The Effects of Acetic Acid Supplement and Voluntary Wheel Running on Gut Microbiome in Ovariectomized Mice

Tan, Yuan; Park, Ki-Woong; Ryu, Byeong-Jun; Lee, Seung-Min; So, Byunghun; Jang, Junho; Park, Jinhan; Kang, Chounghun; Lee, Hanall; Hwang, Moon-Hyon; Cho, Jinkyung; Park, Young-Min; Yuan Tan; Ki-Woong Park; Byeong-Jun Ryu; Seung-Min Lee; Byunghun So; Junho Jang; Jinhan Park; Chounghun Kang; Hanall Lee; Moon-Hyon Hwang; Jinkyung Cho; Young-Min Park

doi:10.15857/ksep.2025.00199

Exerc Sci > Volume 34(3); 2025 > Article

Tan, Park, Ryu, Lee, So, Jang, Park, Kang, Lee, Hwang, Cho, and Park: The Effects of Acetic Acid Supplement and Voluntary Wheel Running on Gut Microbiome in Ovariectomized Mice

Original Article

Exerc Sci. 2025; 34(3): 267-277.

Published online: Aug 28, 2025

DOI: https://doi.org/10.15857/ksep.2025.00199

The Effects of Acetic Acid Supplement and Voluntary Wheel Running on Gut Microbiome in Ovariectomized Mice

Yuan Tan MS^1,^†

, Ki-Woong Park MS^1,^†

, Byeong-Jun Ryu BS¹

, Seung-Min Lee BS¹

, Byunghun So MS²

, Junho Jang MS²

, Jinhan Park MS²

, Chounghun Kang PhD^2,³

, Hanall Lee PhD⁴

, Moon-Hyon Hwang PhD^1,⁴

, Jinkyung Cho PhD⁵

, Young-Min Park PhD^1,⁴

¹Division of Health and Kinesiology, Incheon National University, Incheon, Korea

²Department of Health and Exercise Science, College of Graduate School, Inha University, Incheon, Korea

³Department of Physical Education, College of Education, Inha University, Incheon, Korea

⁴Sport Science Institute & Health Promotion Center, Incheon National University, Incheon, Korea

⁵College of Sport Science, Sungkyunkwan University, Suwon, Korea

Corresponding author: Young-Min Park Tel +82-32-835-8586 Fax +82-32-835-0789 E-mail ypark@inu.ac.kr

†Authors contributed equally as the first author.

*This work was supported by the Incheon National University (2023-0184).

*All experimental procedures were conducted in accordance with the guidelines and regulations set by the Institutional Animal Care and Use Committee of Incheon National University (INU-ANIM-2021-04).

Received Apr 7, 2025 Revised May 14, 2025 Accepted Jun 10, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

PURPOSE

A correlation exists between a healthy physiological state and the diversity of the gut microbiome. Menopause-induced deterioration of whole-body metabolism negatively contributes to microbial diversity. The combined effect of acetic acid supplementation and exercise on the gut microbiome remains unknown. This study aimed to investigate 1) whether menopause negatively influences gut microbial diversity, including Firmicutes abundance, and 2) whether the combined treatment of acetic acid supplementation and exercise rescues microbial dysbiosis, and which specific bacteria are related to this rescue effect.

METHODS

The 15-week study used 8-week-old female C57BL/6J wild-type mice (n=40), which were randomly assigned to sham (SHM), ovariectomy (OVX), ovariectomy with exercise (OVXE), ovariectomy with acetic acid (OVXA), or ovariectomy with exercise and acetic acid (OVXAE) groups. Gut microbial diversity and abundance were assessed using next-generation sequencing (NGS).

RESULTS

Firmicutes were more abundant in the OVX group than in the SHM group. However, OVXA partially protected against the OVX-induced increase in Firmicutes (SHM, 50.6%; OVX, 57.2%; OVXE, 52.0%; OVXA, 50.6%; and OVXAE, 56.5%). In terms of microbial diversity, the acetic acid-supplemented groups (OVXA and OVXAE) showed greater microbial diversity than the SHM and OVX groups (p<.05). The phylogenetic cladogram analysis showed that the OVXA and OVXAE groups had lower levels of UCG009 and Akkermansia (p<.05) than the OVX group.

