Supplementation of sodium acetate improves the growth performance and intestinal health of rabbits through Wnt/β-catenin signaling pathway

Abstract

Acetic acid, which is one of the most abundant short-chain fatty acids (SCFA) in rabbits’ cecum, has been reported to play an important function during various physiological metabolic processes. The present study was conducted to elucidate the effects of sodium acetate on growth performance and intestinal health by evaluating feed intake and efficiency, diarrhea score, serum and cecum metabolites, cecal pH and SCFA, histological staining, nutritional composition of meat and gene expression profile of cecum in rabbits. As a result of sodium acetate supplement, the feed conversion ratio, diarrhea score, and diameter of muscle fiber were significantly decreased (P < 0.05). Additionally, dietary sodium acetate significantly increased in total area of muscle fibers and content of crude ash (P < 0.05). Dietary sodium acetate significantly increased serum glucose, total bile acid, and total cholesterol levels and decreased amylase, lipase, and tCO₂ content (P < 0.05). Further examination suggested that sodium acetate supplementation enhanced the micro-environment of cecum, evidenced by significantly increased levels of total antioxidant capacity, total superoxide dismutase, and glutathione peroxidase, and decreased pH and amylase levels (P < 0.05). According to transcriptome sequencing of cecal tissues, differentially expressed genes were predominantly enriched in cell cycle, ABC transporters, and chemokine signaling pathways. Sodium acetate was further suggested to stimulate the proliferation and migration of rabbits’ cecum epithelial cells by activating Wnt/β-catenin pathway both in vivo and in vitro. In conclusion, dietary sodium acetate supplementation improved growth performance and intestinal health in rabbits.

Dietary sodium acetate improves the growth performance of rabbits by supporting intestinal health and proliferation of cecum epithelial cells.

Graphical Abstract

Graphical Abstract.

Introduction

Herbivores have the most diverse microbiome in the cecum and colon and it is essential for regulating the immune, endocrine, and digestive functions of the intestine. Especially for rabbits, which have a well-developed cecum and the largest proportion of the cecum among all monogastric herbivores, a large number of microorganisms colonize the cecum. For rabbits, the nondigestible polysaccharides intake that are not fully digested in their small intestine will be transferred to cecum and digested by cecal microorganisms to produce short-chain fatty acids (SCFA) (Belenguer et al., 2011). In livestock and poultry production, SCFA are typically used as feed additives in the form of SCFA salts to improve animals’ electrolyte balance, intestinal microflora, and gut barrier function. Acetic acid, which is one of the most abundant SCFA in rabbits’ cecum, has been reported to play an important role in various metabolic processes.

Acetate plays a key role in the regulation of intestinal physiology by acting as an energy source for colonic epithelial cells and maintaining the dynamic balance of intestinal water and electrolytes (Stevens and Hume, 1998). A review suggested that acetate reduced colon electrolyte-related diarrhea in human, mice, and rabbits (Kunzelmann and Mall, 2002). Dietary acetate was also found to play a synergistic role in boosting the growth and stability of the commensal microbiota, counteracting inflammation, and enteric infections (Yap et al., 2021). Furthermore, acetate has been shown to act as a signaling molecule to influence glucose homeostasis and body weight control by reducing insulin resistance and appetite (Frost et al., 2014; Vandenbempt et al., 2024).

Although the effect of acetate in improving the growth performance of rabbits has been suggested in previous study (Fang et al., 2020), a more recent study has shown that high concentration of acetate can protect the intestinal barrier and reduce the negative effect of inflammation to colonic epithelial cells (Deleu et al., 2023). However, much work remains to be done to elucidate the underlying mechanism of how sodium acetate improves the growth performance of rabbits. We hypothesize that sodium acetate is beneficial for growth performance, likely through improving the intestinal health of rabbits. Therefore, the objective of the present study was to assess the effects of dietary sodium acetate on growth performance, meat quality, serum profile, and intestinal health by using in vivo and in vitro approaches. This study enhances our understanding of the effects of sodium acetate on growth performance and meat quality, and ultimately provides strategies for safe feed additives for healthy rabbit cultivation.

Materials and Methods

Animal ethics

All the experimental procedures were performed under the approval of the Institutional Animals Care and Use Committee (IACUC) of the College of Animal Science and Technology of Henan Agricultural University, China (permit number: 11-0085; date: June 13, 2011).

Animal and treatments

A total of 20 weaned male New Zealand White rabbits (35 ± 2 d, 1.70 ± 0.09 kg) were used for this experiment. These rabbits were raised in separate cages and were randomly divided into 2 groups: the control group (n = 10) and the acetate group (n = 10). Rabbits in the control group were fed the basal diet, and rabbits in the acetate group were supplemented with 2 g/kg sodium acetate (Aladdin, Shanghai, China) into the basal diet. Rabbits were provided ad libitum access to commercial pellet diets and water. Composition and nutrient levels of the diet are listed in Supplementary Table S1. Natural lighting was maintained for about 13 h throughout the experiment. After 1 wk acclimatization period, the formal trial lasted for 10 wk. All rabbits were in good health and were not given antibiotics, anticoccidials, probiotics, or prebiotics during the entire experiment.

