Research Article

Combined Herbal and Probiotic Intervention Enhances Behavioral Recovery after Chronic Unpredictable Mild Stress in Mice

Dicky Faizal Irnandi1,2, Evalina Izzatur Rochmah3, Daniel Stenly Chandra4, Nurdiana5, Margarita Maria Maramis6,7, Reny I’tishom8*

1Doctoral Program of Medical Sciences, Faculty of Medicine, Universitas Airlangga, Surabaya, 60131, Indonesia; 2Department of Biochemistry and Biomolecules, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia; 3Master Program of Biomedical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia; 4Bachelor Program of Medicine, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia; 5Department of Pharmacology, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia; 6Department of Psychiatry, Dr. Soetomo General Academic Hospital, Surabaya, 60286, Indonesia; 7Department of Psychiatry, Faculty of Medicine, Universitas Airlangga, Surabaya, 60131, Indonesia; 8Department of Biomedical Sciences, Faculty of Medicine, Universitas Airlangga, Surabaya, 60131, Indonesia; 9Research Group Medical Biology, Faculty of Medicine, Universitas Airlangga, Surabaya, 60131, Indonesia.

Received | December 30, 2026; Accepted | January 21, 2026; Published | August 08, 2026

*Correspondence | Reny I’tishom, Department of Biomedical Sciences, Faculty of Medicine, Universitas Airlangga, Jl. Prof. Dr. Moestopo No.47, Surabaya, 60131, East Java, Indonesia; Email: [email protected]

Citation | Irnandi DF, Rochmah EI, Chandra DS, Nurdiana, Maramis MM, I’tishom R (2026). Combined herbal and probiotic intervention enhances behavioral recovery after chronic unpredictable mild stress in mice. Adv. Anim. Vet. Sci., 14(8): 1803-1811.

DOI | https://dx.doi.org/10.17582/journal.aavs/2026/14.8.1803.1811

ISSN (Online) | 2307-8316

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Stress is defined as a stimulus that impairs physiological homeostasis, whether at the behavioral, cellular, or molecular level. It involves complex neurobiological mechanisms, including dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis, neuroinflammatory responses, neurotransmitter imbalances, and impaired neuroplasticity (Lages et al., 2021; Shirokova et al., 2025). The adverse impact of stress depends not only on its intensity but also on its duration. Although chronic stress lacks an exact definition, it is generally characterized by prolonged and repeated exposure to stressors (Tran and Gellner, 2023). Chronic stress has been shown to adversely affect animal welfare and behavioral performance in both experimental and veterinary contexts. This condition leads to alterations in emotional regulation and adaptive behavior, manifested as anxiety-like and depressive-like symptoms, including social withdrawal, reduced self-care, and altered locomotor activity (Wu et al., 2025; Hu et al., 2017). In translational research, chronic stress been implicated as a contributing factor that may impair translational validity, highlighting the importance of controlling stress-related variables in animal models. While the mechanisms underlying stress are well documented, the extent to which they directly relate to behavioral outcomes varies across models and remains an area of ongoing investigation.

The chronic unpredictable mild stress (CUMS) model is one of the most widely used paradigms for investigating stress-induced behavioral changes in animals due to its reliability in simulating complex cognitive and/or emotional processes. CUMS implies prolonged exposure of animals to a wide range of diverse stressors, which occur unpredictably (Alqurashi et al., 2022; Markov and Novosadova, 2022). Behavioral changes can be measured using several parameters, including the sucrose preference test (SPT) to assess sensitivity to reward and the open-field test (OFT) to assess psychomotor retardation (Hu et al., 2017; Alcantara et al., 2017).

Previous studies have reported that CUMS gradually induces anhedonia, reduced self-grooming, and decreased locomotor activity that persists over time. Selective serotonin reuptake inhibitors, fluoxetine, and citalopram have been shown to ameliorate anhedonia induced by chronic stress. However, several antidepressant treatments are known to further impair sexual function in male rodents, which may already be suppressed by the stressors (Petković and Chaudhury, 2022; Sylvester et al., 2019). Therefore, the exploration of alternative safe and effective therapeutic agents remains necessary for the development of stress-reduction strategies in experimental and veterinary models.