CONCLUSIONS

Ovariectomy increased the relative abundance of Firmicutes. Acetic acid supplementation partially attenuated the increased proportion of Firmicutes in ovariectomized mice. Notably, this study found that acetic acid supplementation showed a significant beneficial effect on microbial diversity in ovariectomized mice, independent of exercise intervention. Future research should explore the potential role of UCG009 in protecting against menopause-related metabolic dysfunction.

Keywords: Ovariectomy, Menopause, Acetic acid, Voluntary wheel running, Gut microbiome

INTRODUCTION

The microbiome refers to the entire genetic information of gut microorganisms, and intestinal microbes significantly contribute to maintaining human health [1]. Gut microorganisms constitute more than 90% of the total human microbiota, and function as symbiotic organisms that interact with high complexity and dynamics within the human [2,3]. Gut microorganisms contribute to maintaining metabolic function by producing short-chain fatty acids through fermentation, and microbial diversity has been reported to have a positive correlation with human health [4,5]. Dysbiosis of gut microorganisms can adversely affect immune and metabolic processes [6].

According to biological classification systems, Firmicutes and Bacteroidetes account for the highest proportions of gut microorganisms [7], and their ratio (Firmicutes/Bacteroidetes, F/B ratio) is closely related to metabolic capacity and can be used as a biomarker for obesity and weight changes [8]. While Firmicutes, as obesity-related bacteria, can contribute to weight gain by promoting energy extraction and storage, Bacteroidetes have been reported to show a significant effect on enhancing short-chain fatty acid metabolism [9,10]. Particularly, a decrease in Ruminococcaceae UCG009, which belongs to Firmicutes, is closely related to reductions in blood glucose and lipids and may also reduce the risk of atherosclerosis development [11]. However, studies on the role of UCG009 in energy metabolism are limited and remain unclear, necessitating further investigation.

Gut microbial diversity is classified into alpha diversity, which represents microbial diversity within individuals or populations, and beta diversity, which reflects differences between individuals or populations [12]. Many postmenopausal women experience obesity and metabolic disorders [13,14], and the menopause-related changes in body composition are related to decreased gut microbial diversity and increased F/B ratio [15-17]. Previous studies reported a negative correlation between obesity and microbial diversity [18] and weight gain and elevated F/B ratio in ovariectomized mouse models [19]. The decrease in estrogen hormone level may act as a factor that reduces gut microbial diversity and increases the F/B ratio in postmenopausal women [20-22]. A positive correlation between estrogen and gut microbial composition was also reported [23]. These previous research findings suggest the significant role of the gut microbial ecosystem in postmenopausal women and emphasize the need for strategic interventions to enhance gut microbial diversity.

Vinegar consumption has shown potential as an effective alternative therapy for improving obesity [24]. The main component of vinegar is acetic acid, and sufficient intake of acetic acid can help improve obesity [24]. Short-chain fatty acids, fermentation byproducts of gut microorganisms, affect various physiological aspects including host metabolism, blood pressure, blood glucose, immunity, and free radical scavenging related to cellular oxidative stress improvement, with acetic acid being the most abundant short-chain fatty acid in vinegar [25,26]. Intragastric administration of vinegar extract to mice showed positive effects on gut microbial composition and homeostasis, increasing the abundance of Bacteroidetes while decreasing Firmicutes [26]. According to previous research demonstrating the effects of vinegar on gut microbial composition using obese mice, the F/B ratio was significantly reduced in the vinegar-treated group compared to the control group [27]. Not only in obesity research but also in studies exploring perimenopausal and postmenopausal women, a negative correlation between serum acetic acid levels and obesity was found [28]. Thus, further investigation is required to determine whether acetic acid intake shows positive effects on gut microbial diversity following menopause and to identify the involvement of specific microorganisms.

