Stesura Seveso Archivio Italiano di Urologia e Andrologia 2023; 95(4):11593 1 ORIGINAL PAPER Numerous animal studies including models of castrated rodents were useful to mimic the hypogonadism medical condition and evaluate the effects of androgen depletion on the cognitive functions (6). Multiple tasks were used to assess the spatial learning and working memory abilities of castrated rodents varying from navigating toward hidden platform in a pool of water in the Morris water maze test (7), to look for displaced objects in the novel object recog- nition and location tests (8). Although, the mechanism by which testosterone influences the working of spatial mem- ory is poorly understood, many of the animal studies showed a positive correlation between high/optimum testosterone level and better cognitive abilities in males including improved spatial memory (9, 10). Moreover, on a physiological level it has been reported that testosterone deficiency caused metabolic disorders including changes in body composition, fat distribution (11) and promoted oxidative stress and inflammation (12). The main treatment for hypogonadism in men is testos- terone replacement therapy (TRT). Therapeutic options for TRT varied from oral and injectable testosterone admin- istration to patches and testosterone gels. Innovations and advances in TRT during the years have enhanced the role and safety of the use of testosterone as a metabolic hormone and had beneficial effects on obesity, cardiovas- cular and hepatic diseases (13). Many research data nonetheless consolidated the long-term side effects of the TRT. In fact, long-term use of TRT has been associated with elevated oncologic risks mainly in the prostate (14) as well as the likelihood of developing obstructive sleep apnea and erythrocytosis (15). Therefore, potential alternatives, mainly plants and their derived natural substances, are being studied to replace and/or minimize the TRT side effects. For instance, onion supplementation was positively correlated to an increase in Luteinizing Hormone (LH) production and has been proven to reduce testis oxidative stress (16). Ginger supplementa- tion effect on intoxicated rats was also hypothesized to reduce oxygen species production and lipid peroxidation in the gonads thus improving testosterone level (17). Arthrospira platensis also commercialized under the name of Spirulina is a cyanobacterium which captured the sci- entists and food industry’s attention during the last few decades for its high nutritional as well as potential thera- Objective: Androgen deficiency is associated with multiple biochemical and behavioral disorders. This study investigated the effects of testosterone replacement and Spirulina Platensis association on testosterone deficiency-induced metabolic disorders and memory impairment. Methods: Adult male rats were randomly and equally divided into four groups and received the following treatments for 20 consecutive days. Control group: non-castrated rats received dis- tilled water. Castrated group received distilled water. Testosterone treated group: castrated rats received 0.20 mg of testosterone dissolved in corn oil by subcutaneous injection (i.p.). Spirulina co-treated group: castrated rats received 0.20 mg of testosterone (i.p.) dissolved in corn oil followed by 1000 mg/kg of Spirulina per os. Results: Data showed that castration induced an increase in plasma ALT, AST, alkaline phosphatase (PAL), cholesterol, and triglycerides level. Castrated rats showed a great elevation in SOD and CAT activities and MDA and H2O2 levels in the prostate, seminal vesicles, and brain. Testosterone deficiency was also associated with alteration of the spatial memory and exploratory behaviour. Testosterone replacement either alone or with Spirulina combination efficiently improved most of these biochemical parameters and ameliorated cognitive abilities in castrated rats. Conclusions: Testosterone replacement either alone or in combi- nation with Spirulina improved castration-induced metabolic, oxidative, and cognitive alterations. KEY WORDS: Castration; Testosterone; Spirulina Platensis; Cognition; Oxidative stress. Submitted 18 July 2023; Accepted 30 July 2023 INTRODUCTION With aging men can develop several cognitive and meta- bolic impairments due to the reduction in endogenous testosterone production (1). Androgen deficiency is referred to as