Calcium chloride (CaCl2), monopotassium phosphate (KH2PO4), sodium lactate, and vitamin C were purchased from R&M Marketing, Essex, UK. AlCl3 and glucose were purchased from Friendemann Schmidt Chemical, USA. Analytical-grade sodium chloride (NaCl), potassium chloride (KCl), magnesium sulfate (MgSO4), and sodium bicarbonate (NaHCO3) were purchased from Bendosen, Malaysia. Sodium pyruvate was purchased from Sigma Chemical, USA. EGCg green tea extract—400-mg capsules—were purchased from Now Foods, USA. Each capsule of EGGg contains 200 mg of EGCG.
Healthy, adult, male SD rats with a weight of 180 ± 20 g were used for the study. The rats were housed and maintained in large, spacious polyacrylic cages at ambient room temperature with a 12-h light/12-h dark cycle. The animals were fed with water and normal rat pellet diet ad libitum. The study was approved by the University Human and Animal Ethics Committee (AUAEC/FOP/2019/09), and the study was conducted according to the Animal Research Review Panel guidelines.
2.3 Experimental design
Healthy, adult male SD rats were used for the study. The animals were divided into six groups with six animals each as follows:
Group I: control
Group II: AlCl3 (10 mg/kg)
Group III: Vitamin C (200 mg/kg)
Group IV: EGCG (50 mg/kg)
Group V: AlCl3 (10 mg/kg) + vitamin C (200 mg/kg)
Group VI: AlCl3 (10 mg/kg) + EGCG (50 mg/kg)
The doses of vitamin C and EGCG were selected based on the available literature [17, 18]. AlCl3 (10 mg/kg) was used to induce oxidative stress . Vitamin C, EGCG, and AlCl3 were dissolved in distilled water for injection and administered intraperitoneally.
All the animals were administered with respective assigned treatment once daily for 28 days. The animals in groups I to VI were administered with drug vehicle, AlCl3, vitamin C, EGCG, vitamin C, and EGCG, respectively. The animals in groups V and VI were additionally challenged with AlCl3 (10 mg/kg) immediately after vitamin C and EGCG treatment. The changes in body weight and behavior were measured at regular intervals . At the end of the study, the blood sample was collected from all the animals, and the serum was separated. The serum samples were used for biochemical analysis. Later, the rats were subjected to bilateral orchiectomy; sperm was collected from the cauda epididymis for microscopic examination [21, 22]. Then, the animals were sacrificed, and the organs such as the brain, lungs, heart, liver, kidney, spleen, and testis were collected for organ weight analysis.
2.4 Body weight analysis
The body weight (BW) of each rat in each group was recorded initially and at 3 days intervals. The change in BW was calculated.
2.5 Behavior and muscular activities
At regular intervals, behavioral (locomotor activity), muscular coordination (rotarod and wire grip test), and memory function (Morris water maze test) were evaluated.
2.5.1 Locomotor activity
The activity of the rats was recorded in a rat activity cage (actophotometer) which is constructed with an acrylic cage and 8 beams of infrared light along both the x and y-axes. The activity of each rat was monitored at room temperature over 10 min.
2.5.2 Muscle coordination (rotarod) test
The rat was placed on a rotarod with a speed of 20 RPM. The time between rat maintains on the rotarod and when it fall was recorded. The procedure was repeated for each rat.
2.5.3 Hanging wire grip test
The string is made up of metallic wire and suspended in mid-air about 30 cm height from the ground. The rat was placed on the center of the wire, and the time taken to fall, i.e., “fall on time,” was noted.
2.5.4 Morris water maze test
The water navigation test is employed as a method to test spatial learning and memory parameters to evaluate spatial learning and memory functions. The training was taken place for 3 consecutive days, with four consecutive trials/day for each experimental rat at the inter-trial interval of 30 min. In the pre-study training sessions, if the animals failed to escape on the platform within 180 s, they will be excluded from the study.
The Morris water maze consists of a round pool, filled with tap water, which is close to 23–26 °C, at a depth of 0.3–0.4 m. The pool was divided into four hypothetical quadrants, with an escape platform placed 1 cm below the water surface at the center. Four different starting points for rats were placed around the perimeter of the pool. The test was performed from day 24 onwards at day intervals. Escape latency time (ELT) to locate the hidden platform in the water maze was noted as an index of learning.
2.6 Quality evaluation of semen
2.6.1 Determination of sperm motility
At the end of the study, sperm samples were collected from the cauda epididymis. The cauda epididymis was isolated and placed in a petri dish contacting Biggers, Whitten, and Whittingham (BWW) [which consists of 95 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM sodium lactate, 5 mM glucose, 0.25 mM sodium pyruvate, and 25 mM NaHCO3, pH 7.4] medium to allow the sperm to swim (swim-up technique). An appropriate volume of spermatozoa was transferred to a preheated cell count plate. From a total of 200 spermatozoa, the active ones were observed using a microscope to calculate sperm motility .
2.6.2 Determination of sperm count
After completing the determination of sperm motility, the rest of the sperm suspension was quickly transferred to 60 °C water bath for 5–10 min to induce loss activation of sperm and then carefully added to the cell count plate. The number of spermatozoa was calculated using the red blood cell counting method .
2.7 Biochemical analysis
At the end of the experiment, a few milliliters (mL) of the blood sample was collected in the micro-centrifuge tube through retro-orbital plexus and the serum was separated by centrifuging at 3000 RPM for 20 min. The serum samples were used for biochemical marker estimation such as glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine and, urea, and lipid parameters such as total cholesterol (TC), triglyceride (TGL), and high-density lipoprotein (HDL) using the Reflotron Plus biochemical analyzer (Roche Diagnostics, Germany) with the help of commercially available Reflotron strips. Very low-density lipoprotein (VLDL) and cholesterol ratio were calculated mathematically .
2.8 Gross pathology and organ weight analysis
At the end of the experiment, all the experimental animals were sacrificed under mild ether anesthesia followed by cervical dislocation. The animals were dissected, and the gross pathology was observed. The organs such as the brain, lungs, heart, liver, kidney, spleen, and testis were harvested; absolute organ weights were measured, and relative organ weights were calculated.
2.9 Statistical analysis
Data were represented as mean ± standard error of the mean (SEM). Statistical analysis was carried out using one-way ANOVA followed by Turkey’s post-hoc test. A value of P < 0.05 shall be considered to be significant.