Perimenopausal and postmenopausal women experience decreased physical activity levels [29]. Physical activity levels have been reported to have a positive correlation with adult fecal bacterial diversity and short-chain fatty acid levels [30,31]. While high-intensity interval training and resistance exercise programs did not affect gut microbial alpha diversity in postmenopausal women, they were reported to have positive effects on microbial species composition [32]. Not only in clinical trials but also in animal studies using C57BL/6J mice, research has shown that F/B ratio decreased after exercise interventions with voluntary wheel running [33]. While previous studies showed positive effects of exercise on gut microbiota, there are also studies indicating that stress from high-intensity exercise can increase gastrointestinal oxidative stress and inflammatory responses [34]. Forced exercise may induce physiological and psychological stress, thereby negatively affecting gut microbial composition [35]. Further research is needed to determine whether voluntary wheel running, which induces lower stress levels, may positively influence gut microbial diversity in menopausal mouse models. The ovariectomized mouse model creates an estrogen-deficient state by removing the sex hormone-secreting organs of female mice and is widely used in research exploring solutions for metabolic syndrome and other diseases occurring after menopause [36,37]. While acetic acid intake and exercise intervention may each have positive effects on gut microbial composition, research on whether the combined intervention of acetic acid and exercise has synergistic effects on gut microbial diversity and composition in ovariectomized mice remains unclear. This study aims to (1) investigate the effects of ovariectomy on gut microbial diversity and Firmicutes abundance in mice, (2) explore whether the combined intervention of acetic acid intake and exercise shows effects in improving gut bacterial dysbiosis in ovariectomized mice, and if so, which specific microorganisms are involved.

METHODS

1. Animals

Forty 8-week-old C57BL/6J female wild-type mice were randomly assigned to 5 experimental groups: (1) sham surgery control (SHM, n=8), (2) ovariectomy (OVX, n=8), (3) ovariectomy with exercise (OVXE, n=8), (4) ovariectomy with acetic acid intake (OVXV, n=8), (5) ovariectomy with combined exercise and acetic acid intake (OVXVE, n=8). The experimental environment was maintained with a 12-hour light/dark cycle, 50-80% humidity, and room temperature at 22±1°C. All mice were housed individually and provided with regular chow or acetic acid-containing chow (Rodent NIH-41KO, Zeigler Bros Inc., USA) and water ad libitum. All experiments in this study were approved by the Institutional Animal Care and Use Committee of Incheon National University (INU-ANIM-2021-04).

2. Experimental design

After the group assignment, dietary intake, body weight, and running wheel distance were measured weekly. Each experimental group (OVXE, OVXA, OVXAE) underwent designated interventions for 13 weeks. Exercise groups were provided with cages having a voluntary wheel running (wheel diameter: 10 cm, cage width: 13 cm, cage length: 23 cm, cage height: 14.5 cm). Running distance was monitored through a computerized monitoring system. After 12 weeks of intervention, body composition (whole-body fat and lean mass) was assessed. On the final measurement day of week 13, overnight-fasted mice were anesthetized using 2.5% tribromoethanol in 0.01 ml per gram of body weight. Approximately 0.5 cm of cecum tissue was excised, rapidly frozen in liquid nitrogen, and stored at -80°C for subsequent gut microbial analyses.

3. Research procedures

1) Ovariectomy surgeries

After anesthetization with 2% isoflurane through an inhalation system (RWD, Life Science Co., CA, USA) for approximately 5 minutes, loss of movement was confirmed by checking reflex activity via foot stimulation. Isoflurane concentration was maintained at 0.5% during surgery. The central dorsal area was shaved and disinfected, and then bilateral incisions of less than 0.5 cm were made in the muscle layer. After ovariectomy, 500 mg/kg acetaminophen was administered, and incision sites were closed using small animal wound clips. Animal status was monitored twice daily during the first week of the recovery period. For the SHM group, the same surgical procedure was performed, but the ovaries were only identified without removal.