hypogonadism, it is a health issue that can occur within men aged from 40 to 80 years old and includes fatigue, cognitive and mood disorders as clinical symptoms (2). It is also associated with common medical conditions such as hypertension, diabetes, and obesity (3). In fact, a very common consequence of testosterone deficiency in men is a decline in some forms of memory such as episodic and working memory (4, 5). Effects of testosterone replacement on lipid profile, hepatotoxicity, oxidative stress, and cognitive performance in castrated wistar rats Oumayma Boukari, Wahid Khemissi, Soumaya Ghodhbane, Aida Lahbib, Olfa Tebourbi, Khemais Ben Rhouma, Mohsen Sakly, Dorsaf Hallegue Laboratory of Integrated Physiology, Department Life Sciences, Faculty of Sciences of Bizerte, University of Carthage, Jarzouna 7021, Bizerte, Tunisia. DOI: 10.4081/aiua.2023.11593 Summary Archivio Italiano di Urologia e Andrologia 2023; 95(4):11593 O. Boukari, W. Khemissi, S. Ghodhbane, A. Lahbib, O. Tebourbi, K. Ben Rhouma, M. Sakly, D. Hallegue 2 peutic values (18-20). The huge interest in Spirulina is essentially due to its high protein level and the protein quality as it contains essential amino acids as well as the availability of vitamins and minerals notably vitamin B12, iron and calcium (21). Many human and animal studies on the effects of Spirulina intakes have been reported, yet their results varied regarding the duration of administra- tion, the doses and target groups. In fact, evidence from animal studies were in favour of a potential reproprotec- tive effect of Spirulina particularly by enhancing antioxi- dant enzymes activities, hence restoring the production of testosterone in bifenthrin-intoxicated mice (22),and mit- igating pro-inflammatory cytokines in furan exposed rats (23). Additionally, a previous study showed that Spirulina supplementation could prevent the memory impairment in senescence-accelerated mice through counteracting oxidative stress damages (24) which calls attention to the possible beneficial effect of Spirulina in mitigating memo- ry and metabolic impairment induced by testosterone deficiency in castrated group. The present study was assigned to analyse the effects of testosterone replacement with or without Spirulina com- bination on castration-induced metabolic, oxidative stress and cognitive alterations in adult male Wistar rats. MATERIALS AND METHODS Animals Male Wistar rats weighing 155-250 gr at the beginning of the experiment, purchased from Pasteur Institute, Tunisia, were housed in separate cages under controlled conditions of temperature (25°C) and a 12:12 light/dark cycle. All animals were provided with water and food ad libitum. All rats were acclimatized 10 days prior to the beginning of the experiment. Animals were cared for in compliance with the Institutional Ethics Committee code of practice for the Care and Use of Animals for Scientific Purposes. The experimental protocols were approved by the Ethics Committee of Faculty of Sciences, Bizerta, Tunisia. Castration surgery The castration surgery was performed under ether anaes- thesia. All rats were bilaterally castrated, each testis was excised through a small incision at the posterior end of the scrotum and then ligated. The testis was exposed by performing a transverse resection on both scrota in the supine position, and the spermatic cord and blood vessels were ligated and resected (25). Experimental design Ten days after surgery, rats were randomly assigned in 4 groups, five animals per group, and treated for 20 consecu- tive days as follows: Group 1 (Control group): non-castrat- ed rats received distilled water orally (10 ml/kg). Group 2: Castrated group (CT) was given distilled water orally after castration (10 ml/kg). Group 3: Testosterone treated group (TT) castrated rats received 5 mg/kg of testosterone (Sigma- Aldrich,Co, St Louis, MO, USA) dissolved in corn oil by sub- cutaneous injection (26). Group 4: Spirulina co-treated group (SP) castrated rats received 5 mg/kg of testosterone dissolved in oil by subcutaneous injection followed by 10 ml/kg orally of Spirulina (1000 mg/kg) (BioAlgues Tunisia). During the experiment period, all rats were monitored daily for body weight. Behavior tests were performed at the end of the treatments. Biochemical analyses Rats were sacrificed by decapitation under slight ether anesthesia. Blood was collected in EDTA tubes and