Tribulus terrestris, a Mediterranean plant belonging to the Zygophyllaceae family, has been traditionally used in Chinese and Indian medical practice to treat various diseases (Sanagoo et al., 2019; Gamal-El-Din, 2018). Previous studies have demonstrated that its beneficial effects on male reproductive function in both humans and animals are largely attributed to its antioxidant properties. Its primary active compound is protodioscin, a furostanol-type steroidal saponin (Haghmorad et al., 2019; Salgado et al., 2017). Interestingly, numerous studies suggest the neuroprotective potential of Tribulus terrestris. The administration of Tribulus terrestris has been associated with the attenuation of inflammatory mediators and cytokines in the prefrontal cortex and hippocampus, an increase in serotonin levels, and improvement of oxidative parameters (Baraka et al., 2025; Pavin et al., 2018). Another study reported alterations in HPA-axis-related hormone levels following Tribulus terrestris treatment, involving changes in corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH) (Wang et al., 2013). These findings indicate that Tribulus terrestris has been associated with neurobiological changes under experimental conditions. However, the relevance of these observations to behavioral outcomes in animals exposed to chronic stress has not been established.

Existing research has highlighted the bidirectional communication between the brain and the intestinal microbiota, known as the “microbiota-gut-brain axis”. This mechanism is responsible for maintaining homeostasis in the gastrointestinal tract and central nervous system, suggesting that disturbances in the microbiota-gut-brain axis may result in neuropsychiatric disorders (Du et al., 2020; Yang et al., 2017). Certain probiotics, referred to as psychobiotics, have been investigated for their specific value in improving behavioral and emotional regulation (Dinan et al., 2013). Among these, Bifidobacterium strains are widely studied psychobiotics whose abundance is significantly reduced in patients with depression (Li et al., 2023; Irnandi et al, 2025). Preclinical studies have explored the potential association between this intestinal probiotic and behavioral outcomes under chronic stress conditions (Huang and Liu, 2024). Importantly, evidence supporting these mechanisms is derived from heterogeneous models, and their direct applicability to the present behavioral model remains limited.

This study aimed to evaluate the effects of Bifidobacterium lactis and Tribulus terrestris, either alone or in combination, in mice exposed to CUMS. However, the present study was designed to evaluate behavioral outcomes only, and no neuroendocrine, inflammatory, or microbiome-related parameters were assessed.

Materials and Methods

Study design

This study employed a two-stage experimental design, consisting of the validation of a chronic stress model using the CUMS protocol, followed by recovery analysis. Animals were divided into six groups: Control, CUMS-only, placebo, Tribulus terrestris (TT), Bifidobacterium lactis (BL), and a combination of Tribulus terrestris and Bifidobacterium lactis (BLTT). To ensure that all groups reached the endpoint simultaneously, a staggered timeline was applied. Groups receiving treatments (placebo, TT, BL, and BLTT) initiated the experiment earlier and underwent intervention after CUMS exposure, whereas the control and CUMS-only groups started two weeks later without post-CUMS treatment. At the end of the experimental day, corresponding to the last stress exposure period in the control and CUMS-only groups, and the last intervention in the treatment groups, all mice underwent behavioral assessments, including the OFT, defecation frequency, coat state measurement, and the SPT to confirm stress-induced deficits.

Experimental animals

BALB/C mice (8-10 weeks old, 28-30 g in weight) were obtained from Balai Penyidikan dan Pengujian Veteriner, Malang. Male mice were used to reduce variability associated with estrous cycle-related hormonal influences on stress responses. They were housed in a clean and well-ventilated laboratory under controlled environmental conditions: temperature 22-24 ºC, relative humidity 50-60%, and a 12-hour light-dark cycle (lights off at 06.00 pm). Mice were maintained in groups of five within plastic boxes lined with wood chip bedding (cage size: 17.5 x 23.75 x 17.5 cm) and were provided free access to a standard diet and water. After seven days of adaptation, mice were assigned to the experimental groups (n = 5 per group). Each group was housed in two cages (2-3 mice per cage) to minimize cage-related bias. All of the protocols related to experimental animals were performed according to the guidelines and approved by the Health Research Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia, with the reference number 356/EC/KEPK-S3/10/2024. All efforts were considered to minimize animal suffering.

