Sexual aggression and prenatal stress lead to poor maternal care and aggressive behaviour in male Wistar rat offspring

Elvis Mbiydzenyuy Ngala; Sian Megan Joanna Hemmings; Jacqueline Samantha Womersley; Thando W Shabangu; Lihle-Appiah Qulu

doi:10.1016/j.ibneur.2026.02.012

. 2026 Feb 17;20:332–351. doi: 10.1016/j.ibneur.2026.02.012

Sexual aggression and prenatal stress lead to poor maternal care and aggressive behaviour in male Wistar rat offspring

Elvis Mbiydzenyuy Ngala ^a,^⁎, Sian Megan Joanna Hemmings ^b,^c, Jacqueline Samantha Womersley ^b,^c, Thando W Shabangu ^a, Lihle-Appiah Qulu ^a

PMCID: PMC12933451 PMID: 41756405

Abstract

Sexual aggression and prenatal stress can exert profound intergenerational effects, disrupting maternal care and enhancing aggression in offspring via alterations to the hypothalamic–pituitary–adrenal (HPA) axis and related neurochemical systems. This study in Wistar rats examined the combined impact of male sexual aggression and prenatal stress on maternal caregiving behaviours, and the neurobiological mechanisms underlying sexually aggressive behaviours in male offspring (F1). Following exposure to sexual aggression, females were mated with group-housed or isolated males and assigned to prenatal stress or control conditions. Maternal care was quantified from postnatal day (PND) 2–8, while F1 male aggression was assessed in resident–intruder and sexual aggression paradigms. Neurochemical analyses measured arginine vasopressin (AVP), corticotropin-releasing hormone (CRH), serotonin, oxytocin, corticosterone, and neurokinin B in the prefrontal cortex, hippocampus, amygdala, and hypothalamus, alongside gene expression profiling. Prenatal stress significantly reduced maternal care—particularly nursing and licking—across PND 2–8, with deficits most pronounced when paired with paternal isolation. High-quality maternal care was associated with reduced F1 aggression, longer attack latencies, and lower CRHR1 expression in the hippocampus, suggesting a neuroprotective role. Prenatal stress increased CRHR1 expression in the amygdala and amplified aggression, anxiety-like behaviours, and reduced sociability. Serotonin correlated negatively with aggression and positively with non-social exploration, while corticosterone correlated positively with aggression. Oxytocin was linked to social behaviours, and CRH to exploratory behaviours, indicating distinct neuromodulatory pathways. These findings highlight the interactive effects of maternal and paternal environments on intergenerational behavioural programming and identify key neurochemical targets for mitigating stress-related aggression.

Keywords: HPA axis, Maternal care, Aggression, Prenatal stress, Sexual aggression, Neuromodulators

Highlights

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Maternal care influenced by litter size and postnatal day, showing adaptability.
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Prenatal stress reduced maternal care and increased offspring aggression.
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Maternal care associated with reduced offspring aggression, buffering effects of stress.
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Gene expression showed neuroprotective effects of maternal care and impact of prenatal stress.
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Neurochemical analysis highlighted serotonin's role in social behaviours and corticosterone's link to aggression.

1. Introduction

Sexual aggression is a complex phenomenon with far-reaching consequences, not only for the immediate victims but also for potentially influencing subsequent generations (Mbiydzenyuy et al., 2024). The intricate dynamics between environmental stressors, such as sexual aggression and prenatal stress, and their effects on behaviour and physiology, are critical areas of research, particularly concerning maternal care and the propagation of aggressive behaviours in progeny. Central to understanding these interactions is the hypothalamic–pituitary–adrenal (HPA) axis, which is integral to stress response regulation and is closely associated with both aggressive behaviours and parental care (Sheng et al., 2021). Disruptions in the HPA axis, triggered by stress or trauma, can lead to significant behavioural and physiological modifications, impacting parenting practices, anxiety, and aggression as noted by Murphy et al. (2022).

Prenatal stress represents a particularly sensitive form of environmental adversity, as it coincides with critical periods of fetal brain development during which neuroendocrine and limbic circuits are being organized. Exposure to elevated maternal glucocorticoids during gestation can alter placental barrier function, fetal glucocorticoid receptor expression, and neural circuit maturation, leading to persistent recalibration of HPA-axis function in offspring (Eick et al., 2020, Mbiydzenyuy et al., 2022, Shapiro et al., 2013, Tanpradit and Kaewkiattikun, 2020, de Kloet et al., 2005). These organizational effects influence basal and stress-induced glucocorticoid secretion later in life, thereby shaping emotional regulation, stress reactivity, and social behaviour in adulthood. Prenatal stress has been shown to exert enduring effects on offspring stress regulation systems and neurodevelopment, with downstream consequences for behavioural outcomes (Bale, 2015, Provencal and Binder, 2015). This has strengthened the concept that maternal stress can exert transgenerational influences on behaviour and physiology (Jagtap et al., 2023). Environmental stressors can precipitate sustained changes in gene expression within the HPA axis and other neurobiological pathways associated with aggression and social behaviour, thereby influencing both maternal care capacity and the development of aggressive phenotypes in offspring (Mbiydzenyuy et al., 2022, de Kloet et al., 2005)

In addition to direct fetal programming, prenatal stress may indirectly shape offspring behaviour through stress-induced alterations in postnatal maternal care. Experimental studies have demonstrated that dams exposed to stress during gestation often exhibit reduced licking, grooming, and nursing behaviours, which in turn modify offspring HPA-axis feedback sensitivity and limbic system reactivity (Dogani et al., 2025, Wu et al., 2024). Such maternal care–mediated pathways provide a critical mechanism by which prenatal adversity is translated into stable behavioural traits, including heightened aggression and altered social responsiveness across the lifespan. Neurochemical systems, including arginine vasopressin (AVP), corticotropin-releasing hormone (CRH), testosterone, serotonin, and oxytocin, along with their respective receptors in distinct brain regions, play pivotal roles in mediating social and aggressive behaviours. AVP and CRH, core regulators of the stress response, have been strongly implicated in the modulation of aggression and social dominance (Beurel and Nemeroff, 2014, Yadawa and Chaturvedi, 2016). Their actions within the amygdala and hypothalamus influence territorial aggression, social hierarchy formation, and stress reactivity in rodents (Ronan et al., 2023, Zuloaga et al., 2020). Testosterone is closely associated with sexual and aggressive behaviours, with its effects mediated through androgen receptors expressed in regions such as the hypothalamus and prefrontal cortex (Batrinos, 2012). Serotonin exerts inhibitory control over aggression through multiple receptor subtypes distributed across cortical and limbic regions, modulating impulse control, risk assessment, and social decision-making (da Cunha-Bang and Knudsen, 2021). Oxytocin, traditionally linked to social bonding and stress buffering, acts via oxytocin receptors in the amygdala and hippocampus and has been shown to regulate social recognition, maternal behaviour, and stress responsivity in both rodents (Fulenwider et al., 2024), and humans (Powell et al., 2019).

Beyond maternal pathways, growing evidence demonstrates that paternal experience prior to conception can exert lasting effects on offspring neurodevelopment, behaviour, and stress responsivity. Across vertebrate taxa, including teleost fish, rodents, and mammals, paternal social environment, dominance status, and stress exposure have been shown to shape offspring aggression, anxiety-like behaviour, and social competence through non-genomic mechanisms such as sperm RNA, epigenetic modifications, and altered seminal signalling (Cavallino et al., 2023, Franks et al., 2023, Hellmann and Rogers, 2024, Hellmann et al., 2015, Hellmann et al., 2020). In fish models in particular, paternal social isolation and instability of social rank robustly predict offspring behavioural phenotypes, highlighting the evolutionary conservation of paternal effects on stress- and aggression-related outcomes (Hellmann et al., 2020). In mammals, paternal stress and social isolation similarly alter vasopressinergic, serotonergic, and HPA-axis–related pathways in offspring, contributing to heightened aggression, impaired social behaviour, and altered anxiety phenotypes (Perkeybile and Bales, 2015, Speranza et al., 2024). Despite this expanding literature, paternal experience is rarely examined alongside prenatal stress and maternal care within a single experimental framework, limiting mechanistic understanding of how maternal and paternal environments interact to shape intergenerational vulnerability or resilience.

From an evolutionary and biological perspective, aggression is a highly conserved behavioural trait that plays a central role in adult survival and reproductive success. In males, aggressive behaviour facilitates mate competition, territorial defence, establishment of dominance hierarchies, and access to reproductive opportunities. While adaptive levels of aggression are essential for these functions, dysregulated or excessive aggression reflects maladaptive neurobiological organization, often arising from early-life stress exposure. Consequently, aggression serves as a sensitive behavioural readout of how prenatal stress reorganizes stress–social interaction networks with direct relevance to mating and competition in adulthood (Levy et al., 2025, Mbiydzenyuy et al., 2024).

The present study aimed to investigate how maternal and paternal stress-related environments jointly shape intergenerational behavioural and neurobiological outcomes. Specifically, we examined the effects of exposure to male sexual aggression and prenatal stress on maternal care behaviours in Wistar rats, and assessed whether such exposure exacerbates sexually aggressive behaviours in male offspring towards non-receptive females. By assessing changes in gene expression associated with the HPA axis and other neurobiological pathways alongside behavioural and neurochemical outcomes, this study sought to elucidate convergent mechanisms underlying the intergenerational transmission of aggressive behaviour and stress-related dysregulation. In addition to maternal prenatal stress, the paternal social environment was incorporated as an independent experimental factor, given its well-established role in shaping male aggression, stress responsivity, and social behaviour. Social housing versus isolation of males was therefore used to systematically modulate the severity of sexual aggression experienced by females prior to and during mating, allowing examination of how paternal social context interacts with prenatal stress to influence maternal care and offspring outcomes. Importantly, by integrating prenatal stress exposure, maternal care assessment, paternal social environment, and offspring aggression within a single experimental framework, the present work extends existing literature and provides a more comprehensive understanding of how early-life adversity shapes biologically meaningful behavioural phenotypes across generations.

2. Methods

2.1. Animals and housing conditions

Peripubertal virgin male (200 g - 230 g) and female (180 g - 200 g) Wistar rats Rattus norvegicus were obtained from Stellenbosch University Animal Breeding Facility. Animals were housed in standard laboratory cages (55 ×35 x 20 cm) with sawdust bedding under a controlled environment with a 12-hour inverted light/dark cycle, (lights off at 06:00 h and lights on at 18:00 h), ambient temperature maintained at 22 ± 2°C, and relative humidity at 55 ± 5 %. Food and water were provided ad libitum. Social housing consisted of 2–3 rats per cage, whereas isolated animals were housed singly to experimentally manipulate the social environment. All experimental procedures adhered to ethical guidelines approved by the Stellenbosch University’s Research Ethics Committee: Animal Care and Use (ACU-2021–13333), following the Guide for the Care and Use of Laboratory Animals.