Body weight and feed intake of each rabbit were measured weekly to calculate the feed conversion ratio (FCR), average daily body weight gain (ADG), and average daily feed intake (ADFI). Ambient temperature and humidity were recorded every morning and evening to calculate temperature-humidity index (THI). The equation for calculating THI was as follows: THI = Td-[(0.31-0.31RH) × (Td-14.4)], where Td represents dry bulb temperature (°C) and RH represents relative air humidity (%) (Marai et al., 2001). In the present study, all rabbits were raised in a relatively comfortable environment without undergoing heat stress over the whole period of experiment (THI ≤ 27.8, Supplementary Figure S1)

Sample collection

At the end of the experiment, all rabbits were sacrificed by using an overdose of isoflurane (Abbot, Chicago, IL, USA), and slaughtered for sample collection. Blood was obtained via jugular venipuncture into ethylene diamine tetra acetic acid (EDTA) tubes to collect whole blood. Samples of cecal tissue, cecal content, and quadriceps femoris muscle tissue were collected and transferred immediately into liquid nitrogen. Weight of heart, liver, spleen, lung, and kidneys were measured to evaluate the performance of carcass traits. Length of intestinal tract (including duodenum, jejunum, ileum, cecum, and colon) was measured. The pH of cecal and duodenum content was measured by using a pH meter (SOUTH RANCH, China). Tissues of cecum and leg muscle were taken and rinsed with physiological saline and immediately stored in 4% paraformaldehyde for histology staining. The cecal content of each rabbit was collected for SCFA analysis.

Diarrhoea score evaluation

Diarrhea score was recorded and evaluated every morning and evening for the whole period of the experiment based on the following scoring standard (Yap et al., 2021): 1 = normal stool, 2 = soft stool, 3 = paste-like, not shaped, 4 = watery diarrhea. Diarrhea scoring was performed by the same person throughout the whole trial period. The final diarrhea score was calculated by the average diarrhea score recorded in the morning and evening for each rabbit in every group.

SCFA analysis

Extraction and measurement of SCFA were performed as previously described with some modifications (Dai et al., 2015). In brief, 100 mg of cecal contents were weighed and used for the measurement of SCFA, 50 μL of 15% phosphoric acid and 125 μg/mL of internal standard (isocaproic acid) solution were added to a 2-mL centrifuge tube. The sample was homogenized with 100 μL of internal standard (isocaproic acid) solution and 400 μL of ether for 1 min, centrifuged at 4 °C for 10 min at 12,000 × g. All samples were analyzed by GC-MS (Thermofisher, ISQ7000) according to the previously published protocol (Dai et al., 2015). Briefly, in the gas chromatographic system, an Agilent HP-INNOWAX capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm) was used. The temperature of the injector was 250 °C, and a sample of 1 µL was injected at the split ratio of 1:10. The mass spectrometric conditions were as follows: the temperatures of MS transfer line and ion source were set at 250 and 300 °C, respectively; electron impact energy was operated at 70 eV.

Histology staining

Haematoxylin and eosin (H&E) staining of muscle and cecal tissues and alcian blue-periodic acid Schiff reaction (AB-PAS) staining of cecal tissue sections were performed according to a previously reported method (Zhu et al., 2017). The prepared slices were observed under an optical microscope (Eclipse Ci-L; Nikon, Japan). Quantification of the staining results was analyzed by using Image-Pro Plus 6.0 (Media Cybemetics, USA) software.

Biochemical measurements

The whole blood of the rabbit was used for serum separation by centrifuging at 4,000 × g for 15 min at 4 °C and stored at −80 °C. The activity of serum albumin (ALB), amylase (AMY), lipase (LIP), and content of serum total bile acid (TBA), total cholesterol (TC), triglyceride (TG), glucose (GLU), total CO₂ (tCO₂), total protein (TP), globulin (GLOB), globulin ratio (A/G), total bilirubin (TB), glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine kinase, creatinine (Crea), uric acid, urine creatinine (UREA), urinary anhydride ratio (U/C), and inorganic phosphorus (PHOS) were measured by using an automatic biochemistry analyzer SMT-120 (Seamaty, Chengdu, China) and animal biochemical reagent plate (Seamaty, Chengdu, China). Content of TG, TP, total antioxidant capacity (T-AOC), malondialdehyde (MDA), and activity of amylase, Na⁺–K⁺ ATPase, and Ca²⁺–Mg²⁺ ATPase of cecal tissues were determined by using assay kits from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China) (Supplementary Table S2). ELISA Kit (Meimian, Jiangsu, China) was used to analyze the content of cytokines in cecal tissue according to the manufacturer’s instructions, these cytokines measured in the present study included glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), intestinal fatty acid-binding protein (iFABP), d-Lactic (d-LA) acid, and flagellin (Supplementary Table S2).

Chemical composition of rabbit quadriceps femoris muscle

Moisture and crude ash were analyzed following the procedures of the AOAC (Quirino et al., 2022). Moisture was measured on the basis of a constant weight in oven-drying at 105 °C. Crude ash was measured by a muffle furnace at 550 °C for 5 h. TP and triglyceride content were measured by using the TP assay kit and triglyceride assay kit (Jiancheng, Nanjing, China).

Transcriptome sequencing of cecal tissue and data analysis

The RNA-seq library was prepared using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). De novo assembly and annotation identified the differentially expressed genes (DEG) between 2 groups. The expression level of each transcript was measured according to the fragments/kb of exon per million mapped reads method. RNA-Seq by Expectation Maximization (RSEM, version 1.3.3) was used to quantify the abundance of genes and isoforms. The R statistical package software EdgeR (version 4.2.0) was used for the analysis of differential expression. Functional enrichment analysis was performed to identify the DEG enriched significantly in GO and metabolic pathways with Bonferroni-corrected P < 0.05 as compared to the whole-transcriptome background. GO functional enrichment and KEGG pathway analyses were performed using Goatools (version 1.2.3) and KOBAS (version 2.0), respectively (Xie et al., 2011; Klopfenstein et al., 2018).