cen- trifuged at 4°C at 4000 rpm for 15 minutes. Plasma was recuperated and stored at -25°C for further biochemical determinations. Organs, brain, prostate, and seminal vesicles were immediately dissected out, washed in saline solution, weighed, and stored for further oxidative stress measurement. Cholesterol, triglycerides, alkaline phos- phatase (PAL), aspartate aminotransferase (AST) and ala- nine aminotransferase (ALT) were determined using com- mercial analysis kits (BioMaghreb, Tunisia) according to the manufacturer’s instructions. Oxidative stress measurement Tissue was homogenized in Tris-buffered saline (TBS). The homogenate was centrifuged at 4°C at 9000 rpm for 10 minutes and supernatants were collected. Protein level was estimated by Bradford method (27). Superoxide dis- mutase (SOD) and catalase (CAT) activities were meas- ured in tissue homogenates according to Misra and Fridovich (28) and Aebi methods (29) respectively. Lipid peroxidation was assessed by measuring the malondialde- hyde (MDA) level according to the Draper and Hadley method (30). Hydrogen peroxide (H2O2) level was meas- ured according to Jabri et al. (31). Behavioural testing Animals were habituated to the arena the day following the 20 days of treatment and then submitted to the object loca- tion test (OLM) (32) and the novel object recognition test (NOR) 24 hours later (33). Both behavioural tests were per- formed between 08:00 am and 03:00 pm (Figure 1). Figure 1. Object location (OLM) and Novel object recognition (NOR) tests. Archivio Italiano di Urologia e Andrologia 2023; 95(4):11593 3 Metabolic and cognitive effects of testosterone replacement Assessment of spatial memory using object location memory test (OLM) The arena was a metal circular area with a 50 cm wall and divided into 1 central and 6 peripheral parts of equal sur- face. Testing consisted of one habituation day and one OLM testing day. On both days, testing was performed between 8:00 am and 04:00 pm and rats were brought to the testing room 15 minutes prior to the start of the test- ing. During the habituation day, rats were free to explore the vacant arena for 15 minutes. On the OLM testing day, two trials were performed, training and testing trials. During the training trial, two identical objects were placed 5 cm away from the wall such that they are counterbal- anced in the arena, each rat was placed then in the centre of the arena and allowed to explore it for 5 minutes. Rats were placed back into their cages following the training trial for a 1h interval between trials. During the testing trial, one of the objects was moved to a quadrant diagonal from the other object, each rat was then replaced in the arena and allowed to explore for 5 minutes. The apparatus was cleaned with 30% alcohol and dried prior to the start of each trial for every rat. Rat movements were tracked and recorded using the Debut video recorder program. A rat is considered exploring an object when its nose was within 2 cm from the object. Touching and sniffing activi- ties were counted as exploration, while sitting on the object was not. Rats who don't meet these criteria were excluded from all analyses. To analyse cognitive performance, the fol- lowing data were measured: the time spent exploring the object moved to a novel place (T1), the object remaining in the familiar place (T2), and the investigation time (%) i.e., which represents the percentage of the time spent in explor- ing the objects relative to the total time of the trial. Indexes measurements were also considered (34). Discrimination index (D1) represented the ability of the rat to distinguish the new object location from the familiar one; this index varies between -1 and +1 with a positive value indicating more preference for the displaced object. D1 is calculated as follows D1 = (T1-T2)/(T1+T2). Recognition index (R1) rep- resented the percentage of time spent exploring the dis- placed object relative to the total exploration time and was calculated as follows: R1=T1/(T1+T2) ×100. Assessment of spatial memory using novel object recognition memory test (NOR) An hour after the OLM last testing trial, the familiar object was replaced with a novel object. Rats were placed in the centre of the arena and allowed to explore it for 5 minutes comprising one old object that was used in the last trial of the OLM test and one novel object. The arena was