2.2. Grouping and Experimental Setup

All animals underwent a 7-day habituation period before experimental procedures commenced. Post-habituation, male rats were randomly divided into group-housed (socials, GHSM, n = 10) and isolation (isolates, ISM, n = 10) groups. This manipulation was implemented to systematically vary the paternal social environment, as social isolation in males is known to enhance aggressive, stress-reactive, and sexually coercive behaviours, thereby allowing controlled modulation of the intensity of sexual aggression experienced by females. A third group of adult male rats (sexually experienced rats, n = 5) were used to check for receptivity in experimental female rats. Another set of adult male rats (n = 5) were used as intruders in the resident intruder test. Isolated male rats were housed singly, while social rats were kept in groups of 2–3 per cage. Female rats were divided into two groups: those exposed to group-housed (GHSF, n = 10) and isolated (ISF, n = 10) males during the SxAT. This design allowed the paternal housing condition to function as an independent experimental variable rather than a confound, enabling assessment of how male social environment interacts with prenatal stress to influence maternal care and offspring behavioural outcomes. All experimental groupings and behavioural assessments are summarized schematically in Fig. 1.

This schematic illustrates the experimental design and temporal sequence used to investigate the effects of male sexual aggression and prenatal stress on maternal care and offspring behaviour across two generations (F0 and F1). In the F0 generation, adult male rats were allocated to six groups: group-housed control males (GHCM), group-housed sexual males (GHSM), isolated sexual males (ISM), isolated control males (ICM), group-housed sexual females (GHSF), and isolated sexual females (ISF) (all n = 10 per group). ISM and ICM males underwent 7 days of social isolation (DAY 8; PND 63), while GHCM and GHSM males remained group housed. All F0 males were subjected to a resident–intruder test (DAY 15; PND 70). Sexual aggression was assessed in GHSM and ISM males using the Sexual Aggression Test (DAY 16; PND 71), followed by a second resident–intruder test (DAY 20; PND 75) to assess post-exposure changes in aggression. Anxiety-like behaviour was evaluated on DAY 21 (PND 76), after which a subset of F0 males was euthanized on DAY 22 (PND 77) for downstream analyses, while the remaining males were retained for breeding. At PND 77, selected F0 males were mated with either GHSF or ISF females to generate the F1 generation. Pregnant females were subsequently divided into prenatal stress and no prenatal stress groups. Prenatal stress was applied during gestational days (GND) 11–22, as indicated in the schematic. In the F1 generation, only male offspring were studied. Maternal care behaviour was assessed during the early postnatal period (PND 2–PND 7), followed by weaning at PND 21. Based on paternal housing condition and prenatal stress exposure, F1 males were categorized into four groups: stressed group-housed (SGH), non-stressed group-housed (NSGH), stressed isolation-housed (SIH), and non-stressed isolation-housed (NSIH) (n = 10 per group). Adult F1 males underwent a resident–intruder test (PND 56), followed by the Sexual Aggression Test (PND 57–PND 60), a second resident–intruder test (PND 61), and an anxiety-like behaviour test (PND 62). All F1 males were euthanized on PND 63 for neurochemical, gene expression, and molecular analyses. Annotations within the schematic denote the exact experimental days and postnatal days (PND) at which each procedure was performed, providing a detailed temporal framework for both generations.

2.3. Sexual aggression paradigm in the F0 generation

2.3.1. Assessment of sexual aggression-like behaviour

The assessment of sexual aggression-like behaviour comprised three critical stages: (a) oestrous cycle monitoring, to categorize female rats into oestrous and non-estrous groups based on vaginal smears; (b) evaluation of sexual receptivity, to confirm sexual receptivity by pairing adult female rats with sexually experienced adult male rats, and (c) the SxAT proper, to evaluate male aggressive responses towards non-receptive female rats.

2.3.2. Estrous cycle monitoring

Sexual receptivity in female Wistar rats was identified through careful assessment of the oestrous cycle stage, employing daily vaginal smear analysis to navigate the reproductive phases. The oestrous cycle, integral to rodent fertility, is separated by four phases, each with distinct vaginal epithelial alterations: proestrus, characterized by follicular proliferation and a predominance of nucleated epithelial cells; estrus, indicative of sexual receptivity through a surge of cornified cells; metestrus, denoted by a leukocyte and cornified cell mixture, signifying post-ovulation; and diestrus, dominated by leukocytes, marking the inter-cycle hiatus (Robert et al., 2021).

Vaginal smears were collected daily between 07:00 and 08:00 h during the dark phase of the inverted light–dark cycle (lights off at 06:00 h; lights on at 18:00 h) to determine the stage of the oestrous cycle. This timing was selected to ensure consistency across days while minimizing disturbance to normal circadian and reproductive rhythms. Sample collection was performed gently using saline lavage and immediately examined under light microscopy to classify cycle stage according to established cytological criteria assessing the proportions of nucleated, cornified, and leukocytic cells to determine the oestrous phase (Yener et al., 2007). This classification divided female rats into 'estrus' and 'non-estrus' groups based on their vaginal cytology, correlating with phases of sexual receptivity and quiescence, respectively (Paccola and Resende, 2018). This approach allowed for the precise selection of sexually receptive females for subsequent behavioural assays assessing sexual aggression in male counterparts.

2.3.3. Assessment of sexual receptivity in female rats during estrus

Sexual receptivity testing was conducted at 11:00 h, corresponding to approximately 5 h after lights-off under the inverted 12:12 h light–dark cycle. This time window coincides with the nocturnal active phase of rats and aligns with established protocols for mating and lordosis assessment, where females exhibit an arched-back posture facilitating copulation, accompanied by darting, hopping, and ear-wiggling, which are recognised as signs of sexual readiness in rodents (Le Moëne & Ågmo, 2019). Receptivity was assessed immediately prior to mating trials to ensure that females were in an appropriate behavioural and hormonal state. To avoid actual mating, females displaying these behaviours were promptly removed from the presence of the males. This process was repeated over four consecutive days, accounting for the cyclical nature of estrus and enabling a thorough evaluation of sexual receptivity across various hormonal states.

2.3.4. The sexual aggression test

The SxAT, a validated behavioural assay, previously described by Oliveira et al. (2022) was used to evaluate sexual aggression and associated behaviours in male Wistar rats. The assessment was performed in a dimly lit environment (under 2 lux) to facilitate stress-free video documentation of the subjects' behaviours while preserving the nocturnal conditions conducive to rat activity. After the daily staging of the oestrous cycle and assessing for receptivity, the test protocol ran as briefly described below:

Following daily vaginal cytology and confirmation of female receptivity, the SxAT protocol proceeded as follows. By 12:00 h, experimental male rats and stimulus female rats were transferred in their home cages, together with their bedding, to the recording room and allowed to habituate for 1 h.

At 13:00, a receptive female rat was introduced into the cage of an experimental male rat to interact. Upon successful intromission, the receptive female rat was removed and replaced by a non-estrous, unreceptive female rat. This male-female interaction was recorded for 10 min.

Sexual aggressive-like behaviours manifested as the male forcefully mounting an unresponsive female, characterized by forepaw placement and pelvic thrusts. Additional behaviours such as forced grooming, keep-down, and threat postures were also noted, alongside neutral and social activities.

All sessions were scored offline from video recordings by a trained observer blinded to group allocation. The frequency of forced mounting and aggressive acts was quantified over the 10-min test period. Video recordings were additionally used to calculate the percentage of time spent in aggressive, social, and neutral behaviours relative to total interaction time. The use of a GoPro camera for the SxAT, in contrast to fixed overhead cameras employed for other behavioural assays (e.g., resident–intruder, open field, and elevated plus maze tests), was intentional, as the SxAT requires higher spatial resolution and flexible camera positioning to reliably capture subtle sexual and aggressive behaviours occurring at close proximity within the home cage.

2.4. Mating procedure and prenatal stress induction

To investigate the impact of sexual aggression and prenatal stress on maternal behaviours and the potential exacerbation of sexually aggressive behaviours in male offspring, an experimental mating protocol was designed. This involved the pairing of female rats, previously subjected to the SxAT, with males categorised as either socially housed or isolated.

This design ensured consistency in prior exposure among the females used for mating. The rationale was to investigate the combined effects of sexual aggression exposure and prenatal restraint stress (PRS) on maternal behaviours and offspring outcomes.

The inclusion of both stressors reflects real-world conditions where individuals may encounter multiple adverse experiences. This cumulative stress model aimed to elucidate how prior sexual aggression interacts with prenatal stress to influence maternal behaviours and neurobiological outcomes in offspring, focusing on the HPA axis and aggression-related pathways.

2.4.1. Mating Protocol

The mating regimen commenced with a thorough monitoring of the females' oestrous cycles through daily vaginal smears, extending over a 4–5-day period to ensure accurate detection of the proestrous phase, which signals peak fertility. Females cohabitated in a communal setting to synchronize their cycles, enhancing the likelihood of successful mating. The introduction of a male to the female enclosure during the proestrous phase facilitated mating, with the presence of a vaginal plug or sperm in a post-mating vaginal smear marking the onset of gestation (designated as gestational day 0) (Norris and Adams, 1981). After mating, males were removed from the enclosure.

Following the SxAT, females were stratified into PRS-exposed and NS groups. The PRS-exposed group underwent a standardized restraint stress protocol during gestation, while the NS group continued gestation under normal conditions. This design enabled the exploration of both individual and combined effects of sexual aggression and prenatal stress on maternal and offspring behaviours. This ultimately gave rise to four experimental F1 male offspring groups, i.e., those born to non-stressed dams and F0 males housed under group conditions (NSGH); non-stressed dams and F0 males housed under isolation conditions (NSIH); stressed dams and F0 males housed under group conditions (SGH); and stressed dams and F0 males housed under isolation conditions (SIH).

2.4.2. Prenatal stress induction in pregnant rats

A well-established prenatal restraint stress (PRS) protocol, described by Maccari et al. (1995), was employed to elucidate the effects of prenatal stress on maternal behaviours and the potential transmission of sexually aggressive behaviours to offspring. This protocol involved subjecting pregnant rats to a defined stress regimen during the critical period of gestation.

Starting from the 11th day of gestation and extending until the onset of parturition (between gestational days 22 and 23), the pregnant rats underwent stress induction. This involved three daily sessions of restraint stress, precisely scheduled at 09:00, 12:00, and 17:00 h. Each session entailed confining the individual pregnant rat within a transparent rodent restrainer for one hour. This method of stress induction was rigorously maintained throughout the latter stages of gestation to ensure consistency in the stress exposure across all subjects within the PRS group.

The control group (non-stressed, NS) of pregnant rats did not undergo the PRS protocol. These rats were allowed to continue their gestation undisturbed within their home cages, serving as a baseline against which the effects of prenatal stress could be measured.