RNA extraction and quantitative real-time PCR

Total RNA of tissues and cells was extracted by using TRIzol reagent (Invitrogen, CA, United States) according to the manufacturer’s instructions. The concentration and integrity of total RNA were measured by using the NanoDrop One spectrophotometer (Thermo Scientific, USA) and agarose gel electrophoresis. The first-strand cDNA was prepared by using a PrimeScript RT Kit (Takara, Tokyo, Japan). RT-qPCR assay was performed by using SYBR Premix Ex Taq II Kit (Takara) to determine mRNA expression of genes on a CFX96 real-time PCR detection system (Bio-Rad Laboratories Inc, Hercules, CA, USA) following the previously published protocols (Lv et al., 2020). Gene-specific primers (Supplementary Table S3) were designed and synthesized by Sangon Biotech Co., Ltd (Shanghai, China), these primers were optimized before the initial screening and quantitative analysis.

Western blot

TP of tissues and cells was lysed with RIPA lysis buffer (PC101, Beyotime, Shanghai) supplemented with phenylmethanesulfonyl fluoride (PMSF, Epizyme Biotech, Shanghai, China) and phosphatase inhibitor cocktail (Epizyme Biotech). BCA kit (Epizyme Biotech) was used for determination of the protein concentration. A 10% SDS-PAGE gels was used to separate proteins and then transferred to PVDF membranes. Then the membranes were blocked with 5% BSA for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. Primary and secondary antibodies used in this study are shown in Supplementary Table S4. Followed by incubation with a secondary antibody for 1 h at room temperature. Blots were visualized using Omni-ECL Femto Light Chemiluminescence Kit (Epizyme Biotech) on QuickChemi 5200 chemiluminescence imaging system (Monad Biotech, Wuhan, China). All protein levels were analyzed with Image J and normalized to β-actin, and expression level of the protein located in the nucleus was normalized to Histone H3.

Culture and treatments of rabbits’ primary cecum epithelial cells

Three healthy 1-d-old New Zealand White rabbits were selected and used for cecum epithelial cells (CEC) isolation as described previously (Duque-Correa et al., 2022). Cecum was cut longitudinally, washed with ice-cold 1 × HBSS (Gibco, Thermo Fisher) containing 1 × penicillin/streptomycin to remove the cecal contents and then cut into small fragments. The small fragment of cecal tissue was digested in 2 mg/mL type IV collagenase (GC305015; Servicebio, Wuhan, China) supplemented with 0.25 mg/mL dithiothreitol (GC305015; Servicebio) and 0.5 mg/mL proteinase E (P5147, Sigma-Aldrich, USA). Digestion was stopped with DMEM/F12 (Gibco, Thermo Fisher) medium containing 20% fetal bovine serum (FBS, Pricella, China), 4 ng/mL epidermal growth factor, 1% insulin–transferrin–selenium-supplement A, 1% heparin sodium, and 1% penicillin–streptomycin. Cells were cultured in cell culture dishes with growth medium (DMEM/F12 supplemented with 20% FBS and 1% penicillin–streptomycin) at 37 °C and 5% CO₂, and cell culture was changed every 24 h. CK18 immunofluorescence examination was performed according to published staining methods (Zhang et al., 2019). Twelve hours before treatments, cells were switched to serum-free medium in which FBS was replaced with FA-free BSA (1 g/L, Solarbio, Beijing, China). In order to screen the suitable treatment condition in response to sodium acetate (S2889, Sigma-Aldrich), CEC were first treated with sodium acetate at 0, 2, 4, 8, 12, and 16 mmol/L (final concentration, dissolved in DMEM/F12) for 0, 24, 48, and 72 h. The final concentration of 2 mmol/L sodium acetate was selected and used for the following experiments. All treatments were performed with 3 replicates.

Nuclear and cytoplasmic protein fractionation

After 48 h of incubation with 0 and 2 mmol/L sodium acetate, CEC were collected for nuclear and cytoplasmic protein extraction using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions, and the nuclear protein was quantified using western blot.

Cell counting kit-8 assay

CEC seeded in 96-well plates (Corning, USA) were incubated with different concentrations (0, 2, 4, 8, 12, 16 mmol/L) of sodium acetate for different times (0 h, 24 h, 48 h, 72 h). After incubation, the plate continued to incubate for 2 h with 10 µL Cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Japan) to each well. After incubation at 37 °C for 1 h, the absorbance was measured at 450 nm using a microplate reader (Synergy LX, BioTek, USA) on 96-well plate readings.

Cell wound healing assay

The CEC was seeded in the 6-well culture plates (Corning) at a density of 5 × 10⁶ cells and incubated at 37 °C in 5% CO₂ until 80% confluence. Then the cells were scratched by a pipette tip and washed with HBSS (Gibco, Thermo Fisher) softly. Then, 2 mmol/L of sodium acetate was added to each well and the plate was incubated at the plate was incubated at 37 °C for 24 h in 5% CO₂. After incubation, the wound area was measured under the fluorescent inverted microscope (AE31, Motic, Xiamen, China) and quantified by Image-Pro Plus 6.0 software.

Statistical analysis

All data are described as at least 3 independent experiments. Among them, the in vitro experiment contains 3 biological replicates, and the in vivo experiments contain at least 3 biological replicates. Each biological replicate contains one rabbit. Statistical significance of results was determined with 2-tailed Student’s t-test as indicated in the figure legends by Statistical Package for the Social Sciences (SPSS, USA) and Prism 9 (GraphPad, USA) software. The relative gene expression levels were normalized to the endogenous RNA control GAPDH, HPRT1, PPIC, and YWHAZ with the 2^−ΔΔCq method. Densitometric quantification of the western blot bands was performed using Image J (National Institutes of Health, USA). The correlation between DEG and phenotypes was analyzed by Pearson’s correlation coefficient. All statistical analyses were defined as significant at P < 0.05 and a trend at 0.05 < P < 0.10.