cleaned with 30% alcohol and air-dried prior to the commence- ment of each trial for every rat. Rat movements were tracked and recorded using the Debut video recorder pro- gram. To analyse cognitive performance, the following data were collected: time spent in exploring the novel object(t1), the familiar object(t2), and the investigation time (%), which represents the percentage of the time spent in exploring the objects relative to the total time of the trial. The indexes that were considered were: the discrimination index (D1) representing the ability of the rat to distinguish the novel object from the familiar one (this index varies between -1 and +1 with a positive value indicating more preference for the novel object); the recognition index (R1) representing the percentage of time spent with the novel object relative to the total exploration time. The indexes were calculated respectively as follows D1 = (t1-t2) /(t1+t2); R1=t1/(t1+t2) ×100 (34). Statistical analyses Statistical analysis of data was performed using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparison. Data were expressed as mean ± standard error of the mean. A value of p < 0.05 was considered statistically significant. Data were analysed using GraphPad Prism 5 software. RESULTS Serum biochemical analyses As shown in Table 1, castrated rats expressed statistically increased plasma levels of ALT, AST, PAL, cholesterol, and triglycerides compared to control group. In contrast, testos- terone replacement (TT) alone or in combination with and Spirulina (SP) restored these parameters to normal levels. Evaluation of antioxidant enzyme activities Data showed that castration increased significantly SOD and CAT activities in the prostate, seminal vesicles, and the brain in comparison with the control group (Table 2). Importantly, testosterone replacement significantly ame- liorated the abnormal levels of the antioxidant enzymes in the three tissues compared to control levels. However, cotreatment with Spirulina did not significantly improve these effects in SP group. Evaluation of hydrogen peroxide (H2O2) and lipid peroxidation levels Figures 2,3 and 4 showed that MDA levels respectively in prostate, seminal vesicles, and brain, were significantly higher in castrated group in comparison with control group. These increases were associated with a significant increase in H2O2 levels as compared with control group. A significant and identical decrease in MDA and H2O2 tis- sue contents was noticed in TT and SP groups as com- pared to castrated rats. Indeed, there were no remarkable changes in these oxidative stress parameters between con- trol, TT and SP groups. Table 1. Biochemical parameters in control, castrated, testosterone and Spirulina treated rats. Parameters Control CT TT SP ALT (U/L) 35.35 ± 3.39 88.90 ± 7.5 * 46.81 ± 6.62 # 41.85 ± 5.28 # AST (U/L) 17.50 ± 0.95 32.38 ± 3.88 * 23;98 ± 2.66 24.33 ± 3.11 PAL (U/L) 18.70 ± 1.92 56.93 ± 7.39 * 30.02 ± 3.45 # 20.43 ± 1.17 # Cholesterol (g/l) 1.09 ± 0.12 1.97 ± 0.13 * 1.29 ± 0.07 # 1.06 ± 0.11 # Triglycerides (g/l) 1.25 ± 0.14 3.10 ± 0.14 * 1.78 ± 0.09 # 1.75 ± 0.15 # Values are expressed as mean ± SEM. CT: Castrated group; TT: Testosterone treated group; SP: Testosterone and Spirulina co-treated group. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; PAL: Alkalin phosphatase. * p < 0.05 as compared to control group. # p < 0.05 as compared to CT group. Archivio Italiano di Urologia e Andrologia 2023; 95(4):11593 O. Boukari, W. Khemissi, S. Ghodhbane, A. Lahbib, O. Tebourbi, K. Ben Rhouma, M. Sakly, D. Hallegue 4 Table 2. Effect of testosterone replacement in combination or not with Spirulina on antioxidant enzymes activities in prostate, seminal vesicles, and brain tissues in castrated rats. Prostate Seminal vesicles Brain SOD CAT SOD CAT SOD CAT (U/mg proteins) (umol/min/mg protein) (U/mg proteins) (umol/min/mg proteins) (U/mg proteins) (umol/min/mg proteins) C 6.73 ± 1.32 74.02 ± 8.65 13.28 ± 1.59 93.65 ± 10.56 12.67 ± 1.82 73.90 ± 4.1 CT 13.4 ± 1.45 * 165 ± 15.13 * 40.72 ± 4.26 * 199.4 ± 23.83 * 54.57 ± 5.67 * 140.8 ± 10.89 * TT 7.45 ± 1.58 130.8 ± 3.35 * 30.49 ± 1.65 * 137.7 ± 18.88 22.28 ± 0.99 # 97.63 ± 3.23 # SP 11.68 ± 1.83 109.2 ± 6.18 # 23.92 ± 1.83 # 124 ± 19.05 # 22.48 ± 1.41 # 96.82 ± 4.31 # Values are expressed as mean ± SEM. C: Control; CT: Castrated group; TT: Testosterone treated group; SP: Testosterone