Abstract
Objective
Chronic sleep deprivation (SD) and caffeine consumption individually impair male reproductive function; however, the combined impact of simultaneous exposure on reproductive neuroendocrinology remains largely unexplored. This study investigated the effects of concomitant exposure to a novel Unpredictable Chronic Sleep Deprivation (CUSD) paradigm and high-dose caffeine on hypothalamic monoaminergic neurotransmission, seminal indices, and hypothalamic-pituitary-adrenal/gonadal (HPA/HPG) axis activity in adult male Long-Evans rats.
Results
Twenty male Long-Evans rats were exposed to either CUSD, caffeine, or a combination of CUSD and caffeine. Combined CUSD and caffeine exposure significantly increased intromission latency and reduced intromission number, mounting latency, and mounting frequency compared to controls. Seminal parameters, including sperm motility, morphology, viability, and count, were markedly impaired in the CUSD and CUSD+caffeine groups. Serum GnRH, testosterone, LH, and FSH were significantly reduced, while corticosterone was significantly elevated across treatment groups, with the most pronounced effects in the CUSD+caffeine group. Hypothalamic serotonin and dopamine were significantly depleted in the CUSD and CUSD+caffeine groups. Nitrergic stress, evidenced by elevated iNOS and NO levels, was observed in hypothalamic and testicular tissues. These findings suggest that simultaneous exposure to CUSD and high-dose caffeine synergistically impairs male sexual performance and fecundity by dysregulating monoaminergic neurotransmission and HPA/HPG axis activity.
Graphical Abstract
Keywords: Caffeine, Unpredictable chronic sleep deprivation, Monoaminergic neurotransmission, Hypothalamic-pituitary-adrenal/gonadal axis
Introduction
Caffeine is the world’s most widely consumed psychoactive substance and functions as a non-selective adenosine A1 and A2A receptor antagonist [1, 2]. Caffeine exerts its neuroactive effects primarily through competitive antagonism of adenosine A1 and A2A receptors, both of which are expressed in hypothalamic nuclei and testicular tissue critical to reproductive neuroendocrine function [1]. While moderate caffeine intake is associated with improved alertness and mood, chronic high-dose exposure has been linked to dysregulation of the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes, impaired semen quality, and neuropsychiatric symptoms [3, 4]. Concurrently, chronic sleep deprivation (SD) is an escalating public health concern driven by modern lifestyle demands. SD disrupts neuroendocrine homeostasis, reduces testosterone levels, impairs dopaminergic signalling, and compromises male sexual and reproductive function [5, 6].
Activation of the HPA axis by stressors such as SD elevates corticosterone, which in turn suppresses HPG axis activity, reduces gonadotropin release, and impairs downstream testicular steroidogenesis [7, 8]. Hypothalamic monoaminergic systems, particularly dopamine and serotonin, play critical roles in regulating GnRH pulsatility and sexual behaviour, making them key mediators of stress-induced reproductive dysfunction [9, 10]. While SD and caffeine have been independently shown to dysregulate these systems, whether their concomitant exposure produces synergistic adverse effects on male reproductive neuroendocrinology and sexual performance has not been examined. This study, therefore, investigated the combined effects of a novel Unpredictable Chronic Sleep Deprivation (CUSD) paradigm and high-dose caffeine on sexual behaviour, seminal parameters, monoaminergic neurotransmission, and HPA/HPG axis activity in Long-Evans rats.
Materials and methods
Animals and ethical approval
Twenty adult male Long-Evans rats (200–250 g) were obtained from the ABUAD Animal Research Centre and housed under standard laboratory conditions with ad libitum access to food and water. All procedures adhered to the NIH Guide for the Care and Use of Laboratory Animals [11]. Ethical approval was granted by the Research Ethical Committee of Afe Babalola University (AB/EC/19/10/15). Twenty adult male Long-Evans rats were assigned to four experimental groups of five animals each (n = 5 per group). Given the exploratory and hypothesis-generating nature of this study, this sample size was determined to provide adequate sensitivity for detecting large effect sizes consistent with those reported.