Results

Effects of sodium acetate supplementation on growth performance and diarrhoea score in rabbits

A 27.12% reduction in rabbit’s FCR was found in acetate group (P < 0.05, Figure 1A). During the 4th to 7th week of the feeding experiment, body weight of rabbits was significantly decreased in acetate group (P < 0.05, Figure 1B). Additionally, rabbits’ ADG in acetate group was significantly decreased during the 3th to 7th week, whereas a higher degree in ADG was determined over the 8th to 10th week (P < 0.05) (Figure 1C). From the 5th week, rabbits in acetate group had significantly lower ADFI (P < 0.05, Figure 1D). The slaughter indexes revealed a significant decrease in the weight of liver, lung, and kidneys in acetate group, while an obvious increase in the weight of stomach, the length of small intestine and cecum (P < 0.05, Supplementary Table S5).

Figure 1.

Effects of sodium acetate supplement on growth performance and diarrhea score of rabbits. Changes of (A) FCR, (B) body weight, (C) ADG, (D) ADFI during the whole period, (E) Diarrhea score of rabbits during the first 4 wk of the experiment. Control = the control group; Acetate = the acetate group. *P < 0.05, **P < 0.01.

During the first 4 wk of the experiment (including 1 wk of pilot experiment), diarrhea score was observed and recorded every day. Rabbits in acetate group showed significant decrease in diarrhea score and means an improvement in stool quality (P < 0.05, Figure 1E).

After treatment with sodium acetate, serum GLU, TBA, and TC were increased and ALB, TP, AMY, LIP, and tCO₂ were decreased (P < 0.05). There was no significant change in the content of GLOB, GGT, AST, ALT, ADH, ALP, and Crea (Table 1 and Supplementary Table S6).

Table 1.

Effects of sodium acetate supplement on hematological parameters in rabbits

Items1	Control group	Acetate group	SEM	P value
ALB, g/L	38.92	33.72	1.55	0.015
TP, g/L	69.72	60.44	3.53	0.033
TBA, μmol/L	6.91	10.90	0.50	<0.001
AMY, U/L	303.73	226.63	26.88	0.049
LIP, U/L	123.80	88.00	5.76	<0.001
GLU, mmol/L	1.35	3.03	0.51	0.011
TC, mmol/L	1.54	2.34	0.18	0.001
tCO2, mmol/L	18.28	13.50	1.78	0.041

¹ALB = albumin, TP = total protein, AMY = amylase, LIP = lipase, TBA = total bile acid, TC = total cholesterol, GLU = glucose, tCO₂ = total CO₂.

Effects of sodium acetate supplementation on meat quality of rabbits

Further analysis was performed to evaluate the effects of sodium acetate on the microstructure and nutritional composition of rabbit meat. Muscle fiber of rabbits treated with sodium acetate were more closely and orderly arranged (Figure 2). As shown in Table 2, there was a significant increase in the total number and total area of muscle fibers by sodium acetate, while the diameter of muscle fibers significantly decreased in acetate group (P < 0.05). Muscle fiber density in acetate group tends to increase (P = 0.080).

Figure 2.

Dietary supplementation of sodium acetate improved meat quality and nutritional composition of rabbits. Control = the control group; Acetate = the acetate group.

Table 2.

Effects of sodium acetate supplement on nutritional composition of quadriceps femoris muscle in rabbits

Items1	Control group	Acetate group	SEM	P value
Moisture, %	76.40	76.58	0.37	0.623
Dry matter, %	24.02	23.42	0.47	0.537
Crude ash, %	1.21	1.31	0.04	0.038
TP, g/L	1.77	1.74	0.03	0.472
TG, mmol/g	0.58	0.41	0.05	0.053
Total number of muscle fibers, n	29.3	34.6	1.59	0.010
Total area of muscle fiber, μm2	49,002.5	57,306.1	1,874.44	0.002
Muscle fiber density, n/mm2	580.2	623.4	27.47	0.080
Diameter of muscle fiber, μm	51.63	46.13	1.44	0.018

¹TP = total protein, TG = triglyceride.

Sodium acetate changed the nutritional composition of rabbit muscle. The crude ash content of quadriceps femoris muscle was significantly increased in acetate group (P < 0.05, Table 2).

Effects of sodium acetate supplementation on cecal microstructure in rabbits

To investigate the mechanism underlying the improvement in rabbits’ diarrhea score after sodium acetate supplementation, the microstructure of cecal tissue was examined. Our results showed that sodium acetate supplement trend to increased the cecal villi height (P = 0.096), and improved the integrity of cecal epithelium (Figure 3A), while number of goblet cells did not change (Figure 3B, Table 3). The figure does not show a measurement of the length of cecum epithelium or the number of goblets cells per unit length—these are stained and they can be visually seen but the actual count of number of goblets is presented in Table 3.

Figure 3.

Effects of sodium acetate on cecal morphology. (A) H&E staining of the cecum; (B) AB-PAS staining of cecum sections. Control = the control group; Acetate = the acetate group.

Table 3.

