Man is exposed to many environmental contaminants at different stages of life and exposure to aluminium (Al) due to industrial and environmental pollution poses severe ecological hazards ( 1 ). It has been adjudged the most widely distributed metal in the environment. It is believed to be the most prevalent metal and the third most crucial abundant element in the earth's crust; only oxygen (49.5%) and silicon (26%) occur more commonly than Al(8%)( 2 ). In biological systems, Al is present only in trace amounts, shown to have no definite biological function and is poorly absorbed but efficiently eliminated when absorption does occur. Al is described mainly in bone, liver, testes, kidney, and brain, while its accumulation in tissues and organs results in dysfunction and toxicity ( 3 ).

The wide distribution of this element ensures the potential to exert harmful effects on human health upon exposure ( 4 ). Al compounds are widely used in medicines as antacids, vaccines, anti-diarrhea, phosphate binders, and allergen injections ( 5 ); food additives and toothpaste ( 6 ); water purification agents ( 7 ). It is still a metal of choice in making various household cookware and storage utensils.

Al contributes to various cognitive impairments in mice, rabbits and rats ( 8 , 9 ). Epidemiological studies have indicated a link between Al in drinking water and Alzheimer’s disease, and a variety of human and animal studies have implicated learning and memory deficits post- Al exposure ( 10 , 11 ). AlCl ₃ can generate reactive oxygen species (ROS) ( 12 ). In another instance, Al has been reported to have induced toxicity in the epididymis, vas deferens, seminal vesicle, and ventral prostrate of mice. AlCl ₃ -induced free radicals and inhibited antioxidant enzymes in blood and seminal plasma, liver, testes, kidney, lung, and brain of rabbits ( 12 , 13 ).

Antioxidants are substances that protect cells against the adverse effects of xenobiotics, carcinogens, drugs, and toxic agents ( 14 ). Since spermatogenesis and Leydig steroidogenesis are vulnerable to oxidative stress, the low oxygen tension that characterizes this issue may be an essential component of the mechanisms by which the testis protects itself from free radical-mediated damage ( 15 ).

The use of plants as medicines predates written human history. Nigeria has a vast repository of medicinal plants. It has thus paved the way for synchronizing traditional medicine into the healthcare delivery system, and the guava plant is one such example ( 16 ). The use of herbs to treat disease is almost universal among non-industrialized societies and is often more affordable than purchasing expensive modern pharmaceuticals ( 17 ).

Psidium guajava, the common guava tree, is widely distributed as a native plant in Africa and America from Mexico to Brazil. Whose leaves have been used in folk medicine for many years in different parts of the globe in managing various disease conditions and overcome severaldisorders, such as;rheumatism, diarrhea, diabetes mellitus, cough, and ulcers ( 18 – 21 ). The plant has also been reported to exhibit antimicrobial, analgesic and anti-inflammatory properties ( 22 , 23 ).

Ethical approval for the study was given by the ethical committee of the College of Health Sciences, University of Ilorin, in line with the globally approved guidelines for the use of animals for scientific studies.

Thirty male adult Wistar rats, weighing between 160 to 250g, were obtained from a private animal farm in Ogbomoso, Oyo State, Nigeria, and maintained in the animal house of the College of Health Sciences, University of Ilorin.They were housed in cages with adequate space to encourage free movement under natural and dark cycles (12hr light and 12hr dark) at room temperature. They were bred on conventional commercial standard rat pellets. They were also supplied with water ad libitum and allowed to acclimatize to experimental conditions for 14 days before commencement of the experiments. Interval weighing of animals was done throughout. Strict hygiene was maintained by clearing their wastes daily.

