Testosterone propionate and Swarna Bhasma treatment modulated D-galactose induced reproductive alterations in male Wistar rats: An experimental study

Abstract Background The male reproductive system undergoes several adverse age-related changes like decreased hormone synthesis, sperm count, and testicular alteration that can impact on fertility. Objective The study aims to investigate the effects of testosterone propionate (TP), and ayurvedic formulation Swarna Bhasma (SB) on D-galactose (D-gal) induced reproductive aging in male Wistar rats. Materials and Methods 60 male Wistar rats were divided into 10 groups of 6 animals. Reproductive aging was induced by D-gal (150 mg/kg Bwt) exposure for 60 days. The rats were then treated by post and combination treatment with TP (2 mg/kg Bwt) and SB (6.75 mg/kg Bwt). Then sperm parameters, reproductive hormones, inflammatory markers, testicular antioxidant enzymes, steroidogenic enzymes, and histological manifestation of testis were evaluated. Results Exposure of D-gal caused significant (p < 0.001) decrease in serum testosterone (T), testicular steroidogenic, and antioxidant enzymes. Administration of TP increased the serum T level, testicular antioxidant enzymes, and spermatogenic profile at a significant level of (p < 0.001) compared to D-gal. Further, the SB treatment significantly (p < 0.001) elevated the serum T level, sperm count, testicular antioxidant enzymes, steroidogenic enzymes, when compared to D-gal. Conclusion Both the treatment of TP and SB treatments recovered the reproductive impairments caused by D-gal. However, exogenous T supplementation via TP administration is associated with various side effects during long-term use. SB is an Ayurvedic formulation having a long history of usage in India. The current findings suggest that the SB may be used as a good alternative for potentiating reproductive function in aging males.


Introduction
Aging is a time-dependent gradual progressive deterioration marked by a deceleration of various physiological functions, leading to increased vulnerability and mortality (1). The reproductive system is one of the first biological systems to show age-related changes with reduced fertility in both women and men. Aging related changes in male reproductive system primarily occurs in testis with increased testicular apoptosis, low-sperm count and decreased secretion of sex hormones ultimately leading to infertility (2,3). D-galactose (D-gal) is a widely used xenobiotic for the experimental induction of aging in laboratory animals (4). Recently, the D-gal at a dose ranging from (100-200 mg/kg) body weight (Bwt) for 6-8 wk has been optimized for investigating male reproductive aging in rodent models. Male reproductive aging has been characterized by decreased testosterone (T) production, altered gonadotropic hormone release, a lower sperm count, increased sperm abnormalities, and testicular degeneration (4,5).
T is a primary male sex hormone that plays an important role in the body by regulating sex drive (libido), sperm production, bone mass, muscle mass, and strength (6,7). As men age, there is a slow and continuous decrease in T levels, leading to a condition called hypogonadism (gonadal failure and hypothalamic-pituitary axis failure) (8). The decline level of T is responsible for age-related impairment in spermatogenesis leading to infertility (9,10). The treatment of hypogonadism or lower androgen concentration consists of testosterone supplementation therapy (TST) to normalize serum T levels (11,12). Testosterone propionate (TP) is a fast-acting testosterone ester injectable compound mainly used as TST agent for the treatment of low-testosterone level in men. Recent studies have suggested that T therapy improves libido, sexual activity, erectile function, and fertility (13). According to some researchers, administering exogenous T in various forms of TST may be harmful to the liver, reduce insulin's effect on lipid metabolism, and increase the risk of cardiovascular disease, stroke, and prostate cancer (14)(15)(16). Therefore, an alternative to TST is required, preferably one that increases endogenous T production to normalize steroidogenic function in aging human male (17).
The use of natural products in potentiating the male reproductive system has been associated with human civilization since antiquity (18). Swarna Bhasma (SB), also known as gold ash, is a metallic formulation in traditional Ayurvedic medicine that contains nano and colloidal gold particles. SB is widely known as a metabolic booster and ayurvedic physicians used to treat different diseases such as tuberculosis, cancer, bronchial asthma, rheumatoid arthritis, diabetes mellitus, anemia, nervous, and reproductive system related disorders (19)(20)(21). Ayurveda claimed that SB improves sperm count, quality and quantity of semen, and sexual activity (20,22). However, to our knowledge, there are no experimental reports available on reproductive system enhancement activity of SB in aging animals/human beings. Hence, the present study was planned to study the effect of SB on various reproductive parameters in D-gal induced aging male Wistar rats, and TP was used as a standard drug.
National Tribal University, Amarkantak, Madhya Pradesh, India. The animals were given food and water ad libitum and were kept in an environment with ambient temperatures of 25 ± 3°C, relative humidity of 60 ± 5%, and a 12-hr light/dark cycle.