Effects of sodium acetate supplement on cecal tissue in rabbits

Items 1	Control group	Acetate group	SEM	P value
Length of cecum epithelium, mm	0.275	0.301	0.035	0.116
Number of goblet cells per unit length	20.332	16.950	2.897	0.329
TP, g/L	2.62	3.27	0.275	0.042
AMY, U/mgprot	0.405	0.163	0.096	0.050
TG, mmol/L	0.092	0.107	0.278	0.609
T-AOC, U/mgprot	4.30	6.11	0.734	0.035
GSH-Px, U/mgprot	8.26	25.66	2.495	0.020
T-SOD, U/mgprot	256.06	321.91	24.96	0.044
MDA, μmol/mgprot	0.043	0.043	0.001	0.890
Na+-K+ ATPase, μmolPi/mgprot/h	9.44	11.26	0.907	0.020
Ca2+-Mg2+ ATPase, μmolPi/mgprot/h	4.84	6.11	0.557	0.050
iFABP, ng/L	352.82	348.26	18.84	0.825
d-LA, μg/L	266.05	254.07	29.22	0.468
Flagellin, pg/mL	45.31	44.67	1.11	0.590

¹TP = total protein; AMY = amylase; TG = triglyceride; T-AOC = total antioxidant capacity; GSH-Px = glutathione peroxidase; T-SOD = total superoxide dismutase; iFABP = intestinal fatty acid-binding protein; d-LA = d-lactic acid.

Table 3 displays the content of metabolic products and immune-related enzymes in cecum of rabbits. In acetate group, significant increments were observed in levels of Na⁺-K⁺ ATPase, Ca²⁺-Mg²⁺ ATPase, and TP (P < 0.05). Additionally, the content of T-AOC, T-SOD, and GSH-Px in cecum was significantly increased, while the content of AMY was significantly decreased (P < 0.05). No significant difference was observed in MDA, flagellin, iFABP, and d-LA levels between 2 groups (Table 3). Changes of SCFA in cecum were exhibited in Table 4. The concentration of acetic acid, propionic acid, and butyric acid were the top 3 abundant types of SCFA in both groups of rabbits, followed by pentanoic acid, isobutyric acid, and isovaleric acid, and the content of caproic acid was the lowest. In cecum of acetate group, the content of acetic acid was significantly increased and the content of butyric acid, propionic acid, and caproic acid was significantly decreased (P < 0.05). Accordingly, the acetate group had significantly decreased pH of both duodenal and cecal content (Table 4).

Table 4.

Effects of sodium acetate supplement on SCFA and pH of cecal contents in rabbits

Items	Control group	Acetate group	SEM	P value
Acetic acid, μg/g	288.16	410.72	24.90	0.046
Propionic acid, μg/g	70.00	57.96	22.92	0.003
Isobutyric acid, μg/g	9.63	10.84	1.44	0.421
Butyric acid, μg/g	36.55	25.74	4.14	0.030
Isovaleric acid, μg/g	8.32	9.69	2.16	0.551
Valeric acid, μg/g	13.73	13.50	2.35	0.110
Caproic acid, μg/g	7.50	4.36	0.99	0.016
pH of duodenum	8.51	7.99	0.08	0.022
pH of cecum	6.96	6.78	0.07	0.003

Transcriptome sequencing analysis identified DEGs in cecum induced by dietary sodium acetate

A total of 48,959,343 and 50,996,516 raw reads with a length of 2 × 150 bp were generated from the cecum samples of the control group and acetate group, respectively. Then, stringent quality assessment and data filtering obtained 44,987,390 and 47,250,458 clean paired-end sequence reads with a Q30 percentage (base quality > 30) over 93% from the control group and the acetate group, respectively (Supplementary Tables S7 and S8). Approximately 81.4% of these clean reads were mapped to the rabbit genome. Based on the normalized data, 17,221 genes were expressed. Fragments Per Kilo bases per Million fragments (FPKM) are shown in Supplementary Figure S2.

The selection of DEG was performed with the general chi-squared test. Between 2 groups, a total of 648 unigenes were identified as DEG, of which 368 were upregulated and 280 were downregulated in acetate group (95% confidence limit and global FDR < 1%, Supplementary Figure S3A and S3B). The heatmap between 2 groups indicating that sodium acetate supplement could greatly influence the transcriptome of the cecum in rabbits (Supplementary Figure S3C). The principal component analysis can be used to cluster these 2 groups (Supplementary Figure S3D). Protein–protein interaction networks were constructed to investigate the interactions between DEG (Supplementary Figure S3E). The point in the figure is the gene (the corresponding protein). Genes related to the cell cycle (TTK, CCNB2, PLK1) were primarily enriched cluster among the upregulated ones, whereas those associated with oxidative phosphorylation (COX3, ATP6, CYTB) were mainly enriched cluster among the downregulated genes.

GO term enrichment analysis and KEGG pathway enrichment

GO and KEGG pathway analyses were performed to enrich the upregulated and downregulated DEG, respectively. According to GO term enrichment results, upregulated DEG enriched in cell cycle, ribonucleotide binding of molecular function and chromosomal region of cellular component in acetate group. However, downregulated DEG were substantially enriched in oxidation–reduction process (Figure 4A).

Figure 4.

Transcriptomic analysis of the cecal tissue in rabbits. Heat map of GO enrichment (A) and KEGG pathways (B) showing different enrichment between the control group and acetate group for up and down terms in cecum of rabbits; (C) Pearson’s correlations between gene expression of transcriptome and host-related parameters in cecal tissue of rabbit in response to dietary sodium acetate. Control = the control group; Acetate = the acetate group. *P < 0.05, **P < 0.01, ***P < 0.001.