CUMS procedure

The CUMS model was established using a modified method by a previous study (Nollet, 2021). Mice in CUMS-only and treatment groups (placebo, TT, BL, and BLTT) were subjected to a series of mild psychological stressors over 4 weeks to induce behavioral and physiological phenotypes associated with depression and anxiety. The stressors were applied according to a varied schedule, with no stressor repeated on two consecutive days to minimize habituation for the animals. The control group did not receive any stressors. The detailed stressor schedule is shown in Table 1.

Treatment administration

Following CUMS exposure, the TT, BL, and BLTT groups received their respective treatments for an additional two weeks. Tribulus terrestris was purchased from Biosan and produced by Metiska Farma, Indonesia. The Tribulus terrestris extract contained 60% protodioscin as the active compound. Tribulus terrestris was dissolved in fresh water at a dose of 2.25 g/kg body weight (Wang et al, 2013). Bifidobacterium lactis was obtained from Green Jeeva LLC, US. The powdered preparation of Bifidobacterium lactis was dissolved in 0.2 mL of NaCl 0.9%, containing approximately 1 × 109 CFU (Aoki et al, 2016). All treatments were administered by daily oral gavage at 07:00 am. Mice in the placebo group were given the same volume of vehicle solution.

 

Table 1: Chronic unpredictable mild stress model schedule.

Week	Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
1	Cage tilting (45º) for 12 hours	Water deprivation for 24 hours	Predator exposure for 15 minutes	Overnight illumination for 24 hours	Reversed light/dark cycle for 24 hours	Cold water swimming (4ºC) for 1 minutes	Restraint stress for 2 hours
2	Food deprivation for 24 hours	Narrow dark space isolation for 4 hours	Cage tilting (45º) for 12 hours	Restraint stress for 2 hours	Cold water swimming (4ºC) for 1 minutes	Predator exposure for 15 minutes	Wet bedding for 24 hours
3	Cage tilting (45º) for 12 hours	Wet bedding for 24 hours	Water deprivation for 24 hours	Predator exposure for 15 minutes	Overnight illumination for 24 hours	Narrow dark space isolation for 4 hours	Cold water swimming (4ºC) for 1 minutes
4	Overnight illumination for 24 hours	Reversed light/dark cycle for 24 hours	Food deprivation for 24 hours	Wet bedding for 24 hours	Narrow dark space isolation for 4 hours	Cold water swimming (4ºC) for 1 minutes	Restraint stress for 2 hours

 

Note: Each stressor was applied once daily according to the schedule shown. C: Celsius.

 

Open-field test (OFT)

The OFT was performed to assess spontaneous locomotor activity and exploratory behavior in animal models. The observation room was kept quiet with low-intensity lighting. Before testing, animals were allowed to habituate to the room for five minutes to minimize the distraction associated with an unfamiliar environment. Each mouse was then placed in the central area of the open-field arena of an uncovered plexiglass box (40 x 40 x 40 cm) with a dark floor. Their exploration was observed for 6 minutes using a video camera and was analyzed using specialized tracking software EthoVision XT. Behavioral parameters recorded included movement trajectories, heatmaps of spatial occupancy, total distance travelled, cumulative time spent in the center zone, and the frequency of center zone entries (CZE).

Defecation frequency was calculated manually during OFT. The equipment was cleaned with a 70% ethanol solution between tests to prevent olfactory effects. The physical condition was also observed by quantifying the coat state to assess the mice’s motivation toward grooming activities. It was measured by examining seven body areas (head, neck, back, abdomen, tail, forepaws, and hind paws) with a score of 0 (good) for smooth and clear fur, 0.5 (moderate) for slightly fluffy fur with some spiky patches, and 1 (bad) for bristling with spikes. The total score was obtained by summing the scores of all body parts (Nollet, 2021).