2.5. Assessment of maternal behaviours in the postnatal period

To evaluate maternal behaviours following birth, a systematic observational protocol was implemented from Postnatal Day (PND) 2 to PND 7, based on the methodology outlined by Orso et al. (2018). This involved conducting detailed observations of maternal interactions at six predetermined intervals during each observation session, specifically at the start and every 3 min thereafter, culminating at the 15th minute. These sessions were scheduled across three distinct time slots daily: 09:00, 13:00, and 16:00 h, resulting in a total of 18 observations per day.

2.5.1. Evaluation criteria for maternal behaviours

Maternal behaviour was assessed during the early postnatal period using a video-based, non-intrusive observational approach conducted in the colony room environment. All behavioural observations were recorded continuously using fixed, ceiling-mounted digital cameras positioned directly above each home cage, allowing unobstructed visualization of the nest area and dam–pup interactions. No experimenter was present in the room during recording sessions, and dams were not handled or disturbed at any point during the observation periods, thereby eliminating observer-induced behavioural modulation:

Nursing (N): This behaviour was noted whenever the dams were observed in the act of nursing their pups, irrespective of the nursing position adopted.

Licking Pups (L): Instances where dams were seen licking their pups were recorded under this category.

Contact with Pups (C): This category included moments when the dams were close to their pups within the nest, maintaining contact without engaging in either nursing or licking behaviours.

Retrieving Pups (R): This behaviour was identified when dams actively transported their pups back to the nest.

In addition to maternal behaviours, instances of non-maternal behaviours were also documented:

No Interaction with Pups (X): This encompassed any dam behaviour that did not involve direct interaction with the pups, excluding feeding or drinking activities.

Eating/Drinking (E): This category was reserved for moments when the dams were observed engaging in eating or drinking behaviours.

2.6. Quantitative analysis of maternal behaviours

For quantitative analysis, maternal and non-maternal behaviours were aggregated to derive total maternal behaviour (MB = nursing + licking + contact) and total non-maternal behaviour (NMB = no interaction + eating/drinking). Pup retrieval was analysed separately and excluded from the maternal care index due to its low and inconsistent frequency across groups. The maternal care behaviour index (MCBI) was calculated as MB / (MB + NMB), as previously described by Orso et al. (2018).

All video recordings were scored offline by trained observers who were blinded to experimental group allocation, ensuring unbiased assessment. Behavioural scoring and temporal quantification were performed using Behavioural Observation Research Interactive Software (BORIS; Department of Life Sciences and Systems Biology, University of Turin, Italy). Offline scoring eliminated any potential influence of observer presence on maternal behaviour and allowed precise time-based quantification of each behavioural category. Inter-observer reliability was established prior to formal scoring, and any discrepancies were resolved by consensus, in accordance with established protocols for maternal behaviour assessment during the postnatal period.

2.7. Selection and housing of male offspring for experimental analysis

Post weaning at 21 days, a selection process was employed to identify male offspring for inclusion in the experimental cohorts. This selection was exclusively confined to litters that presented a balanced sex ratio and comprised 10–14 pups (Zindove et al., 2013). From these qualified litters, a maximum of two male pups were randomly picked, to partake in the subsequent experimental phases, ensuring genetic diversity and minimizing litter-specific biases.

The selected male offspring, representing both the NS and the PRS groups, were subsequently arranged into communal housing conditions. Each housing unit accommodated groups of 3–4 male rats, ensuring social interaction and environmental consistency across all experimental conditions. The initiation of experimental procedures was deferred until the male offspring attained the age of 2 months, to allow for the maturation of physiological and behavioural systems, thereby ensuring the relevance and reliability of the experimental outcomes derived from these cohorts (Sudakov et al., 2021).

2.8. Evaluation of aggression and anxiety-like behaviour in offspring

2.8.1. Behavioural assessment protocol for male offspring

At PND 62, male offspring underwent a series of behavioural assessments similar to those administered to their parental generation. Contrary to the parental assessment protocol, male offspring were not subjected to post-weaning social isolation prior to behavioural testing. This refers specifically to housing history, as offspring were raised and maintained under standard group-housing conditions following weaning, rather than being reared in isolation. Importantly, offspring were tested individually during all behavioural assays. Cage mates were not present during any behavioural testing, including the resident–intruder test, SxAT, Open Field Test (OFT), or Elevated Plus Maze (EPM). Thus, group dynamics did not directly influence behavioural outcomes during testing.

Aggression-like behaviours were assessed using the resident intruder test. Following this, the offspring were exposed to a four-day SxAT protocol against non-receptive females. After the SxAT, they were reassessed for aggression-like behaviours towards an intruder male, and anxiety-like behaviours using the OFT and the EPM. This approach allowed assessment of baseline aggression, aggression following sexual aggression exposure, and anxiety-related phenotypes while avoiding the confounding effects of post-weaning social isolation in the offspring generation.

2.8.2. The resident intruder test

Conducted under dim red-light conditions (<2 lux) to simulate the nocturnal environment of rats, this test began post-12:00, with the actual assessment occurring at 13:00. To enhance territorial salience, bedding in the resident male’s home cage was not changed for three days prior to testing. The bedding of resident males was not changed in the 3 days prior to the test to foster a sense of territorial ownership.

On the day of testing, both resident and intruder males were weighed. A deliberate weight and size advantage was maintained for the resident male, consistent with established resident–intruder paradigms designed to elicit territorial aggression rather than competitive or defensive encounters. Size-matched pairings were avoided because such configurations can promote reciprocal fighting or defensive aggression, thereby reducing interpretability of resident-driven offensive aggression.

Intruder males were selected based on predefined criteria: smaller body weight relative to the resident, group-housed history (i.e., no prior social isolation), and no prior exposure to aggression testing. Intruders had no previous interactions with the resident to ensure social novelty. Each intruder was used only once per testing day, and a minimum washout period of 72 h was maintained before an intruder could be reused in subsequent trials, minimizing habituation or carryover stress effects.

Prior to testing, resident males remained undisturbed in their home cages, while intruders were briefly habituated in a clean holding cage with familiar bedding. The test was initiated by introducing the intruder male into the resident’s home cage, and interactions were recorded for 10 min.

Aggressive behaviours scored for the resident male included latency to first attack, lateral threats, upright postures, clinch attacks, and sustained attacks characterized by biting directed toward the intruder’s neck, back, or flanks. Behavioural outcomes were categorized into aggressive behaviours (e.g., threats, offensive grooming, attacks), neutral behaviours (e.g., exploration, eating, self-grooming), and social behaviours (e.g., non-aggressive sniffing and following). This standardized approach ensured that observed aggression reflected territorial offensive behaviour of the resident rather than size-matched competition or defensive responses.

2.8.3. The sexual aggression test

On PND 64, the adult male rats were assessed for sexual aggression-like behaviours using the SxAT protocol described above. Twenty-four and 48 h following the last SxAT assessment, the adult male offspring were assessed for anxiety-like (PND 68) and aggression-like (PND 69) behaviours, respectively.

2.8.4. The open field test protocol

The OFT was implemented in a 1 square meter black arena with 45 cm high walls to prevent escape, under dim red light conditions (2 lux) to simulate a low-stress environment for the rodents. This setup was in accordance with the methodology outlined by Belovicova et al. (2017). Behavioural monitoring was facilitated by an overhead camera capturing the rats' movements within the arena. The arena is segmented into two zones for analysis: the central zone and the outer zone. The central zone is associated with lower anxiety and increased exploratory behaviour, as indicated by longer durations spent by rats in this area. On the other hand, prolonged presence in the outer zone, characterized by thigmotaxis—the tendency to stay near walls—suggests higher anxiety levels and reduced exploratory activity (Heinz et al., 2021).Each rat was then placed at the centre of the arena, allowing for a 6-minute exploration period. Behavioural data were recorded during the final 5 min to ensure adaptation to the environment. Post-test, the arena was sanitised with a 70 % alcohol solution to eliminate residual olfactory cues.

Behavioural indices of exploration and anxiety, including grooming, freezing, and the proportion of time spent in the center and periphery of the arena, were recorded and analyzed as per Seibenhener & Wooten (2015).

2.8.5. The elevated plus maze test

The EPM test serves as a widely recognised measure of anxiety-like behaviour in rodents, exploiting their aversion to open spaces and elevated platforms. This test delineates anxiety levels based on the preference for enclosed over open arms, balancing the rodent's exploratory drive against innate fear tendencies (Korte and De Boer, 2003, Walf and Frye, 2007). The EPM setup consisted of two open (50 × 10 cm) and two enclosed arms (50 × 10 × 40 cm), elevated 50 cm above the ground, with a central square platform (10 × 10 cm) at their intersection.

Following the protocol detailed by Belovicova et al. (2017), each rat was placed on the central platform facing an enclosed arm and was permitted a 5-minute exploration period. A video camera coupled with Behavioural Observation Research Interactive Software (BORIS- Department of Life Sciences and Systems Biology, University of Turin, Italy) captured and analysed the rats' activities, quantifying the time spent and the frequency of entries into the centre, open, and closed arms. The apparatus was thoroughly cleaned with a 70 % alcohol solution between each session to prevent olfactory bias.

2.9. Gene expression analyses in brain tissue

2.9.1. Brain tissue collection and preservation for gene expression analysis

Following the completion of behavioural assessments, rats from each experimental group were euthanised via rapid decapitation using a precision guillotine, ensuring minimal stress and discomfort. Immediately post-mortem, trunk blood was harvested into serum tubes and subjected to centrifugation at 1200 rpm for 20 min at a temperature of 4 °C. The isolated serum was then aliquoted into microcentrifuge tubes and preserved at –80 °C for future neurochemical evaluations.

Following blood collection, the brains were neatly excised while on an ice-cold dissection tray to maintain tissue integrity. The PFC, HYPO, HIPPO, and AMYG were systematically isolated. These tissue samples were instantly snap-frozen in liquid nitrogen to halt any biochemical processes, thus preserving the RNA integrity for gene expression analysis. The frozen samples were then stored at −80°C, awaiting further molecular analysis to elucidate the gene expression alterations associated with prenatal stress and exposure to sexual aggression.

2.9.2. RNA extraction, cDNA synthesis, and quantitative real-time PCR

The stored brain tissue was homogenised using the lysis buffer included in the RNA isolation kit (Cat No. 83913–1EA) provided by Sigma-Aldrich (St. Louis, MO, USA). Total RNA was extracted using the GenElute™ Total RNA Purification Kit (Sigma-Aldrich). RNA purity was determined using the Nanodrop spectrophotometer (NANODROP ONE, Thermofisher Scientific, Madision, WI, USA) by measuring the ratio of absorbance at 260 nm to 280 nm (A260/A280 ratio). A ratio between 1.8 and 2.0 was considered indicative of pure RNA, free from protein contamination. RNA integrity was assessed via gel electrophoresis on a 1 % agarose gel. The appearance of sharp 28S and 18S ribosomal RNA bands confirmed the integrity of the RNA, with minimal smearing indicating little to no degradation (Aranda et al., 2012). RNA quality was measured using TapeStation (RNA ScreenTape). An RNA Integrity Number (RIN), above 7 (scale from 1 to 10 used to assess RNA quality, with 10 indicating intact RNA) was considered acceptable for sensitive downstream applications such as cDNA synthesis and quantitative real-time PCR (qPCR), ensuring reliable and accurate gene expression analysis.