and Spirulina co-treated group. SOD: Superoxide dismutase; CAT: Catalase. * p < 0.05 as compared to control group. # p < 0.05 as compared to CT group. Figure 2. Effect of testosterone replacement in combination or not with Spirulina on prostate MDA and H2O2 levels of castrated rats. Values are expressed as mean ± SEM. CT: Castrated group; TT: Testosterone treated group; SP: Testosterone and Spirulina co-treated group. MDA: Malondialdehyde; H2O2: Hydrogen peroxide; * p < 0.05 compared to control group. # p < 0.05 as compared to CT group. Figure 3. Effect of testosterone replacement in combination or not with Spirulina on seminal vesicles MDA and H2O2 levels in castrated rats. Values are expressed as mean ± SEM. CT: Castrated group; TT: Testosterone treated group; SP: Testosterone and Spirulina co-treated group. MDA: Malondialdehyde; H2O2: Hydrogen peroxide; * p < 0.05 compared to control group. # p < 0.05 as compared to CT group. Figure 4. Effect of testosterone replacement in combination or not with Spirulina on brain MDA and H2O2 levels in castrated rats Values are expressed as mean ± SEM. CT: Castrated group; TT: Testosterone treated group; SP: Testosterone and Spirulina co-treated group. MDA: Malondialdehyde; H2O2: Hydrogen peroxide; * p < 0.05 compared to control group. # p < 0.05 as compared to CT group. Archivio Italiano di Urologia e Andrologia 2023; 95(4):11593 5 Metabolic and cognitive effects of testosterone replacement Assessment of memory performances In the OLM trial, castrated rats exhibited statistically decreased investigation time as compared to control group, while no significant difference in the investigation time was noticed between the control, TT and SP groups (Table 3). Compared with the other groups, castrated rats displayed less preference for the displaced object in the novel place since they spent equal time exploring the object remained in the familiar location and the displaced one. Moreover, the comparison of the discrimination index between control and castrated group revealed a sig- nificant lower index in CT group as compared to control, TT and SP treated groups. In NOR tests, the investigation time was also significant- ly decreased in castrated group as compared to control group. This effect was totally reversed in testosterone and testosterone plus Spirulina treated groups. Furthermore, control, TT, and SP treated groups showed a clear ten- dency to explore the novel object rather than the familiar object. In fact, these rats showed a significantly higher recognition index in comparison to castrated group and spent more than 75% of their investigation time with the novel object. Castrated rats showed no preference for the novel object as they displayed a decreased recognition index as compared to control group. The comparison of the discrimination index between cas- trated and control group showed that castrated rats had a significantly decreased index as compared to control group which showed a non-distinguish of the novel object. However, treated rats with testosterone alone or in combination with Spirulina displayed a comparable dis- crimination index to the control group and a significant increase in the discrimination index value as compared to the castrated group. DISCUSSION Testosterone is a key hormone that has been known for its major role in carbohydrates, lipids, and proteins metabolism (35, 36). Testosterone deficiency has been linked to an increase in body fat mass, impairment in glu- cose and lipid tolerance, as well as oxidative stress imbal- ance; all these factors can contribute to metabolic disor- ders (37). In this regard, we assessed castration effect on adult male rats and evaluated whether testosterone could mitigate physiological perturbations induced by testos- terone deficiency. In addition, we investigated the possi- ble potential of the filamentous cyanobacterium, Spirulina platensis, to enhance the androgen effect in castrated rats. Our results revealed that plasma levels of cholesterol and triglycerides were remarkably increased after castration in CT rat group in comparison with the control group. Our findings are in harmony with previous studies of testos- terone deficiency effect on lipid profiles in aging male rats (38) and orchiectomized rats (39). Testosterone level appears to have complicated relationship with cholesterol metabolism regulation and its associated