Sucrose preference test (SPT)

The SPT was carried out to analyze the relative preference for sucrose in reward sensitivity (Belovicova et al., 2017). Before the test, each mouse was housed individually and was provided with a 1% sucrose solution only for 48 hours to allow adaptation. Thereafter, the mice were deprived of food and water for 24 hours. For the following period of 1 hour, mice were presented with pre-weighed two bottles containing 1% sucrose solution and tap water simultaneously. The bottle positions were changed several times to minimize side preference. At the end of the test, the sucrose preference was determined by dividing the volume of sucrose consumed by the total volume of fluid intake during the experiment.

Data analysis

All statistical analyses were carried out using SPSS version 25.0. The distribution test and homogeneity of variance test were performed to determine that the data fulfilled the assumptions. To validate stress induction, independent-samples t-tests were used to compare the control and CUMS-only groups. For recovery analysis, only post-stress treatment groups were included. One-way ANOVA was performed to test for overall treatment effects following stress cessation. When the ANOVA was significant, Dunnett’s post hoc test was applied to compare each intervention group against placebo as the spontaneous recovery control, thereby controlling for multiple comparisons. Data were presented as mean ± standard deviation (SD). A value of p < 0.05 was considered statistically significant.

Results

Validation of the CUMS model

CUMS-exposed mice demonstrated reduced exploratory activity and center zone engagement in the open-field area, as illustrated by movement trajectories and heatmaps (Figure 1A, B). These qualitative observations were consistent with previously reported CUMS models and supported the subsequent quantitative analyses. Stress-exposed mice showed a significant reduction in total distance traveled compared with controls, indicating decreased locomotor activity (Figure 2A; p < 0.05). The CUMS group showed significantly reduced cumulative center time and CZE, reflecting decreased exploration drive commonly presented in anxiety-like conditions (Figure 2B, C; p < 0.05). Chronic stress further impaired self-care behavior and increased autonomic stress responses, as shown by deterioration of coat state and increased defecation frequency (Figure 2D, E; p < 0.05). SPT showed a significant decrease in sucrose consumption following CUMS exposure, which suggested a decrease in reward sensitivity (Figure 2F; p < 0.05). Collectively, these findings demonstrated the reliable establishment of chronic stress characteristics, validating the CUMS model used in this study.

 

 

Behavioral outcomes

Behavioral parameters among placebo and treatment groups were compared to assess whether behavioral deficits induced by chronic stress improved spontaneously or were modified by interventions.

Locomotor activity

Placebo-treated mice showed limited outcomes in locomotor activity, as indicated by reduced total distance traveled, whereas all intervention groups demonstrated significantly greater locomotor activity compared with placebo (Figure 3A; Dunnett-adjusted p < 0.05). Increased locomotor activity was accompanied by enhanced center exploration, indicating meaningful behavioral activation rather than nonspecific hyperactivity.

Exploratory behavior

One-way ANOVA across post-stress groups revealed a significant effect of treatments on exploratory behavior as indicated by cumulative center time and CZE (Figure 3B, C; p < 0.05). Dunnett’s post hoc analysis demonstrated that TT, BL, and BLTT groups exhibited greater recovery of center-related exploration compared with placebo, with BLTT interventions showing the greatest improvements.

Self-care and autonomic stress response

Impaired self-care behavior and increased autonomic stress responses persisted in placebo-treated mice following stress cessation. One-way ANOVA across post-stress groups identified a significant treatment effect for both parameters (Figure 4A,B; p < 0.05). Dunnett’s post hoc analysis showed that intervention groups, particularly BL and BLTT, attenuated both coat state score and defecation frequency compared with placebo.

 

 

Anhedonia-like behavior

Sucrose preference remained significantly impaired in the placebo-treated mice following stress cessation. One-way ANOVA demonstrated a significant effect of treatment on sucrose preference (Figure 4C; p < 0.05). Dunnett’s post hoc comparisons revealed that TT, BL, and BLTT groups exhibited significantly greater recovery of sucrose preference compared with placebo. The combined BLTT group demonstrated the greatest improvement, indicating enhanced recovery from stress-induced anhedonia.