Experimental design
As shown in Fig. 1, following two weeks of acclimatisation, rats were randomly assigned to four groups (n = 5): (A) Control received normal saline; (B) Caffeine [CF (Cristalchem, UK)] Group B received caffeine (200 mg/kg, dissolved in normal saline, administered by orogastric gavage) daily for 14 days. This dose was selected based on published evidence that it reliably disrupts HPA and HPG axis function in adult rodents [8] and approximates heavy chronic human caffeine consumption using standard allometric scaling; (C) CUSD group exposed to the CUSD paradigm for 14 days; (D) CUSD + CF received concurrent caffeine (200 mg/kg, oral) and CUSD for 14 days.
Fig. 1.
Experimental design
CUSD paradigm
The Unpredictable Chronic Sleep Deprivation paradigm was developed in-house. Over 14 days, animals were exposed daily to one of five unpredictably rotating sleep-interruption stimuli: gentle handling, 24-hour light-dark reversal, platform-over-water, crowded housing, and stroboscopic light, with no stimulus repeated on consecutive days. The experimental protocol was conducted over 14 days to model subchronic stress exposure in rats. The study employed repeated exposure within this timeframe to assess neurobiological and behavioural outcomes associated with the experimental conditions [12–15].
Sexual behaviour assay
Sexual behaviour was assessed by pairing each male with an oestrous-induced female (cloprostenol 0.5 µg i.p. × 2, three days apart; progesterone 3 mg s.c.) in a clear observation chamber (60 × 60 × 80 cm) for 60 min following a 10-minute acclimatisation period [16, 17]. Intromission latency, intromission number, mounting latency, and mounting frequency were recorded by digital video. Intromission latency and mounting latency reflect sexual motivation and the latency to initiate copulatory behaviour, respectively, whereas intromission number and mounting frequency index the intensity and efficiency of sexual engagement. Together, these parameters provide a comprehensive phenotypic readout of male sexual performance mediated by hypothalamic dopaminergic and serotonergic circuits [16]. Behavioural parameters were quantified from digital video recordings by an observer blinded to group allocation; cage identities were coded and not revealed until after all scoring was completed.
Euthanasia and tissue collection
Animals were deeply anesthetized with ketamine hydrochloride (100 mg/kg, i.p.), and attainment of a surgical plane of anaesthesia was verified by the absence of the pedal withdrawal reflex. Under this condition, euthanasia was performed by cardiac exsanguination, confirmed by cessation of cardiac activity. Blood samples were obtained via cardiac puncture, and the cauda epididymis, testes, and whole brain were promptly harvested. Hypothalamic and testicular tissues were dissected, either snap-frozen in liquid nitrogen or processed immediately for biochemical assays. All animals were euthanized within 30 min of completing the behavioural session, and tissues were stored at − 80 °C until analysis. Biochemical assays were conducted in a single batch.
Seminal analyses
Cauda epididymal sperm suspensions were prepared in phosphate-buffered saline. Sperm motility was graded according to WHO criteria (grades a–d) [18]. Viability was assessed by eosin-nigrosin staining, morphology by Wyrobek and Bruce criteria, and count using a Neubauer haemocytometer (sperm count = total sperm in 5 squares × 50,000 × 100 cells/mL) [19, 20].
Hormonal and biochemical assays
Serum oestradiol, GnRH, testosterone, LH, FSH, and corticosterone were quantified using competitive ELISA kits according to the manufacturer’s instructions (absorbance at 450 nm). Hypothalamic and testicular levels of serotonin, dopamine, iNOS, and nitric oxide (NO) were measured using sandwich ELISA kits (Wuhan Fine Biotech, China), with tissue homogenates prepared in phosphate buffer (pH 7.4), centrifuged at 5000 × g for 10 min, and supernatants retained for analysis. Nitric oxide was quantified as total stable nitrite/nitrate via the Griess reaction (Wuhan Fine Biotech ELISA), an established and widely validated approach for tissue-level assessment of NO production in homogenate-based preparations.