Reverse transcription of RNA to cDNA was performed using the High-Capacity cDNA Reverse Transcription Kit 4368814 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) for 120 min at 37°C, with 10 ng of RNA per sample quantified using the NANODROP ONE (Thermofisher Scientific, Madison, WI). The qPCR conditions were set to 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C using 200 nM of both forward and reverse primers. Primers targeting the AVPR1A, CRHR1, OXTR, AR, and Htr1a genes were sourced from Thermofisher Scientific, which has been designed to ensure specificity (Table 5.2). qPCR was conducted in triplicate on a Quantstudio 5 system with TaqMan® Fast Advanced Master Mix (Applied Biosystems-Life Technologies), adhering to standard protocols.

Table 5.

Significant effects of prenatal stress and F0 male housing on anxiety-like behaviours in F1 male rats.

Behaviour (s)	NSGH Mean ± SD	NSIH Mean ± SD	SGH Mean ± SD	SIH Mean ± SD	p-value	Post-hoc Tukey’s HSD (p < 0.05) Summary
Rearing time	0.90 ± 1.20	10.00 ± 5.96	4.00 ± 2.71	6.60 ± 3.95	< 0.001	NSIH > NSGH, SGH, SIH
Grooming time	4.10 ± 3.60	1.20 ± 1.23	0.80 ± 1.23	1.00 ± 0.67	0.002	NSGH > NSIH, SGH, SIH
Closed arm time	84.20 ± 19.37	105.30 ± 24.89	67.20 ± 19.37	217.60 ± 28.98	< 0.001	SIH > NSGH, NSIH, SGH

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NSGH = non-stressed dam × group-housed male; NSIH = non-stressed dam × isolated male; SGH = stressed dam × group-housed male; SIH = stressed dam × isolated male

To determine the amplification efficiency of each assay and to calculate relative expression levels, standard curves were prepared using serially diluted cDNA samples (gene amplification efficiencies; Avpr1a = 106.382, Gapdh = 93.386, Htr1a = 84.276, Oxtr = 89.487, Ar = 90.123, Chrh1 = 86.345; see Supplementary material). The standard curves were generated using a range of known concentrations of cDNA to assess amplification efficiency and establish the relationship between Ct values and the quantity of target mRNA.

To calculate relative gene expression levels, we adopted the ΔΔ-Ct method as outlined by (Pfaffl, 2001) and (Rao et al., 2013), which ensures robust normalization of target gene expression to a housekeeping gene. The calculations were conducted as follows:

Calculate ΔTarget (Target Gene): For each sample, the Ct value of the target gene for the experimental condition was subtracted from that of the control condition:

ΔTarget= Ct_{Target, experimental} − Ct_{Target, control}

Calculate ΔHKG (Housekeeping Gene): Similarly, the Ct value of the housekeeping gene for the experimental condition was subtracted from that of the control condition:

ΔHKG= Ct_{HKG, experimental} − Ct_{HKG, control}

Normalize ΔTarget to ΔHKG: The ratio of ΔTarget to ΔHKG was calculated to normalize the expression levels of the target gene against the housekeeping gene:

Relative Expression Level= ΔTarget/ΔHKG

Adjust for Amplification Efficiency: To incorporate variations in amplification efficiency, the formula:

E^{Relative Expression Level} was used, where E represents the amplification efficiency for each gene.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as the endogenous control for normalization due to its consistent expression across experimental conditions and tissues (Pessoa et al., 2024). The expression stability of GAPDH ensures reliable normalization, minimizing technical variability in qPCR data.

To ensure unbiased and representative outcomes, samples from different experimental groups and brain regions were randomly distributed across qPCR plates. Randomization mitigates systematic errors and plate-to-plate variability, ensuring the observed differences reflect biological variations.

The recalculated relative gene expression levels using the corrected ΔΔ-Ct method are presented in the results, with primary qPCR data included for transparency and thorough assessment Table 1.

Table 1.

Genes investigated using TaqMan® gene expression assays.

Probes	Gene symbol	Assay ID
Oxytocin receptor	OXTR	Rn00563503_m1
Arginine vasopressin receptor type 1	AVPR1A	Rn00583910_m1
Corticotropin-releasing hormone receptor 1	CRHR1	Rn01463997_m1
Androgen receptor	AR	Rn00664479_m1
5-hydroxytryptamine (serotonin) receptor 1A	HTR1A	Rn00561409_s1
Glyceraldehyde-3-phosphate dehydrogenase	GAPDH	Rn01775763_g1

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2.10. Hormonal assays in serum samples

Serum samples stored at −80 °C were thawed and the concentration of neurochemicals was determined according to the manufacturer's instructions using enzyme-linked immunosorbent assay (ELISA) kits (Elabscience, Houston, TX). This assay involves precoating microplate wells with antibodies specific to the target neuromarker. The neuromarker present in the samples competes with a fixed amount of neuromarker-horseradish peroxidase (HRP) conjugate for binding sites on the antibody. After incubation, unbound conjugates are removed through a series of wash steps, resulting in an inverse relationship between the amount of bound neuromarker-HRP conjugate and the concentration of neuromarker in the sample. The development of the colourimetric signal, initiated by adding a substrate solution, is inversely proportional to the neuromarker concentration, measured by the optical density at 450 nm (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany). The serum concentrations of testosterone, corticosterone, CRH, arginine vasopressin and serotonin, were quantified using rat-specific ELISA kits.

In relation to AVP, it is well established that AVP levels in rat serum are typically low due to the peptide’s rapid clearance from circulation and its restricted release under baseline conditions (Summanen et al., 2018). This characteristic is consistent with AVP's role in regulating water retention and social behaviour, which generally requires tight regulation and low circulating concentrations (Caldwell et al., 2008, Sparapani et al., 2021). Given the low concentration of arginine vasopressin in serum and the potential for interference from other serum components, an extraction procedure was performed before immunoassay analysis (Bankir et al., 2017, Christ-Crain et al., 2022). The extraction involved mixing a 1-part serum sample with 1.5 parts of Extraction Solution, vortexing, and then incubating samples at room temperature for 90 min. The mixture was then centrifuged at 4 °C at 1660 x g for 20 min. The supernatant was transferred to a clean tube and dried using a speed vac at 37°C. The samples were then reconstituted with 250 µL of Assay Buffer. Following extraction, the serum levels of AVP were quantified using a highly sensitive and specific immunoassay kit for AVP (DetectX® Arg8-Vasopressin (AVP) Immunoassay Kit, ARBOR Assays-K049-H), according to the manufacturer's protocol. The analyses of the samples were carried out in triplicates, and the values were estimated from the standard curve produced from the calculated concentrations.

2.11. Statistical Analysis

Statistical analyses were conducted using R version 4.3.1 (Chan, 2018) and Stata 17, employing a comprehensive analytical framework tailored to the experimental design and data structure. Descriptive statistics were calculated and are presented as means ± standard deviations (SD), medians with interquartile ranges (IQR), or frequencies and percentages, as appropriate.

Data distribution was assessed using the Shapiro–Wilk test, and extreme outliers were identified using Grubb’s test prior to analysis. Normally distributed data were analysed using Student’s t-tests for two-group comparisons or one-way analysis of variance (ANOVA) with Tukey post-hoc correction for multiple comparisons. Non-normally distributed data were analysed using Mann–Whitney U tests or Kruskal–Wallis tests, as appropriate. Effect sizes were reported using Cohen’s d or eta-squared (η²) where applicable.

Longitudinal behavioural outcomes, including MCB and aggression metrics across testing days, were analysed using linear mixed-effects models (LMMs) implemented in the lme4 package. Fixed effects included prenatal stress status, F0 male housing condition, test day, and relevant interaction terms, with rat identity included as a random effect to account for repeated measures. Litter size was included as a covariate in maternal care models. Where random slope models failed to converge, simplified random-intercept models were used. Post-hoc contrasts for significant interactions were conducted using estimated marginal means (emmeans) with Tukey correction.

Changes in aggressive behaviour before and after SxAT exposure were evaluated using LMMs and linear regression models incorporating time, housing condition, prenatal stress, and baseline behaviour as predictors. Neurochemical and gene expression data were analysed using multivariate regression models to assess region-specific effects, followed by univariate analyses where multivariate effects were significant. Fold-change values were log₂-transformed prior to analysis, and one-sample t-tests were used to assess deviations from zero. Correlations between behavioural, neurochemical, and gene expression measures were assessed using Pearson’s or Spearman’s correlation coefficients, depending on data distribution.

To control for multiple testing, the Benjamini–Hochberg false discovery rate (FDR) correction was applied. All p-values reported in the Results section represent Benjamini–Hochberg–adjusted p-values (q-values), unless otherwise stated. Statistical significance was defined as q < 0.05. This approach ensured rigorous control of type I error while maintaining sensitivity across multiple behavioural, neurochemical, and molecular outcomes.

3. Results

To improve clarity and focus, figures that provide supporting or component-level analyses have been moved to the Supplementary Material, while figures directly addressing maternal care trajectories, offspring aggression, and key neurobiological mechanisms are retained in the main manuscript.

3.1. Maternal care behaviour

3.1.1. Overview of maternal care across postnatal days 2–8

Maternal care behaviour (MCB) was significantly influenced by prenatal stress, F0 male housing condition, and postnatal day. Stressed dams consistently exhibited reduced MCB compared to non-stressed dams (Fig. 2), with the lowest scores observed on PND 2 and 3.

Fig. 2 — Maternal care behaviour (MCB) trajectories across early postnatal development in stressed and non-stressed dams. Predicted marginal means of the maternal care behaviour (MCB) index (MB / [MB + NMB]) from postnatal day (PND) 2 to PND 8 are shown for dams exposed to prenatal stress and non-stressed controls, with error bars representing 95 % confidence intervals. Estimates were derived from a linear mixed-effects regression model with day treated as a categorical fixed effect, prenatal stress condition as the main predictor, litter size included as a covariate, and dam identity specified as a random intercept to account for repeated observations within dams. A significant overall effect of prenatal stress on MCB was observed (χ²(1) = 86.21, p < 0.0005), indicating persistently reduced maternal care in stressed dams across the early postnatal period. Post-hoc pairwise comparisons (multiplicity-adjusted) revealed significantly lower MCB in stressed dams on PND 2 (Δ = −0.158, 95 % CI: −0.188 to −0.127, χ²(1) = 110.62, p < 0.0001), PND 3 (Δ = −0.107, CI: −0.137 to −0.076, χ²(1) = 56.93, p < 0.0001), PND 4 (Δ = −0.068, CI: −0.098 to −0.038, χ²(1) = 18.43, p < 0.0001), PND 5 (Δ = −0.040, CI: −0.070 to −0.009, χ²(1) = 6.52, p = 0.0106), PND 7 (Δ = −0.060, CI: −0.090 to −0.030, χ²(1) = 15.07, p = 0.0001), and PND 8 (Δ = −0.053, CI: −0.084 to −0.023, χ²(1) = 11.26, p = 0.0008). PND 6 was excluded from pairwise testing due to insufficient observations for stable estimation. Values shown represent model-based predictions adjusted for litter size and within-dam dependence, rather than raw daily means, allowing comparison of maternal care trajectories across conditions while controlling for repeated-measures structure and unequal sampling across days.