Discussion

The present study demonstrates that persistent mild stress induced by CUMS results in multiple disturbances relevant to animal behavior and welfare. CUMS is widely employed as an experimental paradigm to model chronic mild stress in rodents without causing overt physical harm. Unlike acute stress models, CUMS mimics prolonged exposure to unpredictable, low-intensity stressors, making it particularly suitable for studying persistent, subclinical behavioral alterations relevant to animal welfare contexts (Markov and Novosadova, 2022; Strekalova et al., 2022).

Stressed animals consistently show a general decline in psychomotor function, an impaired capacity to meet physiological needs, and inappropriate adaptive responses. Reduced locomotor activity reflects withdrawal, fatigue, or motivational deficits, whereas reduced center engagement represents avoidance under anxiogenic conditions. These results reflect anxiety-like behavior in animals, as their immediate response to a new environment is displayed as excitement, emotion, and stress responses. However, this cannot be interpreted as anxiety per se, since emotional states are not directly assessed (Belovicova et al., 2017; Seibenhener and Wooten, 2015). Grooming represents a basic maintenance activity in animals, and its disruption is considered a sensitive marker of compromised well-being (Nollet, 2021). Increased defecation frequency has been associated with autonomic hyperresponsiveness triggered by fear and emotional arousal. Rodents typically exhibit a preference for sweet foods over other types of diet. The decreased drive for foods represents anhedonia, or the inability to experience pleasure, which is a core feature of depression-like states in animal models (Belovicova et al., 2017; Verharen et al., 2023; Willner, 2005). This study corroborates earlier studies identifying anhedonia as the most commonly reported effect of chronic stress (Alcantara et al., 2017).

Notably, placebo-treated mice, used to represent spontaneous recovery after chronic stress, exhibited limited behavioral improvements. Although cessation of chronic stress resulted in partial spontaneous recovery, placebo-treated mice continued to exhibit residual behavioral deficits. This outcome aligns with previous reports indicating that prolonged stress exposure leads to sustained behavioral alterations even after the stressor is removed. Such persistence may be associated with long-lasting neurobiological and behavioral adaptations. Chronic stress has been shown to induce enduring changes in neural circuitry, synaptic plasticity, and stress-response systems, which may not rapidly normalize once stressors are removed (McEwen et al., 2013; Bloss et al., 2010). From an animal welfare perspective, these findings highlight that prolonged exposure to mild but unpredictable stressors may induce behavioral impairment that extends beyond the stress period itself. Such conditions are relevant in both experimental and managed animal environments, where repeated handling, environmental demands, or procedural interventions may cumulatively generate low-grade stress. The incomplete spontaneous recovery observed in placebo-treated animals underscores the potential need for supportive strategies to promote behavioral resilience.

Administration of Bifidobacterium lactis and Tribulus terrestris was associated with consistently greater behavioral improvements beyond stress cessation compared to placebo. This distinction is critical, as it allows treatment-associated recovery to be distinguished from spontaneous normalization following stress withdrawal, thereby strengthening the interpretation that the observed behavioral improvements are attributable to the interventions. Tribulus terrestris exhibited moderate behavioral benefits, suggesting involvement in regulating stress responses in the present model. However, the outcomes were less marked than those observed with probiotic supplementation. Despite being primarily associated with reproductive and endocrine mechanisms, the observed recovery in anxiety-like behavior points toward broader psychobiological processes that warrant further investigation (Sirotkin and Kolesarova, 2021; Wang et al., 2013).

The probiotic intervention, either Bifidobacterium lactis alone or in combination with Tribulus terrestris, was associated with greater improvements in multiple behavioral measures. The observed effects were not interpreted as treatment superiority but rather as convergent recovery signals across exploratory, self-care, autonomic, and reward sensitivity behaviors. These findings align with growing evidence that implicates the gut-brain axis in stress regulation through the modulation of neuroinflammatory signaling, HPA axis activity, and neurotransmitter availability (Bravo et al., 2011; Chevalier et al., 2020; McVey et al., 2018). Accordingly, gut-associated modulation may influence behavioral responses to stress, supporting the relevance of probiotic-based approaches in animal stress management. In particular, Bifidobacterium lactis has been shown to improve depressive-like and anxiety-like behaviors in rodents exposed to chronic stress (Huang et al., 2022). Although this study was not designed to examine pharmacological synergy, the observed behavioral patterns suggest that interventions with conceptually different targets may be associated with complementary behavioral effects. However, this study was exploratory in nature, and the findings should be interpreted as behavioral associations rather than mechanistic evidence.