2) Acetic acid intake

The acetic acid groups were fed chow containing 5% (g/g, weight of sodium acetate/weight of chow diet) acetic acid (sodium acetate; S2889; Sigma-Aldrich) for 13 weeks [38]. The acetic acid groups had free access to acetic acid-containing chow (Table 1).

3) Body composition

Body composition (whole-body fat and lean mass) was measured using dual-energy X-ray absorptiometry (DXA, GE Medical Systems Ultrasound & Primary Care Diagnostics, LLC, Madison, Wisconsin, USA). Fat and lean mass were measured using average values obtained from three scans performed for each animal using the DXA software program.

4) DNA extraction

Collected fecal samples were stored at -80°C until DNA extraction. For analysis, frozen samples were thawed at 4°C, and DNA was extracted from approximately 200 mg of sample using the DNA kit (SPINeasy DNA Kit for Feces, MP Biomedicals, Irvine, CA, USA) according to the manufacturer's instructions. Fecal samples were homogenized using a bead homogenizer (FastPrep-24TM 5G, MP Biomedicals, Irvine, CA, USA) at 6.0 m/sec for 40 seconds. Extracted DNA was analyzed through 1% agarose gel electrophoresis (AD160, Mupid-One, Takara Bio, Japan), and DNA integrity was confirmed using ChemiDoc (Bio-Rad, Hercules, CA, USA).

5) 16S rRNA-based amplicon sequencing

Extracted DNA was amplified through amplicon sequencing targeting the V4 region of the 16S rRNA gene. V4 region-specific primers and primers containing position-specific overhang sequences were used for PCR, as shown in Table 2. T100 Thermal Cycler (Bio-Rad, USA) was used for V4 region amplification, with reaction mixtures containing 5 ng DNA, 1 μL Amplicon PCR Reverse Primer, 1 μL Amplicon PCR Forward Primer, and 12 μL 2x KAPA HiFi HotStart Ready Mix. PCR thermal cycling conditions were: initial denaturation at 95°C for 3 minutes, followed by 25 cycles (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds), and final extension at 72°C for 5 minutes before storage at 4°C. Index PCR was performed using Nextera XT index kit V2 Set A (Illumina, San Diego, USA) to attach indices and Illumina sequencing adapters. Index PCR thermal cycling conditions used initial denaturation at 95°C for 3 minutes, followed by 8 cycles (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds), and final extension at 72°C for 5 minutes.

4. Data analysis

One-way ANOVA was performed to compare body composition and food intake between groups. Additionally, an independent t-test was performed to compare total running distance between OVXE and OVXAE groups. Raw sequence preprocessing was performed using Quantitative Insights Into Microbial Ecology 2 (QIIME2) v2024.10 pipeline. Denoising, including primer removal, quality filtering, error correction, chimera sequence removal, singleton removal, paired-end read merging, and deduplication, was performed using Divisive Amplicon Denoising Algorithm 2 (DADA2). Amplicon sequence variants (ASVs) were taxonomically classified by comparison with the SILVA 138.1 database at 99% similarity using a pre-trained Naïve Bayes machine learning classifier. ASV and taxonomy tables were converted from tab-separated values (.tsv) to comma-separated values (.csv) through QIIME2 pipeline, enabling sequence processing, statistical analysis, functional prediction, and marker gene data meta-analysis using Microbiome Analyst webbased R analysis platform [39,40]. Total sample reads numbered 551,505, with an average of 19,017 reads per sample (maximum 35,396, minimum 9,005). During data filtering, low count data below 4 was removed, with prevalence threshold set at 20%. Additionally, a low variance filter of 10% inter-quantile range (IQR) was applied to remove 387 low abundance features, and 28 additional low variance features were removed, resulting in 248 features selected for final analysis. Alpha diversity was analyzed at the feature level using Shannon and Fisher indices. Betweengroup comparisons were performed using Kruskal-Wallis post-hoc tests with FDR-adjusted p-values. Beta diversity was evaluated through Principal Coordinates Analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) based on Bray-Curtis index and Unweighted UniFrac distance. Additionally, the Linear Discriminant Analysis Effect Size (LEfSe) method was used to identify significant differences between groups [41]. All statistical significance levels were set at 0.05.