anomalies, in a matter of facts low testosterone levels is associated with pro atherogenic lipid profiles in men (2), particularly lower levels of the high-density lipoprotein cholesterol (HDL-C). As steroid hormones can bind and interact with specific DNA domains it has been suggested that testos- terone is involved in the molecular metabolism of choles- terol within the liver through the upregulation of several genes namely the hepatic lipase (HL), the scavenger B1 receptor (SR-B1) (40), and the nuclear liver X receptor (LXR) (41). Both HL and SR-B1 mediate and facilitate the uptake of HDL into hepatocytes, thereby stimulate the cholesterol uptake and efflux. Studies demonstrated that increased activity of SR-B1 and HL were linked to choles- terol level lowering effect of testosterone administration (40). It was also suggested that testosterone is involved in the liver uptake of low-density lipoprotein cholesterol (LDL- C) through the modulation of the PCSK9-LDLR pathway, thus the clearance of LDL-C from circulation (42). Importantly, our study showed that plasma lipid profile perturbations were reversed by testosterone replacement either alone or in association with Spirulina in castrated rats. Our results are in line with previous studies which have demonstrated that testosterone replacement therapy (43) and Spirulina platensis supplementation (44) amelio- rated serum cholesterol and triglycerides levels respec- tively in castrated or high fat diet-fed rats. Liver enzymes such as transaminases (ALT, AST) and ALP are sensitive biomarkers widely used to assess liver injury (45). Thus, these intracellular proteins are released into the blood upon hepatocyte damage. Our results showed that in castrated rats, these enzymes greatly increased in plasma above normal value. However, supplementation with testosterone or testosterone plus Spirulina were effective in improving these liver damage biomarkers. Oxidative stress is a major mechanism of tissue injury, it is induced by the imbalance between the production of the oxygen reactive species (ROS) and the antioxidant sys- tem (46). It is generally caused by lipid accumulation, and DNA damages and leads to the loss of organ func- tions. The occurrence of oxidative stress is linked to aging, aging-related diseases and diverse clinical condi- tions including diabetes and heart diseases (47, 48). Previously, it has been shown that low testosterone level Table 3. Effect of testosterone replacement in combination or not with Spirulina on castrated rats’ behaviour during OLM and NOR tests. Object location memory test Novel object recognition test Control CT TT SP Control CT TT SP Investigation time (%) 11 ± 0.83 5.61 ± 0.37 * 8.22 ± 0.97 8.67 ± 0.83 14.47 ± 1.19 3.94 ± 0.57 * 11.80 ± 0.44 # 11.94 ± 0.43 # Recognition index 78.31 ± 0.43 38.50 ± 3.15 * 77.51 ± 0.46 # 77.85 ± 0.97 # 81.27 ± 1.65 34.35 ± 2.43 * 75.66 ± 1.37 # 77.85 ± 1.95 # Discrimination index 0.56 ± 0.008 -0.23 ± 0.07 * 0.55 ± 0.009 # 0.56 ± 0.01 # 0.63 ± 0.03 -0.31 ± 0.04 * 0.51 ± 0.02 # 0.55 ± 0.03 # Values are expressed as mean ± SEM. CT: Castrated group; TT: Testosterone treated group; SP: Testosterone and Spirulina co-treated group. * p < 0.05 as compared to control group. # p < 0.05 as compared to CT group. Archivio Italiano di Urologia e Andrologia 2023; 95(4):11593 O. Boukari, W. Khemissi, S. Ghodhbane, A. Lahbib, O. Tebourbi, K. Ben Rhouma, M. Sakly, D. Hallegue 6 is correlated to an imbalance of the oxidative stress status. In the study of Mancini et al. (49), sixteen patients with hypogonadism were compared to ten healthy patients to investigate the role of testosterone in the oxidative stress mechanism showing that a lipid antioxidant enzyme Coenzyme Q10 (CoQ10) was reduced in the hypogo- nadism condition and that the testosterone replacement therapy resulted in an increase in CoQ10 serum level (49). Furthermore, in vitro assays showed that low testos- terone treatment could decrease lipid peroxidation and the ROS production in TM3 Leydig cells (50). In the present study we revealed that castrated rats showed a remarkable increase in SOD and CAT activities in the prostate, seminal vesicles, and the brain in com- parison with the control group. Whereas testosterone replacement result in a significant increase in the level of these antioxidant enzymes. However, cotreatment with Spirulina did not significantly improve these