As suggested by KEGG pathway results, upregulated DEG in acetate group were primarily enriched in the process of cell cycle, cellular senescence, regulation of actin cytoskeleton, P53 signaling, DNA replication and folate biosynthesis signaling pathway, while downregulated DEG were significantly enriched in retinol metabolism and apelin signaling. In addition, these identified DEG were also enriched in chemokine signaling, natural killer cell-mediated cytotoxicity, T cell receptor signaling and ABC transporters (Figure 4B), suggesting that the immune function and inflammatory response of cecum may be stimulated.

Correlation analysis between DEG of RNA-seq and phenotypes in rabbits

Spearman rank correlation analysis was performed to evaluate the potential relationship between gene expression of transcriptomic sequencing and host-related parameters. In the correlation analysis, DEG enriched in GO and KEGG were selected and compared with cecum-related biochemical and host-related parameters. As shown in Figure 4C, correlation analysis was also conducted on 27 DEG involved in the process of cell cycle, ABC transporters, regulation of actin cytoskeleton, natural killer cell-mediated cytotoxicity, chemokine signaling, retinol metabolism, PPAR signaling, apelin signaling and folate metabolism. The content of propionic acid and butyric acid in cecal contents was negatively correlated with ABC transporters (ABCA8), cell cycle (PLK1), and retinol metabolic (CYP26B1, ADH4, and AOX1), whereas that of acetic acid and GSH-Px was positively correlated with the above-mentioned genes and pathways. Aside from that, the content of ATPase in cecum was positively correlated with ABC transporters (ABCC10), while the content of serum and cecal AMY was negatively correlated with ABC transporters (ABCA8 and ABCC10). Together, there was a negative correlation between ADFI, FCR, diarrhea score and ABC transporters (ABCA8 and ABCC10), regulation of actin cytoskeleton (DIAPH3) and cell cycle (CDC25B), while a positive correlation between retinol metabolism (CYP26B) and PPAR signaling (FABP6, FABP3, and ACSL5). Goblet cells numbers in cecum were negatively correlated with natural killer cell-mediated cytotoxicity (PRKCQ) but positively correlated with PPAR signaling (FABP6, FABP3, ACSL5). Also, the content of serum TG was negatively correlated with folate metabolism (SHMT1) and cell cycle (CCNA2 and E2F2), while positively correlated with apelin signaling (PLCB1).

Validation of RNA-seq by RT-qPCR and western blot

As a verification of the accuracy of RNA-seq results, RT-qPCR and western blot analysis of the cecal tissues were performed. The gene expression pattern determined by RT-qPCR was consistent with the RNA-seq results (Figure 5A and B). In addition, western blot analysis revealed that Wnt/β-catenin, mTOR, P53, BCL-2, and BAX were activated (Figure 5C and D). Although the fold change in the expression patterns of RNA-seq (Supplementary Table S9) and RT-qPCR was slightly biased, this was probably owing to methodological differences, indicating the reliability of RNA-seq.

Figure 5.

Relative quantification by RT-qPCR of rabbits’ cecal tissue after treatment with sodium acetate. Relative mRNA expression of proliferation (A) and apoptosis (B) related genes. Control = the control group; Acetate = the acetate group. *P < 0.05, **P < 0.01.

Sodium acetate promoted the proliferation of CEC in rabbits

The underlying mechanism of sodium acetate treatment on growth performance and immune function in rabbits was further uncovered by isolating, culturing, and treating rabbit primary CEC with sodium acetate. An inverted microscope was used to observe the morphological features of CEC (Supplementary Figure S4A). After incubation for 24 h, clusters of colonies formed, followed by a regular pavement-like pattern. CK18 was used to stain cultured cells (shown in red, Supplementary Figure S4B). It was confirmed that the isolated cultured cells were CEC by immunofluorescence staining.

After treatment with sodium acetate for 12 and 24 h, the wound area of CEC was significantly decreased, as shown by healing assay (P < 0.05, Figure 6A). As determined by CCK-8 assay, addition of 2 mmol/L sodium acetate for 48 h significantly contributed to the proliferation of CEC (P < 0.05, Figure 6B). Besides, sodium acetate supplement significantly upregulated the mRNA expression of genes responsible for cell proliferation (PCNA, MKI67, CCNB2, and others) (P < 0.05, Figure 6C). Sodium acetate treatment of CEC significantly decreased the content of p-β-catenin and the ratio of p-β-catenin/β-catenin and significantly increased the content of β-catenin in the nucleus (P < 0.05), while the content of total β-catenin did not change significantly (Figure 6D). It was demonstrated that CEC treated with sodium acetate activated the Wnt/β-catenin pathway and nuclear translocation of β-catenin in the cytoplasm occurred, stimulating the proliferation and migration of CEC.

Figure 6.

Dietary sodium acetate promoted the proliferation of CEC in rabbits. (A) Healing assay of CEC after treatment with sodium acetate, (B) CCK-8 assay analysis after treatment with different concentrations of sodium acetate, (C) The protein expression of β-catenin by western blot after treatment with sodium acetate, (D) Relative mRNA expression changes of cell proliferation related genes of CEC in response to sodium acetate, (E) Model summarizing the overall effects of dietary sodium acetate on rabbits. Values are represented as means ± SEM (n = 3), *P < 0.05, **P < 0.01.