RESULTS

1. Metabolic characteristics

No significant differences in body weight and lean mass were found between all groups. For fat and epididymal fat mass, SHM showed significantly lower values in comparison to other groups (p=.048). In food intake, OVX was significantly lower than SHM, and OVXAE was significantly higher than OVX and OVXA (p<.05). For total running distance, OVXAE was significantly higher than OVXE (p<.001, Table 3).

2. Abundance of firmicutes and bacteroidetes

The relative proportions of Firmicutes (Bacillota) to total gut microbiota in each group were: SHM 50.6%, OVX 57.2%, OVXE 52.0%, OVXA 50.6%, OVXAE 56.5%, and the relative proportions of Bacteroidetes to total gut microbiota were: SHM 40.3%, OVX 33.7%, OVXE 38.3%, OVXA 39.1%, OVXAE 33.6% (Fig. 1). OVX, OVXE, and OVXAE showed a trend toward increased Firmicutes composition compared to SHM, but OVXA showed similar levels compared to SHM (p>.05).

3. Alpha diversity-Shannon index

Shannon Index represented α-diversity, including species richness and evenness of gut microbiota in each group (Fig. 2). Comparison of both species richness and evenness showed no statistically significant differences between groups (H=3.156, p=.532).

4. Alpha diversity-Fisher index

Fisher Index represented α-diversity of gut microbiota in each group under different environmental or experimental conditions (Fig. 3). Comparison results showed no statistically significant differences between groups (H= 4.894, p=.298).

5. Beta diversity―Bray-Curtis index

In principal coordinate analysis based on the Bray-Curtis index, Axis 1 and Axis 2 represented 13.2% and 11.0%, respectively, of the total variation of 24.2%, visually representing β-diversity between groups. PERMANOVA analysis showed significant differences in microbial species composition (p=.008, R²= 0.18602, Fig. 4). While analysis failed to show any group differences in gut microbial species composition between SHM and OVX, acetic acid treatment groups, OVXA and OVXAE, showed significant differences from SHM and OVX (SHM vs. OVXA, p=.032; SHM vs. OVXAE, p=.012; OVX vs. OVXA, p=.005; OVX vs. OVXAE, p=.01).

6. Beta diversity-Unweighted UniFrac index

In principal coordinate analysis based on Unweighted UniFrac index, Axis 1 and Axis 2 represented 12.3% and 10.2%, respectively, of the total variation of 22.5%, visually representing β-diversity between groups. PERMANOVA analysis showed significant differences in microbial species composition (p=.021, R²= 0.18065, Fig. 5). In gut microbial species composition, acetic acid treatment groups, OVXA and OVXAE, showed significant differences compared to OVX (OVX vs. OVXA, p=.009; OVX vs. OVXAE, p=.01).

7. Phylogenetic cladogram analysis

According to analysis results of specific gut microbial abundance changes between groups, no differences were found between SHM and OVX, but OVXA contained lower levels of UCG009 and Akkermansia than OVX (UCG009: OVX vs. OVXA, p=.031; Akkermansia: OVX vs. OVXA, p=.003; Fig. 6A). Similarly, OVXAE had lower levels of UCG009 and Akkermansia than OVX (UCG009: OVX vs. OVXAE, p=.03; Akkermansia: OVX vs. OVXA, p=.015; Fig. 6B).

