d 14 (PERMANOVA,
P=0.082), became highly significant by d 42 (
P=0.001), indicating that the modulatory effects of BETP on ileal microbial structure accrue over time. In comparison to the low-energy and low-protein group, the supplementation of 1000 and 1500 mg/kg BETP to the low-energy and low-protein diet could enhance the community richness and diversity, however, the 2000 mg/kg group did not exhibit the corresponding changes, which are consistent with the elevated serum MDA levels and impaired intestinal barrier function. Our results showed that the low-energy and low-protein group exhibited an enrichment of
Firmicutes members, particularly those from the genus
Lactobacillus and the class
Bacilli. These members have the potential to improve intestinal health by regulating inflammatory pathways and producing SCFAs, thereby enhancing epithelial barrier integrity and modulating mucosal immunity
[39], which was in line with the serum DAO and ileal pro-inflammatory cytokines. The supplementation of 1000 mg/kg BETP reshaped this profile by enriching fiber-degrading and butyrate-producing taxa within the
Bacteroidota,
Oscillospirales,
Ruminococcaceae, and
Lachnospiraceae. It indicated that the host relies more heavily on microbial fermentation of dietary fiber and other nutrients for energy salvage to compensate for the reduced availability of dietary amino acids as an energy substrate under the low-energy and low-protein condition, finally improving feed efficiency. Furthermore, butyrate is critical for both intestinal health and muscle development, which may partly explain the improvements in intestinal health and carcass performance observed in the BETP1000 group
[40,41]. The supplementation of 1500 mg/kg BETP identified a shift toward protein-fermenting bacteria within
Romboutsia,
Peptostreptococcaceae,
Clostridiaceae, which indicated that the output of the ileal microbiota transitions from fiber-degrading toward proteolytic fermentation. This shift may represent an adaptive microbial strategy to enhance endogenous nitrogen recycling under the low-energy and low-protein condition. However, excessive intestinal protein fermentation is frequently linked to the release of deleterious metabolites such as ammonia
[42], which could undermine gut integrity, a premise corroborated by the numerically elevated serum MDA and DLA relative to the BETP1000 group. Taken together, these findings suggest that 1500 mg/kg BETP may represent an approximate upper effective level under the present experimental conditions, with higher inclusion rates eliciting significant increases in DAO, DLA, MDA, and pro-inflammatory cytokines in the present study. These results suggested that a low-energy and low-protein diet with 1000 mg/kg BETP supplementation could reshape the ileal microbiota toward a fiber-degrading and butyrate-producing community, thereby improving feed efficiency, intestinal health, and carcass performance under low-energy and low-protein conditions. A schematic summary of these proposed mechanisms is presented in
Fig. 9.
Conclusion
In conclusion, dietary supplementation with 1000 mg/kg BETP is the optimal supplemental level under a low-energy and low-protein diet in this study. This supplementation level improved feed efficiency and carcass performance by improving energy metabolism and enriching fiber-degrading and butyrate-producing ileal microbiota.
Abbreviations
ADFI Average daily feed intake
AME Apparent metabolic energy
AMPK Adenosine monophosphate-activated protein kinase
CPT1 Carnitine palmitoyltransferase 1
EPEF European Production Efficiency Factor
FCR Feed conversion ratio
FGF21 Fibroblast growth factor 21
GSH-Px Glutathione peroxidase
HE Hematoxylin-eosin staining
PPARα Peroxisome proliferator-activated receptor alpha
T-AOC Total antioxidant capacity
TNF-α Tumor necrosis factor-alpha
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