3.1.1.1. Effects of prenatal stress and F0 male housing on maternal behaviours

Component analyses of maternal caregiving behaviours revealed that the observed reduction in composite MCB in prenatally stressed dams was reflected across multiple individual caregiving domains. Specifically, stressed dams exhibited reduced nursing and licking behaviours across early postnatal development, with additional modulation by F0 male housing condition. Detailed analyses of nursing, licking, pup contact, and non-interactive behaviours are provided in the Supplementary Results (Figures S1–S2, S3).

3.1.1.2. Modulatory effects of F0 male housing

In the absence of prenatal stress, F0 male housing condition did not significantly influence MCB across the early postnatal period (Fig. 3a). Predicted MCB trajectories were stable across postnatal days, and no significant housing × day interaction was detected in non-stressed dams. In contrast, under prenatal stress, F0 male housing exerted a time-dependent modulatory effect on maternal care (Fig. 3b). Dams mated with isolated males exhibited higher predicted MCB early (PND 2) and late (PND 8) in the postnatal period; however, this advantage diminished or reversed on intermediate days, resulting in a significant housing × day interaction among stressed dams.

3.1.1.3. Day-specific patterns and behavioural components

Postnatal day emerged as a strong independent predictor of MCB (Table 2). Model-based estimates indicated a progressive increase in MCB from PND 2 through PND 5, followed by a plateau across later postnatal days. This temporal pattern was consistent across multiple caregiving components, including nursing, licking, and pup contact (Supplementary Figure S3), suggesting coordinated maturation of maternal behaviours during early postpartum development.

Table 2.

Effects of litter size and postnatal day on maternal care behavior.

Variable	Beta estimate	Std. Error	tvalue	p-value
Litter size	0.017	0.004	4.043	0.001**
Postnatal day 4	0.059	0.017	3.521	0.001***
Postnatal day 5	0.083	0.017	4.912	< 0.000***
Postnatal day 6	0.049	0.017	2.915	0.005**
Postnatal day 7	0.084	0.017	4.969	< 0.000***
Postnatal day 8	0.088	0.017	5.192	< 0.000***

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Significant postnatal day effects are reported relative to postnatal day 2, which served as the reference category in the model. **p < 0.01; ***p < 0.001.

In contrast, pup retrieval exhibited greater day-to-day variability and group-specific fluctuations, indicating a less stable temporal trajectory relative to other caregiving behaviours. Non-maternal behaviours, particularly self-grooming and pup neglect (“no interaction”), were more pronounced in prenatally stressed dams mated with isolated males, consistent with behavioural disengagement under combined prenatal stress and adverse paternal housing conditions (Supplementary Figure S4).

3.1.2. F1 male aggressive behaviour in the resident intruder test

3.1.2.1. Pre- and post-SxAT behavioural outcomes

Behavioural analysis of F1 males during the resident–intruder test revealed marked group differences in aggressive, social, and neutral behaviours both before and after exposure to the SxAT (Table 3). The SIH group exhibited the highest levels of aggression, characterized by increased forced mounting, threat displays, attack duration, and attack frequency, along with reduced latency to first attack relative to other groups.

Table 3.

Descriptive summary of aggressive and social behavioural metrics in F1 male rats during pre- and post-SxAT resident–intruder tests.

Behavioural metric	NSGH	NSIH	SGH	SIH
Forced mounting (s)	Pre: 2.9 ± 1.5 Post: 39.3 ± 13.3	Pre: 3.8 ± 2.5 Post: 49.7 ± 14.1	Pre: 3.4 ± 2.6 Post: 61.6 ± 13.6	Pre: 13.3 ± 4.3 Post: 87.4 ± 11.7
Threat displays (s)	Pre: 10.9 ± 3.9 Post: 17.8 ± 6.1	Pre: 12.6 ± 7.6 Post: 44.2 ± 11.0	Pre: 20.4 ± 7.7 Post: 47.2 ± 10.7	Pre: 22.0 ± 8.4 Post: 67.5 ± 17.5
Total aggression duration (s)	Pre: 65.0 ± 11.6 Post: 115.8 ± 9.1	Pre: 138.0 ± 19.9 Post: 202.0 ± 22.9	Pre: 137.9 ± 32.3 Post: 215.3 ± 13.3	Pre: 216.5 ± 29.0 Post: 268.7 ± 23.9
Aggression time (% of test)	Pre: 10.9 ± 2.1 Post: 19.5 ± 1.6	Pre: 23.1 ± 3.1 Post: 33.7 ± 3.8	Pre: 23.0 ± 5.3 Post: 35.8 ± 2.4	Pre: 36.2 ± 4.9 Post: 44.9 ± 4.0
Latency to first attack (s)	Pre: 425.7 ± 107.2 Post: 427.7 ± 87.8	Pre: 120.1 ± 22.4 Post: 96.0 ± 11.1	Pre: 202.4 ± 86.3 Post: 151.4 ± 40.2	Pre: 50.1 ± 20.9 Post: 34.8 ± 14.1
Attack frequency (count)	Pre: 4.0 ± 1.3 Post: 5.2 ± 1.9	Pre: 7.6 ± 4.2 Post: 10.2 ± 4.4	Pre: 7.5 ± 3.0 Post: 9.4 ± 2.2	Pre: 14.5 ± 2.1 Post: 23.1 ± 6.9
Social behaviour time (% of test)	Pre: 31.4 ± 6.6 Post: 39.8 ± 4.7	Pre: 37.9 ± 2.9 Post: 19.9 ± 4.0	Pre: 31.4 ± 6.6 Post: 20.2 ± 5.3	Pre: 28.2 ± 4.7 Post: 19.9 ± 2.3

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Note: Values represent mean ± SD (n = 10 per group). NSGH = non-stressed dam × group-housed male; NSIH = non-stressed dam × isolated male; SGH = stressed dam × group-housed male; SIH = stressed dam × isolated male. Durations are reported in seconds unless otherwise indicated. Metrics shown represent key aggressive and social behaviours used to contextualize inferential analyses presented in the main text. Additional behavioural measures (including grooming, exploration, and rearing) are provided in the Supplementary Material (Table S1-S2).

Social behaviours, including anogenital sniffing and overall interaction time, were reduced in SIH males following SxAT, whereas NSGH and NSIH groups showed higher baseline sociability and a less pronounced decline post-exposure. Neutral behaviours, such as grooming and exploratory activity, were lowest in SIH males after SxAT, consistent with a behavioural profile dominated by heightened aggression. Descriptive values summarizing these behavioural outcomes are provided in Table 3.

3.1.2.2. Impact of prenatal stress, F0 male housing, and time

Across aggression-related outcomes, F1 male behaviour was jointly influenced by dam prenatal stress, F0 male housing condition, and exposure to the Sexual Aggression Test (SxAT). Measures of forced mounting, threat display, attack duration, latency to first attack, and attack frequency consistently differentiated offspring of stressed dams and isolated F0 males from their respective controls (Fig. 4, Fig. 5, Fig. 6.

Fig. 4 — Forced mounting and threat behaviours across pre- and post-SxAT. Predictive margins of forced mounting and threat durations in F1 male rats assessed before and after exposure to the Sexual Aggression Test (SxAT), stratified by dam prenatal stress and F0 male housing condition. Estimates were derived from linear mixed-effects models, with error bars representing 95 % confidence intervals. (a) Forced mounting duration showed a significant stress × time interaction (p < 0.0001). Offspring of stressed dams exhibited a greater increase from pre- to post-SxAT (mean difference = 66.15 s; 95 % CI: 60.81–71.49) compared with offspring of non-stressed dams (mean difference = 41.15 s; 95 % CI: 35.81–46.49). Post-SxAT values were significantly higher in the stressed group (mean difference = 30.00 s; 95 % CI: 24.38–35.62; p < 0.0001). (b) A significant time × F0 male housing interaction was also observed (p < 0.0001). F1 males sired by isolated F0 males showed a steeper increase in forced mounting duration (mean difference = 60.00 s; 95 % CI: 54.66–65.34) compared with those sired by group-housed males (mean difference = 47.30 s; 95 % CI: 41.96–52.64). Post-SxAT differences between housing conditions were significant (mean difference = 18.10 s; 95 % CI: 12.48–23.72; p < 0.0001). (c) Threat duration displayed a significant stress × time interaction (p < 0.0001), with a larger increase in offspring of stressed dams (mean difference = 36.15 s; 95 % CI: 30.34–41.96) than in non-stressed controls (mean difference = 19.25 s; 95 % CI: 13.44–25.06). (d) Post-SxAT threat duration differed significantly by F0 male housing (p < 0.0001), with offspring of isolated sires exhibiting higher values than those of group-housed sires (mean difference = 23.35 s; 95 % CI: 17.54–29.16). No significant housing differences were observed pre-SxAT (p = 0.578).

Fig. 5 — Attack duration before and after SxAT. Predictive margins of attack duration (seconds) in F1 male rats assessed before and after SxAT exposure, stratified by dam prenatal stress (a) and F0 male housing condition (b). Estimates were obtained from linear mixed-effects models; error bars indicate 95 % confidence intervals. (a) A significant main effect of dam prenatal stress was observed (p < 0.0001). Offspring of stressed dams exhibited longer predicted attack durations than those of non-stressed dams both pre-SxAT (mean difference = 36.35 s; 95 % CI: 26.82–45.88) and post-SxAT (mean difference = 21.80 s; 95 % CI: 12.27–31.33). Attack durations decreased from pre- to post-SxAT in both stress groups. (b) F0 male housing significantly influenced attack duration (p < 0.0001). Offspring of isolated F0 sires displayed higher predicted attack durations than those of group-housed sires pre-SxAT (mean difference = 49.15 s; 95 % CI: 39.62–58.68) and maintained a smaller but significant difference post-SxAT (mean difference = 9.90 s; 95 % CI: 0.37–19.43; p = 0.0418). Time-dependent reductions were evident across both housing conditions.