DISCUSSION

This study investigated 1) whether menopause models induced by ovariectomy in C57BL/6J mice negatively affect gut microbial diversity and abundance of Firmicutes and Bacteroidetes, 2) whether combined intervention of acetic acid intake and exercise intervention positively affects microbial diversity changes caused by ovariectomy, and which specific gut microorganisms are involved. According to our results, ovariectomy showed a trend toward increased Firmicutes abundance compared to controls, but acetic acid intake protected against ovariectomy-induced increases in Firmicutes abundance. While overall gut microbial diversity assessment failed to show differences between ovariectomy and control groups, beta diversity assessment revealed that acetic acid intake had positive effects of increasing gut microbial diversity. Phylogenetic analysis of specific gut microorganisms showed that levels of UCG009, one of the Firmicutes, were statistically decreased in acetic acid intake groups. Taken together, our findings failed to demonstrate positive, synergistic effects of combined acetic acid intake and exercise intervention on gut microbiota of ovariectomized mice. However, acetic acid intake increased gut microbial diversity and decreased Firmicutes such as UCG009 in ovariectomized mice, demonstrating the potential role of acetic acid in improving gut microbial dysbiosis after menopause.

Numerous microorganisms exist in the human intestine, and these microorganisms play a major role in digesting foods that are not easily digestible. Firmicutes and Bacteroidetes are predominant bacteria in the intestine and play important roles in caloric intake from food and metabolic processes [8]. Firmicutes, as obesity-associated bacteria, can contribute to weight gain by promoting energy extraction and storage [9,10]. Bacteroidetes are important microorganisms that assist carbohydrate metabolism, particularly transforming complex carbohydrates such as polysaccharides and dietary fiber into short-chain fatty acids, and protecting the intestinal mucosal mucus layer to suppress infection [10,42]. Research has shown that obese mice have lower Bacteroidetes abundance but relatively higher Firmicutes abundance than lean mice [43]. According to our results, ovariectomy showed a trend toward increased Firmicutes abundance compared to controls. Postmenopausal women may experience increased risk of obesity due to decreased metabolic function, which may be closely related to decreased estrogen hormone levels or gut microbial imbalance [20,21,44]. Decreased blood estrogen is a major factor that reduces gut microbial diversity and increases F/B ratio [20-22], suggesting a negative correlation between estrogen and Firmicutes. Non-clinical studies using ovariectomized mice have also confirmed weight gain and increased Firmicutes composition following ovariectomy [19], and the results of these previous studies support our findings of increased Firmicutes abundance after ovariectomy.

Regular physical activity of moderate amount and intensity is closely associated with increased adult fecal bacterial diversity and increased short-chain fatty acids [30], and several studies have reported the beneficial effects of physical activity on gut microbial beta diversity [45,46]. These findings suggest that exercise intervention is essential for postmenopausal women because they may experience decreased levels of physical activity with decreased exercise motivation, which can adversely affect gut microbial diversity [29]. According to our results, body weight and body fat mass in exercise intervention groups (OVXE and OVXAE) were not significantly different from the ovariectomy group (OVX). Previous studies have reported no differences in body weight and energy expenditure between exercise and ovariectomy groups after voluntary wheel running intervention [36]. This partially explains our results and suggests that ovariectomized mice may have reduced exercise motivation due to decreased metabolic rate from estrogen deficiency. Indeed, a previous study reported that high-intensity interval exercise interventions showed positive effects on gut microbial species composition in postmenopausal women [32]. However, contrary to these previous research results, exercise alone in our beta diversity assessment did not significantly affect gut microbial diversity, and the group with combined exercise and acetic acid intake also showed no significant changes in increasing gut microbial diversity compared to the acetic acid intake-only group. Our initial hypothesis predicted that the combined intervention of exercise and acetic acid intake would provide positive, synergistic effects that the combined intervention group would show the highest gut microbial diversity among all experimental groups. However, our findings failed to match the initial hypothesis. While previous studies exist showing decreased Firmicutes abundance after voluntary wheel running exercise intervention in intact mice [33], no studies have examined Firmicutes changes after wheel running exercise intervention alone or combined with acetic acid intake in ovariectomized mice. More in-depth investigation is needed for a comprehensive potential underlying mechanisms that may inhibit the positive effects of exercise on gut microbiota in ovariectomized mice.