Discussion

Recently, SCFA have been evidenced to function as key players in the interactions between the micro-environment and metabolic processes in animals. These SCFA play important roles in regulating the process of energy metabolism and bio-synthesis, which will ultimately affect body weight of animals. In this paper, we systematically and comprehensively evaluated how acetate affected the growth performance, meat quality, and chemical composition in rabbits. In the cecum of New Zealand White rabbits, the predominant SCFAs were approximately 65 to 87 mmol/100 mL for acetic acid, 6 to 28 mmol/100 mL for butyric acid, and the lowest for propionic acid, at approximately 3 to 11 mmol/100 mL, with a ratio of approximately 60:20:20 (Marty and Vernay, 1984; Padilha et al., 1995; Flint et al., 2008). Previous studies and dissection of the cecum of weanling rabbits have concluded that the cecum has a capacity of approximately 280 cm³ (Snipes, 1978). The concentration of sodium acetate supplemented to the rabbit diet in this study was determined based on rabbit studies previously published (Fu et al., 2018). In summary, dietary sodium acetate (2 g/kg body weight) raised the acetic acid concentration in the cecum. We suspect that acetic acid produced by microorganisms did not have a significant impact in this study. Previous studies have indicated that endogenous acetic acid does not significantly affect the finishing weight in rabbits (Fang et al., 2020; Wang et al., 2023). In acetate group, there was a significant reduction in FCR of rabbits during the feeding experiment. This reduction may be partially attributed to significant decrease in total feed intake, because addition of sodium acetate was reported to reduce feed intake of mice in previous research (Jiao et al., 2021). SCFAs regulate feed intake by influencing peripheral organ activity and promoting the production of appetite hormones, which then enter the brain via the parasympathetic nervous system, forming a gut–brain neural circuit mechanism (Perry et al., 2016). Several reviews have shown that acetic acid can act directly on the nerve centers in the hypothalamus that regulate appetite and can affect energy metabolism through effects on lipids and insulin (Frost et al., 2014; Felix et al., 2021; van der Hee and Wells, 2021). There was no significant change in GGT, AST, ALT, ADH, ALP, and Crea, suggesting that sodium acetate had no pathological impact on the liver and kidney functions. Despite that dietary manipulation of sodium acetate is beneficial for rabbits’ growth performance, our findings suggest that long term (>8 wk) treatment might diminish this effect since the value of ADG and ADFI changed from the 8th week of feeding experiment. As a result, short-term (≤8 wk) addition of sodium acetate to weaned rabbits is suggested to be a more effective strategy to improve growth performance of rabbits.

SCFA have well-established effects on whole-body energy homeostasis through the gut-muscle axis in animals and humans (Morrison and Preston, 2016). Acetate addition is reported to enhance muscle mass and physical performance by shifting the microbial community (Liu et al., 2021). Meat quality improvement was associated with increased insulin sensitivity and glucose utilization in muscle tissue of obese rats and mice (Canfora et al., 2015). In obese rats, acetate injection for 6 mo improved the expression of GLUT4 as well as AMPK activity in skeletal muscle (Yamashita et al., 2009). Acetate was also shown to indirectly affect muscle insulin sensitivity and glucose metabolism through gut-derived GLP-1 secretion and modulation of muscle microvascular blood volume and flow (Fushimi et al., 2001). This study found that dietary sodium acetate substantially increased the total number of muscle fibers in rabbits’ quadriceps femoris muscle. In acetate group, the muscle fibers were closely and orderly arranged, and the muscle fiber density was increased. Tenderness and flavor of meat depend strongly on the density and diameter of muscle fibers, and finer and denser muscle fibers produce better flavor (Wood et al., 1999). It is possible that SCFA will not affect skeletal muscle mass in an energy surplus situation. However, they are beneficial to skeletal muscle mass in mice under conditions of metabolic stress or increased metabolic demand, such as during energy restriction, growth, or advanced age (Frampton et al., 2020). In this study, addition of sodium acetate greatly improved the growth performance of rabbits, without significantly changing the chemical properties of rabbits’ quadriceps femoris muscle. This is probably because the standard diet used in feeding experiment fully met the nutritional needs of rabbits in both groups. In view of this, effects of sodium acetate on skeletal muscle fiber development and composition are therefore complex and influenced by many other factors.

Mammalian digestive tract not only plays a crucial role in nutrient absorption but also serves as the largest peripheral lymphoid organ (Takeuchi et al., 2024). Their absorptive capacity is mainly determined by the activity of digestive enzymes and the integrity of mucous membrane (Pluske et al., 2018). SCFA serve as energy-suppliers that stimulate intestinal mucosal growth and increase colonic blood flow, thus contributing to gut homeostasis and epithelial integrity (Blottière et al., 2003). In the present study, we observed a greater degree of integrity in cecum villus tissue in rabbits of acetate group. Furthermore, acetate group showed higher activities of Na⁺–K⁺ ATPase and Ca²⁺–Mg²⁺ ATPase of cecal tissue, while a substantial decrease in the content of AMY. Digestive enzymes directly influence nutrient digestibility and growth performance. The digestion of fiber is completed under the synergistic action of α-amylase, trypsin, and chymotrypsin, and changes of the activity of any enzyme could affect its decomposition. In the present study, amylase and lipase activities were found to decrease in serum or cecum tissues, whereas bile acids, protein, cholesterol, and glucose contents increased in serum, suggesting that sodium acetate improves FCR in rabbits. A previous study found that probiotic additives enhanced the activity of digestive enzymes and decreased FCR (Huang et al., 2021). However, as mentioned above, sodium acetate decreases FCR in a different pathway than probiotics and butyrate.