Fig. 6 — Latency to first attack and attack frequency. Predictive margins of latency to first attack and attack frequency in F1 male rats assessed before and after SxAT exposure, stratified by dam prenatal stress and F0 male housing condition. Estimates were derived from linear mixed-effects models; error bars represent 95 % confidence intervals. (a) Latency to first attack was significantly reduced by both prenatal stress and F0 male isolation pre-SxAT (stress: −146.65 s, p < 0.0001; isolation: −228.95 s, p < 0.0001) and post-SxAT (stress: −168.75 s, p < 0.0001; isolation: −224.15 s, p < 0.0001). Over time, latency reduction in offspring of stressed dams approached significance (p = 0.0502), with no significant isolation × time interaction. (b) Attack frequency was significantly increased by prenatal stress and F0 male isolation both pre-SxAT (stress: +5.2 attacks, p < 0.0001; isolation: +5.3 attacks, p < 0.0001) and post-SxAT (stress: +8.55 attacks, p < 0.0001; isolation: +9.35 attacks, p < 0.0001). Over time, males from stressed–isolated lineages showed significant increases in attack frequency, whereas other groups remained relatively stable.

Forced mounting and threat behaviours increased markedly following SxAT, with the largest post-test elevations observed in offspring of prenatally stressed dams and those sired by isolated males (Fig. 4). Similarly, attack duration was greater in these groups both before and after SxAT exposure, indicating a persistently heightened aggressive phenotype (Fig. 5).

Latency to first attack was shortest, and attack frequency highest, in males from stressed–isolated (SIH) lineages, reflecting increased readiness and escalation of aggression (Fig. 6). While most groups showed stable or increased aggression following SxAT, SIH males exhibited a relative reduction compared to their own pre-test levels, although aggression remained higher than in all other groups (Fig. 7). This pattern suggests a ceiling or saturation effect in aggressive expression rather than normalization.

Fig. 7 — Prenatal stress × F0 housing × time interaction on aggression duration. Predictive margins of total aggression duration (seconds) in F1 male rats assessed before and after SxAT exposure, stratified by dam prenatal stress and F0 male housing condition. Predictions were obtained from linear mixed-effects models; error bars represent 95 % confidence intervals. Offspring from stressed dams sired by isolated males (SIH) exhibited significantly higher aggression durations than all other groups both pre-SxAT (stress: +72.9 s, 95 % CI: 54.86–90.94, p < 0.0001; isolation: +73.0 s, 95 % CI: 54.96–91.04, p < 0.0001) and post-SxAT (stress: +50.8 s, 95 % CI: 36.15–65.45, p < 0.0001; isolation: +64.0 s, 95 % CI: 49.35–78.65, p < 0.0001). A significant three-way interaction (p = 0.01) indicated that SIH males uniquely showed a reduction in aggression post-SxAT relative to their own baseline (−38.4 s; 95 % CI: −67.69 to −9.11), while aggression increased in all other groups.

These findings indicate that prenatal stress and paternal social isolation interact to shape a robust and distinct aggression phenotype in F1 males, with SxAT exposure amplifying group differences rather than homogenizing behavioural outcomes.

3.1.2.3. Maternal care effects on F1 aggressive behaviour

Higher maternal care was associated with significantly lower aggression duration (β = –794.39, p < 0.001), fewer attack bouts (β = –36.38, p = 0.017), and longer latency to first attack (β = +17.59, p < 0.001) in F1 males (Fig. 8a–c).

Fig. 8 — Associations between dam maternal care behaviour index (MCBI) and behavioural outcomes in F1 adult male rats during the resident-intruder test, stratified by F0 male housing condition (group vs. isolation). (a) Aggression duration: Higher MCBI was significantly associated with shorter aggression durations (β = –794.39, SE = 129.56, t = –6.13, p < 0.001). (b) Latency to first attack: Higher MCBI predicted longer attack latencies (β = 17.59, SE = 3.31, t = 5.32, p < 0.001). (c) Attack frequency: Higher MCBI was associated with fewer attacks (β = –36.38, SE = 14.53, t = –2.51, p = 0.017). F0 male isolation showed a marginal association with attack frequency (β = 33.91, SE = 17.07, t = 1.99, p = 0.055) but no significant effects on aggression duration or latency.

3.1.2.4. Changes in behaviour pre- and post-SxAT

SxAT exposure led to a significant increase in aggressive behaviour duration in F1 males from isolated F0 males and those receiving lower maternal care. However, the interaction between F0 male housing and maternal care reversed this trend, suggesting that maternal care buffered the pro-aggressive effects of paternal social isolation (Fig. 9).

Fig. 9 — Relationship between dam maternal care behaviour index (MCBI) and change in aggressive behaviour duration (post–pre SxAT) in F1 male rats, stratified by F0 male housing condition (group vs. isolation). Isolated F0 male housing was significantly associated with reduced aggressive behaviour duration (β = –8.663, SE = 3.152, t = –2.747, p = 0.0093), and higher MCBI was also linked to reduced aggression (β = –5.561, SE = 2.681, t = –2.075, p = 0.0452). The interaction between MCBI and F0 housing condition was significant, with increased aggression duration in isolated males at higher MCBI (β = 9.910, SE = 3.683, t = 2.691, p = 0.0107).

3.1.3. F1 male sexual aggressive behaviour in the sexual aggression test

Across the four days of the Sexual Aggression Test (SxAT), prenatal stress and F0 male isolation independently and additively increased aggression-related behaviours in F1 males, with stressed–isolated offspring (SIH) consistently exhibiting the most pronounced phenotype. SIH males showed the highest durations of offensive grooming (Fig. 10a), keep-down behaviour (Fig. 10b), total aggression duration (Fig. 10c), forced mounting (Fig. 10d), and threats (Fig. 10e) across test days.

On Day 4, SIH males reached peak aggression duration (239.2 ± 32.3 s) compared with non-stressed group-housed controls (NSGH: 95.9 ± 16.2 s; Table 4; Fig. 10c). Forced mounting was likewise maximal in SIH males on Day 4 (40.8 ± 8.1 s vs. 8.9 ± 2.1 s in NSGH; Table 4; Fig. 10d), accompanied by elevated threat behaviour (Fig. 10e). These findings indicate a strong amplification of offensive aggression under combined prenatal stress and paternal isolation.

Table 4.

Key aggressive and social behaviour measures of F1 male rats during the Sexual Aggression Test (SxAT) on Days 1 and 4.

Measure	Day	NSGH	NSIH	SGH	SIH
Aggression duration (s)	1	85.6 ± 8.9	133.5 ± 12.1	155.4 ± 18.7	244.5 ± 24.3
	4	95.9 ± 16.2	148.4 ± 16.1	139.3 ± 15.1	239.2 ± 32.3
% Time in aggression	1	14.3 ± 1.4	22.2 ± 1.9	25.8 ± 3.2	40.6 ± 3.9
	4	16.0 ± 2.9	24.8 ± 2.7	23.3 ± 2.5	39.9 ± 5.5
Forced mounting (s)	1	11.0 ± 2.3	23.3 ± 5.7	22.4 ± 3.6	35.2 ± 9.6
	4	8.9 ± 2.1	33.0 ± 5.5	16.8 ± 4.9	40.8 ± 8.1
Threats	1	7.3 ± 1.3	11.6 ± 3.3	11.7 ± 3.3	15.4 ± 6.4
	4	5.9 ± 2.9	13.2 ± 4.4	8.6 ± 1.9	17.5 ± 7.9
Social behaviour duration (s)	1	295.5 ± 34.3	263.1 ± 32.3	233.7 ± 25.8	154.1 ± 24.8
	4	337.3 ± 31.9	239.4 ± 20.1	203.6 ± 32.7	212.1 ± 23.5
% Time in social behaviour	1	49.2 ± 5.6	43.8 ± 5.4	39.1 ± 4.3	25.7 ± 4.1
	4	56.2 ± 5.3	39.9 ± 3.3	34.1 ± 5.6	35.4 ± 3.9
Latency to first attack (s)	4	—	—	—	15.1 ± 6.9

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Values are mean ± SD. NSGH = non-stressed dam × group-housed male; NSIH = non-stressed dam × isolated male; SGH = stressed dam × group-housed male; SIH = stressed dam × isolated male

Social behaviours were concurrently suppressed in SIH males. On Day 1, SIH animals spent substantially less time in social behaviours (154.1 ± 24.8 s) than NSGH controls (295.5 ± 34.3 s; Table 4), with corresponding reductions in the percentage of time spent in social interaction (Table 4; Fig. 10, social behaviour panels).

Copulation latency was significantly reduced by F0 male isolation and prenatal stress, with the shortest latencies consistently observed in SIH offspring across test days (Fig. 11), indicating heightened sexual impulsivity in this group.

Fig. 11 — Adjusted predictions of copulation latency (in seconds) across four days in the SxAT, derived from mixed-effects regression models. Significant effects of dam stress (χ²(1) = 16.78, p < 0.001) and F0 male isolation (χ²(1) = 51.02, p < 0.001) were observed. F1 males of stressed dams and isolated F0 males exhibited shorter copulation latencies compared to their non-stressed and group-housed counterparts.

Associations between maternal care behaviour and adult sexual aggression were explored separately and are presented in the Supplementary Materials (Supplementary Figure 5).

3.1.4. F1 male anxiety-like behaviour

Assessment of anxiety-like behaviour using the open field test (OFT) and elevated plus maze (EPM) revealed clear group differences associated with prenatal stress and F0 male housing (Table 5). In the OFT, NSIH rats exhibited significantly greater rearing time compared with NSGH, SGH, and SIH groups (all p ≤ 0.004), suggesting increased exploratory activity. Grooming time was highest in NSGH rats and was significantly reduced in NSIH, SGH, and SIH animals (p ≤ 0.003).

In the EPM, SIH rats spent markedly more time in the closed arms than all other groups (all p < 0.001), indicating heightened anxiety-like behaviour. Together, these findings demonstrate that prenatal stress combined with paternal social isolation produces a distinct anxiety-like phenotype in F1 males, characterized by increased avoidance behaviour in anxiogenic environments (see also Supplementary Table 2).

3.1.5. Gene expression analyses

Gene expression analyses revealed robust, region-specific transcriptional differences associated with prenatal stress and F0 male housing, providing molecular plausibility for the observed behavioural phenotypes. Significant effects were concentrated in the hypothalamus, hippocampus, amygdala, and prefrontal cortex (PFC), particularly for stress- and social behaviour–relevant genes (CRHR1, OXTR, AR, AVPR1A).

In the hypothalamus, CRHR1 expression was higher in SGH compared with NSGH offspring, whereas NSIH offspring showed reduced expression relative to NSGH. OXTR expression was consistently reduced across NSIH, SGH, and SIH groups relative to NSGH, indicating a broad disruption of oxytocin signalling following either prenatal stress or paternal isolation. In the PFC, OXTR expression was similarly reduced in SGH offspring, while AR expression was higher in NSGH compared with SGH, suggesting stress-related modulation of androgen signalling within frontal regulatory circuits.