In our beta diversity assessment, acetic acid intake showed effects of increasing gut microbial diversity independent of exercise intervention. This demonstrates the potential ability of acetic acid intake to improve gut microbial dysbiosis in ovariectomized mice. Previous research supporting our results showed that vinegar administration containing acetic acid contributes positive effects to gut microbial homeostasis [26], and not only vinegar intake but also acetic acid intake has been reported to demonstrate beneficial effects on gut microbial composition (microbial species and diversity) in obese model mice [27]. Currently, no previous studies have investigated the role of acetic acid intake and its potential contribution to gut microbial diversity in postmenopausal women or ovariectomized rodent models. Only one cohort study exploring perimenopausal and postmenopausal women reported a negative correlation between serum acetic acid levels and obesity, and that decreased serum acetic acid levels are closely related to gut microbial imbalance [28]. Our study demonstrated the potential effectiveness of acetic acid intake to improve gut microbial dysbiosis in ovariectomized mice, and our findings may suggest future clinical studies aimed at determining whether acetic acid intake may have positive effects on gut microbial diversity changes following human menopause.

Ruminococcaceae UCG-009 belongs to Firmicutes and breaks down plant polysaccharides to produce glucose and short-chain fatty acids [47]. Excessive increases in UCG-009 levels can increase circulating blood glucose and lipids, potentially causing diabetes and obesity. According to our results, UCG-009 levels in OVXA and OVXAE were lower than in the OVX group, suggesting that acetic acid intake has a down-regulatory effect on UCG-009 levels independent of exercise intervention. As mentioned earlier, it has already been reported that dietary acetic acid intake can increase gut microbial diversity and decrease relative levels of Firmicutes and F/B ratio. To our knowledge, these findings are the first research suggesting the potential involvement of Ruminococcaceae UCG-009, providing initial insights into how acetic acid intake may influence specific gut microorganisms in ovariectomized mice.

Similar to UCG-009 results, we found that acetic acid intake also decreased Akkermansia levels in the current study. Akkermansia interacts with the colonic mucosa to perform functions such as mucin degradation, mucus layer protection, and intestinal immune regulation [48], and previous reports exist that Akkermansia species can constitute more than 1% of cecal microbial communities in mice [49]. According to research on transplanting fecal extracts from young mice to aged mice, Akkermansia levels, which were rarely found in aged mice before transplantation, significantly increased in abundance in aged mice following transplantation [50]. Interestingly, intestinal acetic acid levels also increased in aged mice following transplantation. While appropriate levels of Akkermansia can contribute positive effects to metabolism and increase intestinal acetic acid, reports exist that excessive proliferation of Akkermansia is associated with the development of obesity, diabetes, and inflammatory bowel disease [51]. Indeed, Akkermansia levels were significantly increased in mice consuming high-sugar diets as compared with controls, suggesting a potential negative role of Akkermansia in metabolic diseases. More research is needed on whether the abundance of UCG-009 and Akkermansia can be altered by female menopause or ovariectomy and whether exercise can restore these specific microbial levels to normal.

The first limitation of the current study is that we did not control exercise intensity and amount between animals during voluntary wheel running exercise intervention. Since physiological and psychological stress from forced exercise using a treadmill can increase gastrointestinal oxidative stress and inflammatory responses [34], this study used voluntary wheel running as the exercise intervention method to avoid inducing such stress. However, differences in exercise motivation and aerobic capacity may exist between animals, potentially causing large differences in exercise amount or intensity between individuals, even within the same group when using wheel running exercise. Due to the limitation, our study may have failed to demonstrate statistically significant effects of exercise intervention on gut microbiota. Follow-up studies are needed to determine whether exercise intervention, controlling exercise intensity and amount between animals through treadmill exercise intervention, contributes to changes in gut microbiota in ovariectomized mice. Research including sham surgery and exercise groups is also considered necessary to verify robust effects of exercise intervention. Second, in the current study, acetic acid was administered to animals by freely supplying chow containing acetic acid. Therefore, animals with low food intake may have consumed less acetic acid. Future follow-up studies using regular and quantitative administration of acetic acid via gavage are needed. Third, the current study analyzed gut microbial diversity and abundance through cecal feces. The cecum is a part of the large intestine with less fecal movement, and incomplete digestion due to indigestion may have affected gut microbial analysis results. Therefore, follow-up studies need to analyze gut microbiota from feces expelled outside the body or from other intestinal locations and present various indices that can indicate alpha diversity such as Chao1 and ACE (asexuality). Finally, this study showed positive effects of acetic acid intake on gut microbiota in ovariectomized animals through pre-clinical experiments. Although our findings provide foundational information for perimenopausal and postmenopausal women, careful interpretation is required before clinical application.