Fresh leaves of P. guajava were obtained from a private farm at Tanke, a suburb of Ilorin, Kwara State-Nigeria. Aqueous extraction of guava leaf was achieved according to an existing procedure ( 24 ). In this procedure, fresh guava leaves were harvested from the botanical garden of the University of Ilorin, washed thoroughly, air-dried and pulverized using an electric blender. Two hundred grams (200g) of the powder obtained was macerated with 600mls of distilled water for 72 hours at 30±4 ^o C. The mixture was sieved and filtered with Whatman No. 1 filter paper and the filtrate was concentrated in an electrically regulated oven at 45°C. Finally, concentrated crude aqueous extract was dried to a solid mass. The testing samples were prepared by dissolving the solid mass with normal saline to obtain a final concentration of 250 mg/kg body weight (bw) and 500 mg/kg body weight (bw) used for the experiment. Laboratory grade AlCl ₃ was locally sourced from Dennis and Dennis Laboratory, Taiwo Road, Ilorin-Nigeria. Other reagents & chemicals were of analytical grade. The thirty ( 30 ) adult male Wistar rats were divided randomly into six ( 6 ) groups of five ( 5 ) animals per group. Group A was orally administered with 0.5 ml of normal saline from the 1 ^st to 14 ^th day and served as the control group; Group B was orally administered with 0.5 ml of 100 mg/kg body weight of AlCl ₃ only from the 1 ^st to 14 ^th day. Group C was orally administered with 0.5 mls of 250mg/kg body weight of Psidium gaujava leaf extract from the 1 ^st to 14 day; Group D was orally administered with 0.5mls of 500 mg/kg body weight of Psidium guajava leaf extract from 1 ^st to 14 ^th day, Group E was orally administered with 0.5 mls of 100 mg/kg body weight of AlCl ₃ from 1 ^st to 14 day, followed by the oral administration of 0.5 mls of 250 mg/kg body weight of Psidium guajava leaf extract from 15 ^th to 28 day, Group F was orally administered with 0.5mls of 100 mg/kg body weight of AlCl ₃ from 1 ^st to 14 ^th day, followed by the oral administration of 0.5mls of 500 mg/kg body weight of Psidium guajava leaf extract from 15 ^th to 28 ^th day. All administration was achieved via oral gavage.

While all the rats from Groups A to D were sacrificed on the 15 ^th day of the experiment, Groups E and F were sacrificed on the 29 ^th day of the experiment. At the end of the treatment protocols, the animals were euthanized with intramuscular injection of ketamine hydro-chloride (20 mg/kg). The testes were identified, removed, cleared of fat, and weighed using a sensitive analytical weighing balance and were placed in a solution of Bouin’s fluid and processed for histological light microscopic examination using routine hematoxylin and eosin, and immunohistological examination using Ki67 marker.

The right testes were placed in 0.25 mM of sucrose solution in the ratio of 1:4. Each of these tissues was separately transferred to a glass homogenizer containing the cold sucrose solution and were homogenized using an electrical homogenizer (Remi 8000 RPM). The unbroken cells and cell debris were removed by centrifuging at 3000 RPM for 15 minutes. The obtained supernatant(s) were used for the enzymatic estimations (antioxidant enzyme levels). Determination of follicle stimulating hormone (FSH), luteinizing hormones (LH), and testosterone were quantitatively measured by adopting an enzyme-linked immunosorbent assay (ELISA) technique using the Accu-Bind Microwell kit. Lake Forest, CA 92630, USA.

The effect of the oral administration of aqueous extract of P. guajava leaves on body weight of Alcl3-induced testicular toxicity is presented in Table 1. The weight of the experimental animals was measured once a week, and the analysis of the data obtained throughout the experiment indicates an increase in body weight in the control group and the P. guajava groups (250mg/kg and 500 mg/kg bw). The extract showed no significant changes (p<0.05) in body weight at all doses administered compared to the control group. However, the animals orally administered Alcl ₃ for 14 days recorded the lowest body weight, which was reversed when treated with the aqueous extract of guava leaves (250 mg/kg and 500mg/kg bw). However, the reversal effect was dose-dependent.