AVPR1A displayed a distinct and region-dependent pattern. Expression was elevated in the hypothalamus and PFC of NSIH offspring relative to NSGH, but reduced in the amygdala, hippocampus, and PFC of SGH animals. These findings indicate that paternal isolation and prenatal stress exert overlapping but non-identical effects on vasopressin signalling, depending on brain region and developmental context. All statistically significant multivariate effects are summarised in Table 6, while full descriptive statistics and non-significant comparisons are provided in Supplementary Tables S3–S4.

Table 6.

Significant multivariate regression results for gene expression across brain regions in F1 male rats.

Group	Gene	Region	β (95 % CI)	p-value
SIH	log2_chrh1	HYPO	0.174 (0.035, 0.313)	0.018
	log2_oxtr	PFC	-0.252 (-0.457, −0.048)	0.019
	log2_avpr1a	HYPO	-0.556 (-0.901, −0.212)	0.004
NSIH	log2_oxtr	HIPPO	0.172 (0.028, 0.315)	0.022
	log2_avpr1a	HIPPO	-0.555 (-1.073, −0.037)	0.037
	log2_avpr1a	PFC	0.748 (0.230, 1.266)	0.007
SGH	log2_chrh1	HYPO	0.230 (0.084, 0.376)	0.004
	log2_chrh1	HIPPO	0.172 (0.026, 0.318)	0.024
	log2_chrh1	PFC	0.178 (0.033, 0.324)	0.020
	log2_oxtr	_cons	-0.144 (-0.241, −0.046)	0.007
	log2_avpr1a	HYPO	0.579 (0.250, 0.909)	0.002
	log2_avpr1a	PFC	0.588 (0.259, 0.918)	0.002
SIH	log2_avpr1a	PFC	0.673 (0.062, 1.284)	0.033

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Multivariate regression results showing only statistically significant (p < 0.05) differences in gene expression across brain regions—hypothalamus (HYPO), hippocampus (HIPPO), and prefrontal cortex (PFC)—in F1 male rats from four experimental groups: NSGH, NSIH, SGH, and SIH. β values represent the change in log₂-transformed gene expression relative to the amygdala (reference region), with 95 % confidence intervals in parentheses.

Maternal care behaviour further modulated gene expression in a region- and stress-dependent manner (Fig. 12). Higher maternal care was associated with reduced CRHR1 expression in the hippocampus of non-stressed offspring (Fig. 12a), consistent with a buffering or anxiolytic effect. In the amygdala, increased maternal care was linked to lower HTR1A (Fig. 12b) and AVPR1A (Fig. 12c) expression in non-stressed animals, whereas these associations were absent following prenatal stress exposure. Prenatal stress altered the direction of the maternal care–CRHR1 relationship in the amygdala; however, this interaction did not reach statistical significance.

3.1.6. Neurochemical analyses

3.1.6.1. Neurochemical profiles across experimental groups

Neurochemical analyses revealed pronounced group-dependent alterations consistent with the behavioural phenotypes observed (Table 7). Serotonin and corticotropin-releasing factor (CRF) concentrations were significantly higher in NSGH and SGH offspring compared with NSIH and SIH (both p < 0.001), indicating reduced serotonergic and CRF signalling in offspring exposed to paternal isolation and/or combined stress. In contrast, neurokinin B (NKB) levels were elevated in SIH compared with NSGH (p = 0.003) and in NSIH compared with SGH (p = 0.009), suggesting stress- and housing-specific modulation of tachykinin pathways.

Table 7.

Neurochemical profiles of F1 male offspring by maternal prenatal stress and paternal housing conditions.

Neurochemical	NSGH (n = 7)	NSIH (n = 7)	SGH (n = 7)	SIH (n = 7)	F-value	p-value
Serotonin (ng/mL)	1043.30 ± 189.38	565.40 ± 134.95	784.26 ± 156.65	257.64 ± 146.28	45.67	< 0.001***
CRF (pg/mL)	24.09 ± 4.53	13.74 ± 2.58	34.88 ± 3.99	18.51 ± 3.41	38.12	< 0.001***
Neurokinin B (pg/mL)	23.54 ± 3.06	29.26 ± 2.28	27.51 ± 6.68	31.78 ± 3.14	5.89	0.003**
Corticosterone (µg/dL)	8.93 ± 1.72	15.71 ± 4.14	14.87 ± 3.18	21.38 ± 2.95	12.45	< 0.001***
Arginine vasopressin (pg/mL)	2.51 ± 0.84	1.98 ± 1.03	1.00 ± 0.95	3.29 ± 0.44	4.78	0.007**
Oxytocin (pg/mL)	340.49 ± 127.04	690.70 ± 59.49	216.59 ± 96.23	503.64 ± 96.98	18.92	< 0.001***
Testosterone (ng/mL)	1.11 ± 0.44	1.44 ± 1.05	1.35 ± 0.30	0.95 ± 0.32	2.35	0.091

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Data are presented as mean ± SD. NSGH = non-stressed dam × group-housed male; NSIH = non-stressed dam × isolated male; SGH = stressed dam × group-housed male; SIH = stressed dam × isolated male. p-values are derived from two-way ANOVA (prenatal stress × paternal housing), with Tukey’s HSD used for post-hoc pairwise comparisons where appropriate. ** p < 0.01; *** p < 0.001.

Corticosterone levels were markedly increased in SIH offspring relative to both NSGH (p < 0.001) and SGH (p = 0.002), indicating heightened HPA-axis activation following combined prenatal stress and paternal isolation. Arginine vasopressin (AVP) concentrations were also elevated in SIH compared with NSGH (p = 0.005). Oxytocin levels were higher in NSIH offspring relative to NSGH and SGH (both p < 0.001), consistent with altered social neuropeptide regulation following paternal isolation in the absence of prenatal stress. Testosterone levels did not differ significantly between groups.

All statistically significant neurochemical group differences are summarised in Table 7.

3.1.6.2. Neurochemicals and behaviour following SxAT

Neurochemical variation was associated with post-SxAT behavioural expression. Combined prenatal stress and paternal isolation increased neutral behaviour duration relative to NSGH offspring (p = 0.035). Higher serotonin concentrations were associated with longer neutral behaviour durations in stressed groups (p = 0.021), while higher CRF levels were linked to reduced social behaviour duration, particularly in SIH compared with NSGH offspring (p = 0.012). Paternal isolation independently amplified stress-related changes in social behaviour relative to group-housed controls (p = 0.021).

3.1.7. Neurochemicals and aggression-related behaviours

Serotonin showed robust inverse associations with multiple aggression-related measures, including attack frequency (r = −0.585, p = 0.001), threats (r = −0.681, p < 0.001), forced mounting (r = −0.661, p < 0.001), and total aggression duration (ρ = −0.745, p < 0.0001). Conversely, serotonin was positively associated with non-social exploration and neutral behaviour (both p = 0.004). Corticosterone concentrations were positively correlated with attack frequency (ρ = 0.756, p < 0.0001) and negatively correlated with latency to first attack (ρ = −0.853, p < 0.0001), supporting a link between HPA-axis activation and heightened aggression. Oxytocin was positively associated with offensive grooming (p = 0.029) and inversely associated with attack latency (p = 0.022).

3.1.8. Neurochemicals and anxiety-like behaviours

Neurochemical–behaviour interactions were further evident in anxiety-related assays. In the open field test (OFT), prenatal stress increased centre-zone time (p = 0.019). Higher NKB concentrations were associated with reduced centre-zone time (p = 0.015); however, a significant prenatal stress × NKB interaction increased centre time in stressed offspring (p = 0.010). In the elevated plus maze (EPM), a prenatal stress × AVP interaction increased open-arm time (p = 0.037), while a three-way interaction with paternal housing reduced open-arm exploration (p = 0.039). Offspring of isolated F0 males spent more time in closed arms (p = 0.030), whereas prenatal stress × AVP reduced closed-arm time (p = 0.030).

These interaction-driven effects are visualised in supplementary figure 6 and provide mechanistic context for the anxiety-like phenotypes observed.

4. Discussion

This study demonstrates that prenatal stress, paternal social isolation, and their interaction exert profound effects on maternal care behaviour, aggression, sociality, anxiety-like behaviour, and neurobiological markers in F1 male Wistar rats. The integration of behavioural, neurochemical, and gene expression data reveals an intergenerational stress phenotype mediated through HPA axis dysregulation, altered oxytocinergic and vasopressinergic signalling, and region-specific transcriptional changes. By simultaneously modelling maternal, paternal, and offspring-level factors, this work extends prior single-hit stress paradigms and provides a more ecologically valid framework for understanding cumulative developmental adversity.

PRS consistently reduced MCB across postnatal days 2–8, particularly in licking, nursing, and pup-contact behaviours. The largest deficits occurred on PND 2–3, a critical period for establishing maternal–offspring bonding. Although paternal isolation exerted only transient effects on MCB in non-stressed dams, its combination with PRS further disrupted caregiving, especially pup retrieval, indicating an additive effect of maternal and paternal stress environments. These findings are consistent with reports linking PRS to impaired oxytocin-dependent maternal behaviours (Li et al., 2021, Nolvi et al., 2023). Importantly, the present results demonstrate that paternal stress exposure can amplify PRS-related caregiving deficits, an effect that has been underexplored in earlier maternal-centric models of intergenerational stress. Larger litter size and later postnatal days were associated with increased MCB, suggesting compensatory responses that nevertheless failed to normalise caregiving deficits in stressed groups.

In the Resident-Intruder Test, PRS and paternal isolation independently elevated aggression—shorter latency to first attack, longer attack durations, and higher attack frequency—while reducing social behaviour post-SxAT. SIH males consistently showed the highest aggression, likely reflecting cumulative dysregulation of vasopressin and corticosterone signalling (Brunton and Russell, 2010, Nguyen et al., 2018). While some studies report stress-induced aggression primarily following maternal adversity (Adeline Dorothy and Rajan, 2025, Ramos et al., 2025), our findings indicate that paternal isolation alone can prime offspring aggression, and that combined parental stress produces a qualitatively more severe phenotype. MCB acted as a protective factor, with higher MCBI predicting reduced aggression duration, fewer attacks, and longer attack latency. However, in the SIH group, this buffering effect was attenuated, suggesting that under compounded adversity, maternal care is insufficient to fully counteract pro-aggressive programming.

Across the Sexual Aggression Test, PRS and paternal isolation additively increased offensive grooming, forced mounting, threats, and reduced social interaction. Aggression duration peaked on Day 4 in SIH males, accompanied by the shortest copulation latencies. These patterns suggest that PRS establishes a heightened baseline of sexual aggression, while paternal isolation amplifies it through AVP–corticosterone mechanisms (Beurel & Nemeroff, 2014). Differences from studies reporting habituation or attenuation of aggression across repeated sexual encounters may reflect the compounded stress exposures modelled here, which likely impair adaptive behavioural modulation. MCB reduced sexual aggression metrics across groups, but its protective effect was weakest in SIH males, mirroring trends in the Resident-Intruder Test.