CONCLUSION

This study is the first research to investigate the effects of the combined intervention of acetic acid intake and exercise intervention on gut microbial abundance and diversity in ovariectomized rodent models. According to our results, ovariectomy showed a trend toward increased Firmicutes abundance, but acetic acid intake partially protected against the ovariectomy-induced increase in Firmicutes. Contrary to our hypothesis, the combined intervention of acetic acid intake and exercise failed to demonstrate synergistic effects on gut microbiota in ovariectomized mice. However, acetic acid intake significantly increased gut microbial diversity and showed decreases in Firmicutes such as UCG009 in ovariectomized mice, which suggests the potential ability of acetic acid to improve gut microbial dysbiosis in postmenopausal women.

Notes

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

Conceptualization: YM Park, J Cho; Data curation: Y Tan, KW Park, BJ Ryu, SM Lee; Formal analysis: B So, J Jang, J Park; Resources: C Kang, YM Park; Visualization: B So, Y Tan, H Lee; Writing - original draft: YM Park, Y Tan, KW Park; Writing - review & editing: H Lee, MH Hwang, YM Park, J Cho; Project administration and supervision: YM Park; Funding Acquisition: YM Park.

Fig. 1.

Phylum level differences between groups.

Fig. 2.

Alpha diversity-Shannon Index.

Fig. 3.

Alpha diversity-Fisher Index.

Fig. 4.

Beta diversity-Bray Curtis Index.

Fig. 5.

Beta diversity-Unweight UniFrac Index.

Fig. 6.

(A) Phylogenetic cladogram analysis of gut microbiome between OVX vs. OVXA. (B) Phylogenetic cladogram analysis of gut microbiome between OVX vs. OVXAE.

Table 1.

Dietary composition

Ingredient	Con	ACE
Calorie (kcal/kg)	4,078.87	4,078.63
Carbohydrates (%)	57.44	59.49
Cellulose (%)	3.00	1.96
Protein (%)	18.99	17.09
Fat (%)	4.32	3.46
Ash (%)	6.84	8.12

Table 2.

V4 region-specific primer sequences

515F-5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-GTGCCAGCMGCCGCGGTAA-3

806R-5’-GTCTCGT GGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCT-AAT-3

Table 3.

Animal characteristics

	SHM	OVX	OVXE	OVXA	OVXAE	p-value
Total mass (g)	23.0±0.38	24.0±0.63	24.1±0.70	24.6±0.53	24.5±0.80	NS
Fat mass (g)	1.0±0.35	2.3±0.55^a	2.4±0.50^a	3.1±0.37^a	2.0±0.61^a	.048
Lean mass (g)	21.6±0.62	21.3±0.59	22.0±0.51	20.9±0.37	21.8±0.68	NS
Food intake (g)	42.57±0.87	38.81±0.84^a	40.34±0.98	40.95±0.76	43.99±1.62^b,c	.027
Epi fat mass (mg)	274.69±31.96	824.89±108.09^a	923.28±158.59^a	860.09±83.54^a	660.49±114.47^a	<.05
Total running distance (km)	-	-	188.41±118.40	-	310.93±139.68^c	<.001

All data are represented as Mean±SE. NS: no significant difference between groups, p >.05; Epi fat: epididymal fat;

^a p<.05 versus SHM;

^b p<.05 vs. OVX

^c p<.05 vs. OVXE.

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