A key strength of this study is the consistent improvement observed across several behavioral parameters. This consistency across behavioral measures makes it less likely that the observed effects are attributable to nonspecific increases in general activity or arousal. By integrating OFT, coat state, defecation frequency, and sucrose preference assessments, the study offers a more comprehensive view of behavioral recovery following chronic stress. In addition, this study used Dunnett’s test to compare the treatment groups against placebo as the reference, which minimizes inflation of false-positive findings while specifically addressing recovery beyond stress cessation.

Several limitations should be acknowledged. The relatively small sample size limits generalizability. Although statistically significant differences were observed, effect estimates should be interpreted with caution and confirmed in larger studies. Animals were housed in multiple cages per group to reduce potential cage-related bias; however, residual cage effects cannot be completely ruled out. Due to the staggered study design, mice in the treatment groups were older at endpoint testing than those in the control and CUMS-only groups. Although recovery analyses were conducted among groups assessed at the same endpoint, age-related influences on behavioral outcomes, particularly locomotor activity, cannot be fully excluded. The study focused primarily on exploratory behavioral outcomes and did not include direct assessments of neuroendocrine, inflammatory, or microbiome-related mechanisms. Consequently, these findings remain preliminary rather than confirmatory and should be interpreted cautiously when considering their relevance beyond experimental animal models.

Despite these limitations, the current study provides valuable insights into the persistence of stress-induced behavioral deficits and the potential for probiotic and herbal interventions to support recovery. The consistent benefits observed with Bifidobacterium lactis and Tribulus terrestris suggest that integrative approaches targeting both gut-related and phytochemical pathways could be relevant in such conditions. Future studies should aim to elucidate the underlying mechanisms, extend behavioral assessments, and evaluate dose-response relationships. These efforts may further define the potential role of combined probiotic and herbal strategies in mitigating the behavioral impact of chronic stress.

Conclusion

Persistent mild stress induced by a CUMS protocol produces multidimensional behavioral disturbances in mice that are only partially resolved following stress cessation. Probiotic supplementation of Bifidobacterium lactis combined with Tribulus terrestris was associated with enhanced behavioral improvements across locomotor activity, exploratory, self-care, autonomic, and reward-related parameters. These findings support further investigation of integrative, non-pharmacological strategies for mitigating stress-related behavioral disturbances in animal and veterinary contexts.

Acknowledgements

The authors would like to express their gratitude to the Center for Higher Education Funding and Assessment and the Indonesian Endowment Fund for Education for funding this study through Beasiswa Pendidikan Indonesia (Indonesian Education Scholarship) under contract number 3173.2/3/UN10.F08/PN/2023.

Novelty Statement

This study presents novel evidence by integrating in silico and in vivo approaches on the interaction between probiotic and its metabolites with stress-related neuroendocrine and reproductive parameters. Using a stress-induced animal model, our work extends previous findings and offers mechanistic insights into how probiotics may influence cortisol, testosterone, and reproductive function.

Authors Contribution

DFI: Conceptualization, funding acquisition, investigation, writing original draft, project administration, data curation, formal analysis. EIR: Investigation, writing original draft, writing review and editing. DSC: Investigation, software, visualization. N: Methodology, resources, supervision. MMM: Supervision, resources, validation. RI: Conceptualization, methodology, supervision, validation. All authors have read and approved the final manuscript.

Generative AI and AI-assisted technology statement

The authors declare that artificial intelligence (AI) tools were used solely for spelling and grammar checking. AI was not used to generate any scientific content, data, interpretations, or conclusions. All content in this manuscript remains the full responsibility of the authors and was carefully reviewed under the authors’ supervision.

Conflict of interest

The authors have declared no conflict of interest.

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