Statistical analysis
Data are expressed as mean ± SEM. Group comparisons were performed using one-way ANOVA with Tukey post-hoc tests (GraphPad Prism; significance threshold p < 0.05).
Results
Sexual behaviour
Concomitant CUSD and caffeine (Group D) significantly increased intromission latency compared to controls (**p < 0.01; F(3,16) = 7.162). Intromission number was significantly reduced in Groups B (*p < 0.05), C (****p < 0.0001), and D (****p < 0.0001; F(3,16) = 21.93). Mounting latency was significantly reduced in Groups C (*p < 0.05) and D (**p < 0.01; F(3,16) = 6.671). Mounting frequency was reduced across all treatment groups: B (*p < 0.05), C (*p < 0.05), and D (***p < 0.001; F(3,16) = 10.21) (Fig. 2).
Fig. 2.
Evaluation of male sexual performance behavioural indices: intromission latency (A); intromission frequency (B); mounting latency (C); and mounting frequency (D) following exposure to chronic SD and high-dose caffeine. Data are presented as mean ± SEM (n = 3). Legend: A = Control; B = Caffeine; C = Sleep Deprivation; D = Sleep Deprivation plus Caffeine
Impairment of seminal parameters following exposure to chronic sleep deprivation and high-dose caffeine in Long-Evans rats
Sperm motility was significantly reduced in Groups C (*p < 0.05) and D (***p < 0.001; F(3,8) = 15.53). Sperm morphology was impaired in Groups C (**p < 0.01) and D (***p < 0.001; F(3,8) = 26.41). Viability scores were significantly decreased in Groups C (**p < 0.01) and D (***p < 0.001; F(3,8) = 23.26). Sperm count was most severely reduced in Group D (****p < 0.0001) and Group C (***p < 0.001; F(3,8) = 44.57). Group B (caffeine alone) did not significantly alter sperm motility, viability, or count compared to controls (Fig. 3).
Fig. 3.
Evaluation of seminal parameters: sperm motility (A); sperm morphology (B); sperm viability (C); and sperm count (D) following exposure to chronic SD and high-dose caffeine. Data are presented as mean ± SEM (n = 3). Legend: A = Control; B = Caffeine; C = Sleep Deprivation; D = Sleep Deprivation plus Caffeine
Dysregulation of serum reproductive hormones and corticosterone following exposure to chronic SD and high-dose caffeine treatment in male Long-Evans rats
GnRH was significantly reduced in Groups C (*p < 0.05) and D (**p < 0.01; F(3,8) = 7.577). Testosterone was significantly reduced in Groups B (*p < 0.05), C (*p < 0.05), and D (**p < 0.01; F(3,8) = 9.478). LH was significantly decreased in all treatment groups (F(3,8) = 10.02). FSH was significantly reduced across all groups with a dose-response pattern: B (**p < 0.01), C (***p < 0.001), D (****p < 0.0001; F(3,8) = 32.03). Corticosterone was significantly elevated in Groups B (**p < 0.01), C (*p < 0.05), and D (**p < 0.01; F(3,8) = 10.04), with the highest levels in Group D (Figs. 4 and 5).
Fig. 4.
Estimation of serum reproductive hormones: GnRH (A); testosterone (B); LH (C), and FSH (D) following exposure to chronic SD and high-dose caffeine. Data are presented as mean ± SEM (n = 3). Legend: A = Control; B = Caffeine; C = Sleep Deprivation; D = Sleep Deprivation plus Caffeine
Fig. 5.
Estimation of serum corticosterone levels following exposure to chronic SD and high-dose caffeine. Data are presented as mean ± SEM (n = 3). Legend: A = Control; B = Caffeine; C = Sleep Deprivation; D = Sleep Deprivation plus Caffeine
Dysregulation of corticosterone activity following exposure to chronic SD and high-dose caffeine treatment in male Long-Evans rats
Corticosterone was significantly elevated in Groups B (**p < 0.01), C (*p < 0.05), and D (**p < 0.01; F(3,8) = 10.04), with the highest levels in Group D (Figs. 4 and 5).