effects towards TT group. Testosterone deficiency also signifi- cantly increased both MDA and H2O2 levels in prostate, seminal vesicles, and brain, in comparison with control group. Interestingly, administration of testosterone either alone or in combination with Spirulina restored these changes induced by castration. Our results are consistent with previous studies that have showed that oral admin- istration of Spirulina prevented repro-toxicity by mitigat- ing lipid peroxidation in testis of furan-intoxicated rats (23) and counterbalancing the perturbation of antioxi- dant enzymes in cadmium-intoxicated mice (51). However, the fact that Spirulina cotreatment did not enhanced the positive effects of testosterone in castrated rats suggested that androgen actions might involve other mechanisms unrelated to oxidative stress. The loss of bioavailable testosterone in male is associated with a dysfunction in androgen responsive tissues includ- ing the brain. A vast majority of human and animal stud- ies demonstrated that testosterone deficiency causes a decline in cognitive performance (52) and affective behaviour in males (53). Some data reported conflicting results and supported a non-consistent effect of andro- gens (54). Studies on molecular mechanism of androgen action on the brain indicated that testosterone have a direct impact on glial cells thereby can modulate the myelinisation mechanism, synapse, and dendritic branching number as well as neuron growth (55). It has also been shown that even though gonadotropic neurons do not express androgen receptors, testosterone can mod- ulate these neurons through a neuropeptide called kisspeptin which is not only expressed in the hypothalam- ic-pituitary-gonadal axis (HPG) but also in the limbic regions of the brain implicated in the emotional and cog- nitive behaviour (Mills et al., 2018. However, the exact causality of the relationship between testosterone levels and brain functions is still not firmly established. In our study we performed OLM and NOR cognitive tests to assess the spatial memory performance in rats. Our data demonstrated that castration caused less interest in exploring objects in both the OLM and NOR trials. Likewise, the discrimination index of CT group displayed a negative value which indicate the incapacity of castrat- ed rats to distinguish the novel object and the novel loca- tion of the object. These findings are along with previous results of Pintana et al. (56), that showed a cognitive decline in rats with testosterone deprivation. Whereas rats who received testosterone replacement of 0.20 mg/kg spent significantly more time exploring objects than cas- trated group and showed a significantly greater prefer- ence for the displaced and the novel object in comparison to castrated rats. Coherently, testosterone replacement displayed a positive discrimination index and a higher recognition index in comparison to CT group. Our find- ings contradicted the results of Borbélyovà et al. (57) who reported no effect of low testosterone concentration nei- ther acute testosterone treatment on the exploratory behaviour and memory performance assessed by the Open Field test in aged and castrated male rats. This dif- ference in results can be explained by several limitations namely the difference in the treatment period and the behavioural tests used. Castrated rats who received Spirulina co-treatment also exhibited significantly more time exploring the objects in the arena, they displayed a higher discrimination and recognition index as compared to castrated group. In agreement with our results, previous data showed that Spirulina could improve memory deficit induced by scopolamine in rats through modulation of oxidative stress imbalance (58). Further, Wang et al. 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Correspondence Oumayma Boukari Oumayma.boukari@gmail.com Wahid Khemissi w.khemissi2007@gmail.com Soumaya Ghodhbane ghodhbanes@yahoo.fr Aida Lahbib lahbib.aida@gmail.com Olfa Tebourbi tebourbi.olfa@gmail.com Khémaïs Ben Rhouma k.benrhouma2015@gmail.com Mohsen Sakly (Corresponding Author) mohsensakly@gmail.com Dorsaf Hallegue dorsafhallegue@yahoo.fr Laboratory of Integrated Physiology, Department Life Sciences, Faculty of Sciences of Bizerte, University of Carthage, Jarzouna 7021, Bizerte, Tunisia Conflict of interest: The authors declare no potential conflict of interest.