PRS and paternal isolation interacted to produce severe anxiety-like phenotypes, particularly in the SIH group, which spent the least time in EPM open arms and the most in closed arms. In the OFT, rearing was highest in NSIH males, potentially reflecting heightened novelty-seeking in the absence of maternal stress, while grooming was lowest in stressed–isolated offspring, consistent with maladaptive stress coping. These outcomes align with literature showing PRS-induced disruption of stress-regulatory circuits and serotonergic–CRH signalling (Bale et al., 2002). Notably, the dissociation between anxiety-like behaviour and exploratory rearing in NSIH males suggests that paternal isolation may selectively alter motivational rather than affective domains, a distinction not captured in simpler stress models.

Significant gene expression changes revealed region- and group-specific transcriptional patterns. In the amygdala, AVPR1A was upregulated in NSIH but downregulated in SGH, implicating vasopressin signalling in social aggression and anxiety modulation (Rigney et al., 2023b). Hypothalamic AVPR1A was reduced in NSGH but elevated in NSIH, consistent with paternal isolation enhancing stress sensitivity. Hippocampal AVPR1A downregulation in NSIH and SGH reinforces its role in stress modulation. PFC AVPR1A upregulation in NSIH suggests heightened aggression propensity (Veenema and Neumann, 2008). Across regions, OXTR downregulation—particularly in SGH—implies reduced oxytocin-mediated prosociality. These regionally divergent effects help explain inconsistencies in prior reports that assessed single brain regions or single stressors in isolation.

MCB was inversely associated with hippocampal CRHR1 and amygdalar HTR1A and AVPR1A expression, supporting its role in dampening stress-reactive neurocircuitry. PRS altered these associations, notably reversing the MCB–CRHR1 relationship in the amygdala, indicating that maternal care may moderate rather than fully reverse PRS effects. This context-dependent modulation offers a mechanistic explanation for why enhanced maternal care does not uniformly confer resilience across all stress backgrounds.

Neurochemical analyses showed that SIH males had the lowest serotonin and highest corticosterone, AVP, and NKB, a profile strongly linked to heightened aggression and anxiety (da Cunha-Bang and Knudsen, 2021). CRF was highest in SGH and lowest in NSIH, suggesting differential HPA-axis adaptation. Oxytocin was elevated in NSIH, possibly reflecting a compensatory mechanism under paternal isolation. These neurochemical profiles correlated with behavioural outcomes: serotonin inversely with aggression and positively with exploration; corticosterone positively with attack frequency; and oxytocin positively with offensive grooming but negatively with attack latency. The inclusion of neurokinin B extends existing models of stress-related aggression by implicating tachykinin signalling in cumulative stress sensitivity, a pathway rarely examined in intergenerational paradigms.

4.1. Neurophysiological Model of Stress-Related Behavioural Outcomes

Building on these findings, we propose a neurophysiological model linking prenatal stress, paternal isolation, and maternal care deficits to behavioural outcomes in F1 male offspring. Dysregulation of vasopressin, oxytocin, corticotropin-releasing hormone, serotonin, and neurokinin B constitutes a convergent pathway driving heightened aggression, reduced sociality, and maladaptive stress responses.

Upregulation of AVPR1A in the amygdala of SIH offspring promotes aggression and anxiety, consistent with vasopressin’s role in stress-induced behavioural pathologies (Rigney et al., 2023). Elevated CRHR1 following prenatal stress reflects HPA-axis hyperactivation, while maternal care attenuates CRHR1 expression, reducing stress reactivity (Hartman et al., 2023). In the hypothalamus, AVPR1A upregulation under paternal isolation exacerbates stress sensitivity, whereas CRHR1 upregulation in socially housed groups may reflect adaptive HPA engagement (Veenema and Neumann, 2008).

Downregulation of AVPR1A in the hippocampus links vasopressin dysregulation to impaired stress inhibition, while OXTR upregulation in NSIH rats suggests oxytocin-mediated resilience under paternal isolation (Lee et al., 2015). In the prefrontal cortex, AVPR1A upregulation compromises emotional regulation, whereas OXTR downregulation reduces social bonding, aligning with observed behavioural deficits ((Parise et al., 2023). Suppressed serotonergic signalling and elevated corticosterone in SIH rats further reinforce a pro-aggressive, hyperaroused phenotype (Cao et al., 2017). Importantly, elevated neurokinin B in SIH offspring highlights its emerging role in HPA-axis dysregulation and anxiety-like behaviour under cumulative stress exposure (Grachev et al., 2014).

Together, these pathways suggest that prenatal stress disrupts maternal oxytocin signalling and caregiving, while paternal isolation compounds offspring vulnerability through vasopressin-, serotonin-, and NKB-dependent mechanisms, likely mediated by epigenetic transmission via the paternal germline (Zucchi et al., 2013, Álvarez-Mejía et al., 2025) Compensatory oxytocin increases under isolated paternal conditions may partially buffer stress effects but are insufficient under combined parental adversity.

5. Conclusion

This study demonstrates that prenatal stress, paternal social isolation, and maternal caregiving behaviours interact to shape neurobehavioural outcomes in F1 male Wistar rats. PRS and paternal isolation independently and additively amplified aggression, anxiety-like behaviours, and reduced sociability, mediated by dysregulation of key neuroendocrine systems—vasopressin, oxytocin, corticotropin-releasing hormone, serotonin, and neurokinin B.

Maternal care emerged as a partial protective factor, attenuating stress-related gene expression changes and improving behavioural outcomes. However, its buffering capacity was context-dependent, offering limited protection under compounded adversity, particularly in offspring of stressed dams and isolated sires.

These findings highlight the complex interplay between maternal and paternal influences in intergenerational stress transmission and resilience. The proposed neurophysiological model links prenatal and early-life stress to persistent neurochemical dysregulation and potential epigenetic programming, providing mechanistic insight into the origins of stress-related behavioural deficits and identifying potential targets for prevention and intervention.

CRediT authorship contribution statement

Qulu-Appiah Lihle: Writing – review & editing, Validation, Supervision, Methodology, Funding acquisition, Conceptualization. Jacqueline Samantha Womersley: Writing – review & editing, Visualization, Validation, Formal analysis, Data curation. Thando W. Shabangu: Validation. Sian Megan Joanna Hemmings: Writing – review & editing, Validation, Supervision. Elvis Mbiydzenyuy Ngala: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Animal care and experimental procedures were conducted in strict compliance with the South African National Health Research Ethics guidelines and were approved by the Stellenbosch University Research Ethics Committee (ACU-2021–13333). All experimental procedures were approved by the Institutional Animal Care and Use Committee and conducted in accordance with international ethical standards for the care and use of laboratory animals.

Funding

This study was funded by the South African Medical Research Council (Grant No. SAMRC MB2022/EIP026) through its Division of Research Capacity Development under the Early Investigators Program from funding received from the South African National Treasury. The funder was not involved in the study design; collection, analysis and interpretation of data; writing of the report; and decision to submit the article for publication.

Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the author(s) used ChatGPT (OpenAI, GPT-4) to assist with improving clarity, language clarity and readability. After using this tool, the author(s) reviewed, verified, and edited the content as needed and take full responsibility for the content of the publication.

Declaration of Competing Interest

The authors declare no conflict of interest.

Footnotes

^{Appendix A}

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ibneur.2026.02.012.

Contributor Information

Elvis Mbiydzenyuy Ngala, Email: 24467189@sun.ac.za.

Sian Megan Joanna Hemmings, Email: smjh@sun.ac.za.

Jacqueline Samantha Womersley, Email: jsw1@sun.ac.za.

Thando W. Shabangu, Email: 27825027@sun.ac.za.

Lihle-Appiah Qulu, Email: qulul@sun.ac.za.

Appendix A. Supplementary material

Figures S1

Supplementary material

mmc1.docx^{(536.8KB, docx)}

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PERMALINK

Sexual aggression and prenatal stress lead to poor maternal care and aggressive behaviour in male Wistar rat offspring

Elvis Mbiydzenyuy Ngala

Sian Megan Joanna Hemmings

Jacqueline Samantha Womersley

Thando W Shabangu

Lihle-Appiah Qulu

Abstract

Highlights

1. Introduction

2. Methods

2.1. Animals and housing conditions

2.2. Grouping and Experimental Setup

Fig. 1.

2.3. Sexual aggression paradigm in the F0 generation

2.3.1. Assessment of sexual aggression-like behaviour

2.3.2. Estrous cycle monitoring

2.3.3. Assessment of sexual receptivity in female rats during estrus

2.3.4. The sexual aggression test

2.4. Mating procedure and prenatal stress induction

2.4.1. Mating Protocol

2.4.2. Prenatal stress induction in pregnant rats

2.5. Assessment of maternal behaviours in the postnatal period

2.5.1. Evaluation criteria for maternal behaviours

2.6. Quantitative analysis of maternal behaviours

2.7. Selection and housing of male offspring for experimental analysis

2.8. Evaluation of aggression and anxiety-like behaviour in offspring

2.8.1. Behavioural assessment protocol for male offspring

2.8.2. The resident intruder test

2.8.3. The sexual aggression test

2.8.4. The open field test protocol

2.8.5. The elevated plus maze test

2.9. Gene expression analyses in brain tissue

2.9.1. Brain tissue collection and preservation for gene expression analysis

2.9.2. RNA extraction, cDNA synthesis, and quantitative real-time PCR

Table 5.

Table 1.

2.10. Hormonal assays in serum samples

2.11. Statistical Analysis

3. Results

3.1. Maternal care behaviour

3.1.1. Overview of maternal care across postnatal days 2–8

Fig. 2.

3.1.1.1. Effects of prenatal stress and F0 male housing on maternal behaviours

3.1.1.2. Modulatory effects of F0 male housing

Fig. 3.

3.1.1.3. Day-specific patterns and behavioural components

Table 2.

3.1.2. F1 male aggressive behaviour in the resident intruder test

3.1.2.1. Pre- and post-SxAT behavioural outcomes

Table 3.

3.1.2.2. Impact of prenatal stress, F0 male housing, and time

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

3.1.2.3. Maternal care effects on F1 aggressive behaviour

Fig. 8.

3.1.2.4. Changes in behaviour pre- and post-SxAT

Fig. 9.

3.1.3. F1 male sexual aggressive behaviour in the sexual aggression test

Fig. 10.

Table 4.

Fig. 11.

3.1.4. F1 male anxiety-like behaviour

3.1.5. Gene expression analyses

Table 6.

Fig. 12.

3.1.6. Neurochemical analyses

3.1.6.1. Neurochemical profiles across experimental groups

Table 7.

3.1.6.2. Neurochemicals and behaviour following SxAT

3.1.7. Neurochemicals and aggression-related behaviours

3.1.8. Neurochemicals and anxiety-like behaviours

4. Discussion

4.1. Neurophysiological Model of Stress-Related Behavioural Outcomes

5. Conclusion

CRediT authorship contribution statement

Consent for publication

Ethics approval and consent to participate