Alteration of hypothalamic monoaminergic neurotransmission following chronic SD and high-dose caffeine treatment in Long-Evans rats
Hypothalamic serotonin was significantly reduced in Groups C (*p < 0.05) and D (**p < 0.01; F(3,8) = 8.138); Hypothalamic dopamine was significantly reduced only in Group D following combined CUSD and caffeine exposure (F(3,8), p < 0.05; Fig. 6).
Fig. 6.
Estimation of hypothalamic serotonin (A) and dopamine (B) levels following exposure to chronic SD and high-dose caffeine. Data are presented as mean ± SEM (n = 3). Legend: A = Control; B = Caffeine; C = Sleep Deprivation; D = Sleep Deprivation plus Caffeine
Nitrergic responses in the hypothalamic-testicular axis of Long-Evans rats following simultaneous exposure to CUSD and high-dose caffeine treatment
Hypothalamic iNOS was significantly elevated in Group D (*p < 0.05; F(3,8) = 6.376). Hypothalamic NO was significantly elevated in Groups B (**p < 0.01), C (*p < 0.05), and D (**p < 0.01; F(3,8) = 10.78). Testicular iNOS was significantly elevated in Group C (*p < 0.05) and Group D (**p < 0.01; F(3,8) = 12.45). Testicular NO was elevated in Groups C (*p < 0.05) and D (**p < 0.01; F(3,8) = 10.74). Group B showed no significant changes in testicular iNOS or NO (Fig. 7).
Fig. 7.
Determination of hypothalamic nitrergic stress – iNOS (A), NO (B), as well as testicular nitrergic stress – iNOS (C), NO (D) following exposure to chronic SD and high-dose caffeine. Data are presented as mean ± SEM (n = 3). Legend: A = Control; B = Caffeine; C = Sleep Deprivation; D = Sleep Deprivation plus Caffeine
Discussion
This study demonstrates that concomitant exposure to CUSD and high-dose caffeine synergistically impairs male sexual performance, seminal quality, and reproductive neuroendocrinology in Long-Evans rats, with consistently greater effects in the combined group than either stressor alone. These findings are consistent with the known capacity of both SD and caffeine to independently dysregulate the HPA and HPG axes [4, 21], and extend this evidence to their combined exposure.
The impairment in sexual behavioural indices, particularly the pronounced increase in intromission latency and reduction in mounting frequency in the combined group. likely reflects the cumulative disruption of the neural circuits that regulate sexual motivation and performance. Limbic structures, including the hypothalamus, amygdala, and hippocampus, are critical for neuroendocrine regulation of sexual behaviour [22], and both chronic SD and sustained high-dose caffeine have independently been shown to alter neuroplasticity in these regions [23, 24]. Our observation that caffeine alone impaired mounting frequency but not intromission latency, while CUSD alone affected both, suggests that the two stressors engage partially overlapping but mechanistically distinct pathways, whose convergence amplifies behavioural deficits.
The seminal impairments parallel the behavioural findings. CUSD alone and CUSD+caffeine significantly reduced sperm motility, viability, morphology, and count, while caffeine alone had no significant effect on most seminal parameters. The sparing of seminal parameters in the caffeine-only group at this dose and duration is consistent with reports of a threshold-dependent and dose-sensitive effect of caffeine on spermatogenesis [25, 26]. The more severe impairment in the combined group is likely mediated by compounded activation of the HPA axis. Corticosterone was significantly elevated across all groups, most markedly in the combined group, which suppresses HPG axis activity by inhibiting GnRH and gonadotropin secretion [27], thereby reducing the gonadal hormonal milieu necessary for normal spermatogenesis.