The effect of the oral administration of aqueous extract of P. guajava leaves on testicular weight of Alcl _3- induced testicular toxicity is presented in Table 2. There was an increase in testicular weight in the control and P. guajava groups (250 and 500 mg/kg). The extract showed no significant changes (p<0.05) in testicular weight at all doses administered compared to control. However, the animals given oral administration of AlCl ₃ for 14 days recorded the lowest testicular weight, which was reversed when treated with an aqueous extract of guava leaves (250 and 500 mg/kg); however, the reversal effect was dose-dependent.

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treated rats were normal all through the experiment, depicting that aqueous extract of guava leaves ameliorated the inactiveness caused by aluminum chloride. The inactiveness observed in the aluminum-treated rats might be due to the induction of oxidative stress. Oxidative stress is recognized as the major mode of action of aluminum exposure as this has been observed in vitro in several types of cells and also in vivo in soft tissues such as the liver, kidney, brain, lung, and testes of experimental animals ( 7 ). Guava leaves, with its antioxidant properties, were observed to have ameliorated the effect induced by AlCl ₃ , as all the groups that took 250 mg/kg and 500 mg/kg bw aqueous extract of the leaves appeared active and healthy. Also, the ultimate effect of testicular damage on body weight indicates its toxicity to health ( 32 ). However, there have been conflicting reports of correlations between testicular damage and weight changes in the body. In this current study, the weight of the experimental animals was measured once a week. Analysis of the data obtained from animal body weights monitored throughout the experiment indicates that animals in all the experimental groups gained weight. The group that gained weight most was the control group, which had a weight gain of 103.33±3.71. An increase was observed in the group given 250 mg/kg and 500 mg/kg BW of Psidium guajava leaves extract only, which had a rise of 100.67 ±8.19 and 101±3, respectively. A similar increase was observed in the treated groups given 250 mg/kg and 500 mg/kg BW of Psidium guajava leaves extract after a 100 mg/kg dosage of Alcl ₃ for 14 days. The last increase in weight was that of the group given 100 mg/kg of Alcl ₃ only for 14 days. However, the animals given oral administration of Alcl ₃ only for 14 days recorded the lowest body weight gain; this result is in tandem with previous findings that reported that during three months of observation of rats receiving AlCl ₃ , a decrease in water and food intake and transient diarrhea occurred, which led to lowering of final body mass of animals compared to controls ( 33 ).The low body weight was reversed when treated with an aqueous extract of guava leaves (250 mg/kg and 500 mg/kg bw); however, the reversal effect was dose-dependent, and the weight gained showed no changes. This could be attributed to Psidium guajava leaf extract exerting its antioxidant effect; hence, it lessened the effect of AlCl ₃ on weight. This result corroborates the findings of another study, which also reported an increase in the weight of rats when administered 250 mg/kg, 500 and 750 mg/kg, aqueous extract of guava leaves for 14 days ( 34 ). Oral administration of AlCl ₃ significantly decreased testicular weight with 100 mg/kg bw. At 14 days compared to the control, the groups were administered 250 and 500 mg/kg bw. Aqueous extract of guava leaves. These findings aligned with another study where a decrease in absolute and relative testes and seminal vesicle weights after AlCl ₃ ingestion was reported ( 35 ). The decrease in the reproductive organs' weights could be due to the decrease in testosterone that may be attributed to oxidative damage induced in the rat testes. The decrease in testicular weight was noted to have been reversed when treated with an aqueous extract of guava leaves (250 and 500 mg/kg); however, reversal effect was dose-dependent. These findings are in tandem with another independent study where oral administration of 250 mg/kg and 500 mg/kg of aqueous extract of guava leaves increased body weight ( 36 ).

Oral administration of AlCl ₃ distorted the testicular histomorphology of Wistar rats, causing testicular atrophy associated with degeneration of germ cells within seminiferous tubules, enlarged lumen with fewer spermatids compared with the non-treated animals. Aqueous extract of guava leaves was able to reverse the damage caused by AlCl ₃ , reversing the distorted seminiferous tubules and presenting lumen with densely packed primary spermatocytes. A reversal in weight reduction caused by AlCl ₃ was also established.