GPR43 activated by 300 mmol/L acetate has been shown to facilitate mice gut homeostasis through the regulation of Nod-like receptor protein 3 inflammasome, thus repairing the gut mucosal barrier (Macia et al., 2015). It has previously been demonstrated that luminal acetate exerts protective barrier effects in the intestine both directly through epithelial cells and indirectly through immune cells (Akiba et al., 2015). As shown by Laffin et al., the susceptibility to chemically induced colitis increased after 2-d exposure to high-sugar diets depleted SCFA in mice with colitis. Acetate significantly attenuated colitis in mice by restoring permeability, independent of changes in microbial community composition (Laffin et al., 2019). Furthermore, SCFA stimulate the absorption of sodium and water in the colon by regulating the nutrient and ion transporters of SGLT-1 and GLUT2, which is helpful in preventing diarrhea associated with antibiotic-associated diarrhea (Tappenden et al., 2003; Xu et al., 2024). Similarly, after 4 wk of sodium acetate addition, we found significant reductions in the pH of duodenum and cecum content, and obvious improvements in diarrhea score. The addition of sodium acetate led to a decrease in intestinal pH, increased bioavailability of iron, calcium, and phosphorus, enhanced abundance of beneficial Lactobacillus spp., and reduced abundance of Salmonella enteritidis, thereby improving intestinal health (Van Immerseel et al., 2004; Duncanson et al., 2024). It has already been established that microbial metabolites play an important role in maintaining immune function and this highlights the importance of understanding the role of acetate. Acetate may upregulate glycolysis in CD8⁺ T cells and subsequently enhance immune function (Wang et al., 2024). In our study, sodium acetate addition improved the antioxidant capacity of cecum. In view of this, short-term supplement of sodium acetate to weaned rabbits is also suggested to be a better strategy to improve cecal environment and enhance nutrient absorption.

To investigate the mechanism of how acetate mediates rabbits’ growth and intestinal homeostasis, we performed transcriptomic analysis of cecal tissue after sodium acetate to measure changes in gene expression profile. As a result, significant changes were observed in the expression of genes associated with cell cycle. As a further verification for the mechanism of acetate on cell cycle, we isolated CEC of rabbits and treated them with sodium acetate. The results of in vitro experiments suggest that sodium acetate is capable of promoting the proliferation of CEC and activating cell cycle of cecum. In this study, the expression of BAX and p-P53 in cecum tissues was elevated, indicating that sodium acetate concurrently induced apoptosis, thereby facilitating the renewal of cecal epithelial cells. A previous study pointed out that SCFA (acetate, propionate, and butyrate) treatments stimulate epithelial cell proliferation and differentiation in rats (Frankel et al., 1994). In mammals when intestinal damage is repaired, there is an increase in cell proliferation and migration. Much research has acknowledged that activation of Wnt/β-catenin and mTOR pathway can promote the proliferation of intestinal epithelial cells in pigs, mice, and chicks (Wang et al., 2019; Li et al., 2022; Zhang et al., 2023, 2023; Yu et al., 2024). Moreover, since mTOR serves as a receptor for nutrients, its activation suggests elevated energy levels and enhanced capacity of the cecum to receive growth factors. Consistent with previous reports, our results also indicate that sodium acetate likely stimulates the proliferation and migration of CEC in vitro by activating Wnt/β-catenin signaling pathways. Another study also confirmed that acetate and butyrate promote intestinal stem cell proliferation by activating the Wnt/β-catenin signaling pathway in mice (Pearce et al., 2020; Xie et al., 2022). As other research has shown, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/β-catenin in intestinal epithelial cells (ISCs) (Li et al., 2022). Based on our results and previous studies, we speculate that dietary sodium acetate is beneficial to intestinal health. Hence, promoting the periodic cycle of CEC significantly improved rabbit growth performance and intestinal health. A brief schematic summary of the effect of sodium acetate on rabbit is shown in Figure 6E. Despite this, it is still necessary to further investigate the mechanism of monocarboxylic acid transporter-1, G-protein-coupled receptors, insulin secretion, and AMPK by which SCFA regulate intestinal cell cycle and meat quality (Perry et al., 2016; Liu et al., 2021; van der Hee and Wells, 2021).

Conclusion

Taken together, this study demonstrated that dietary sodium acetate improved the growth performance of rabbits by regulating intestinal health and the proliferation of CEC. The supporting evidence indicated that sodium acetate reduced the FCR and improved diarrhea score, thereby enhancing the integrity of the cecal epithelium in rabbits. Moreover, in vivo and in vitro assays revealed that the proliferation and migration of CEC were promoted by sodium acetate via the Wnt/β-catenin signaling pathway. This research provided important insights into how sodium acetate affected rabbits’ growth performance and intestinal health.

Glossary

Abbreviations

AB-PAS

alcian blue-periodic acid schiff reaction

ADFI

average daily feed intake

ADG

average daily body weight gain

AMY

amylase

CCK-8

cell counting kit-8

CEC

cecum epithelial cells

FCR

feed conversion ratio

GLU

glucose

GSH-Px

glutathione peroxidase

H&E

hematoxylin and eosin

iFABP

intestinal fatty acid-binding protein;

LIP

lipase

MDA

malondialdehyde

RT-qPCR

quantitative real-time PCR

SCFA

short-chain fatty acids

T-AOC

total antioxidant capacity

TC

total cholesterol

TG

triglyceride

THI

temperature-humidity index

TP

total protein

T-SOD

total superoxide dismutase

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

Mengke Ni (Writing—original draft, methodology and visualization), Hui He (Data curation), Mengjuan Chen (Software), Zhichao Li (Conceptualization), Hanfang Cai (Formal analysis), Zhi Chen (Supervision), Ming Li (Project administration and funding acquisition), and Huifen Xu (Investigation and writing—review & editing). All of the writers have read the work and approved its publication.

Availability of Data and Materials

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.