The reductions in GnRH, LH, FSH, and testosterone across all treatment groups, with graded severity in the combined group, are mechanistically coherent. Testosterone, produced by Leydig cells under LH stimulation, is the primary androgen driving spermatogenesis and sexual function [28]. Its reduction, alongside elevated corticosterone, is consistent with glucocorticoid-mediated suppression of Leydig cell steroidogenic enzyme activity [29]. The suppression of hypothalamic dopamine and serotonin in the CUSD and combined groups adds a further upstream mechanism: both monoamines regulate GnRH pulsatility and sexual motivation [10, 30], and their depletion following CUSD and caffeine co-exposure likely compounds HPG axis suppression.
The elevation of iNOS and NO in hypothalamic and testicular tissues of the combined group indicates nitrergic/oxidative stress at both the central and gonadal levels. Sustained NO overproduction, particularly via inducible iNOS, is cytotoxic and has been associated with impaired spermatogenesis, reduced sperm motility, and DNA damage [31, 32]. This finding provides a plausible effector mechanism linking HPA axis hyperactivation and monoaminergic dysfunction to the observed seminal and sexual performance deficits.
Limitations
The biochemical and hormonal analyses were conducted on a small sample (n = 3–5 per group), which limits statistical power and the generalisability of the findings; these results should therefore be interpreted as preliminary and hypothesis-generating. The CUSD paradigm was developed in-house and has not been externally validated; no polysomnographic confirmation of sleep disruption was obtained to verify model efficacy. No histological examination of testicular or epididymal tissue was performed, which would have strengthened the seminal findings. Oxidative stress biomarkers, including malondialdehyde, superoxide dismutase, and catalase, were not measured; their inclusion would have better characterised the proposed nitrergic and oxidative stress mechanisms. Only a single time point (end of the 14-day exposure) was assessed, and recovery or reversibility following cessation of caffeine or sleep deprivation was not evaluated. Finally, inclusion of female animals in future studies would allow sex-specific comparisons that the present male-only design did not cover.
An Open Field Test was not performed in the current study; consequently, we cannot formally exclude the possibility that altered ambulatory activity contributed to the observed changes in sexual behaviour parameters. Future studies should incorporate locomotor assessments to delineate motivational and performance components of caffeine- and sleep deprivation-induced sexual behaviour impairments.
Nitric oxide was estimated as total nitrite/nitrate rather than real-time free NO; future studies employing DAF-FM diacetate fluorescence in tissue slices would provide superior spatiotemporal resolution of nitrergic signalling.
In addition, the adenosine receptor system, particularly A1 and A2A subtypes, was not characterised in this study despite their fundamental role in mediating caffeine’s neuroactive effects. Future studies should incorporate Western blot or quantitative PCR analysis of A1 and A2A receptor expression in hypothalamic and testicular tissues to directly interrogate the mechanistic basis of caffeine’s contribution to the observed reproductive neuroendocrine dysregulation.
Conclusion
Concomitant exposure to CUSD and high-dose caffeine synergistically impairs male sexual performance and fecundity in Long-Evans rats, mediated through dysregulation of hypothalamic monoaminergic neurotransmission, suppression of the HPG axis, HPA axis hyperactivation, and nitric stress in the hypothalamic-testicular axis. These findings highlight the potential reproductive hazard of combined chronic sleep loss and high caffeine intake and underscore the need for larger, well-powered confirmatory studies.
Acknowledgements
The authors acknowledge the facilities and material support provided by Afe Babalola University, Ado-Ekiti.
Author contributions
EEE: Conceptualization, methodology, project administration, writing — original draft, review and editing. COA: Methodology, formal analysis, review and editing. KEN, CFD: Investigation, Resources, Data Collection. KFC: Data curation, review and editing. LAE: Validation, review and editing.
Funding
No external funding was received for this work.
Data availability
The datasets generated during this study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
All animal procedures were approved by the Research Ethical Committee of Afe Babalola University (AB/EC/19/10/15) and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated during this study are available from the corresponding author on reasonable request.