Chemicals
The D-gal was purchased from Himedia Pvt. Ltd., India, whereas the TP and sesame oil used in the current study were procured from Sigma-Aldrich, US. A high-graded SB (Batch number 47), honey, and trikatu were acquired from Shree Baidyanath Ayurved Bhawan Pvt. Ltd., Jhansi, India.

Experimental schedule
A total of 60 rats were randomly divided into 10 groups of 6 animals based on their Bwt. The dose of D-gal and TP were selected based on earlier studies (1,5,23). Further, the dose of SB for the current study was calculated from the therapeutic dose of SB for human, using dose conversion factor from human to rats (24). D-gal and TP were administered daily by subcutaneous injection, whereas SB was administered orally by gavaging daily for 60 days. The normal saline, trikatu in honey and water, and sesame oil were used as a vehicle for administering D-gal, SB, and TP, respectively. The detailed experimental schedule is given in (Table I).
The behavioral and Bwt changes were monitored throughout the experiment. At the end of the experiment, the animals were humanely sacrificed by halothane inhalation. The testis and other accessory reproductive organs (epididymis, prostate, and seminal vesicle) were quickly dissected and washed with ice-cold 0.9% normal saline. The organs were weighed and transferred to -70°C for further study. Blood samples were obtained by cardiac puncture, and serum was isolated and stored at -20°C till further analysis.

Sperm parameters
The epididymal sperm count assessment was done using the hemocytometer method with certain modifications (25). Briefly, the outer covering of cauda epididymis was removed, minced, and homogenized in 1 ml of 0.9% NaCl and 0.05% triton-X solution.
The resultant sperm suspension is then diluted 10 times and centrifuged at 8000 RPM for 2 min. Homogenate (10 µl) was placed in the red blood cell chamber of hemocytometer and sperms were counted at 400× magnification.
For the sperm abnormalities assessment, a piece of the cauda epididymis was minced, 1 ml of 0.9% saline and 1 ml of 10% neutral buffer saline were added. For the assay, the above suspension was further diluted with water, and 1 ml of eosin stain (1%) was added to it. The above solution was further incubated at room temperature for 1 hr. The sperm smear was prepared by pouring a drop of the suspension on the slide and examined at 400× magnification using a trinocular light microscope (Olympus Microscopes, Tokyo, Japan). On each slide, 200 sperm were analyzed for various head and tail abnormalities, and results were expressed as percentage abnormalities. In order to measure sperm motility, the distal end of the epididymis was removed and minced in 2 ml of Dulbecco's phosphate buffer saline and was kept at 36-38°C for further analysis. The minced cauda was placed in a water bath and kept for 1-5 min to disperse the sperms. The sperm suspension (5-10 µl) was placed into the WBC counting chamber, and the number of motile sperms were counted. The hemocytometer was then placed at 40-50°C for 1 min to kill the sperm, and the total number of dead sperms were counted. The results were presented as percentage motility (26).

Hormone assay
The serum T level, luteinizing hormones (LH) and follicle-stimulating hormone (FSH), were measured using rat-specific enzyme-linked immunosorbent assay (ELISA) Kits (Elabscience, China). The procedure for the assay was followed according to the manufacturer's instructions.

Antioxidant enzyme assay
A 10% (w/v) phosphate buffer (pH 7.4 + 150 mM KCl) ice-cold solution was used to homogenise the testis. The lipid peroxidation (LPO) and reduced glutathione (GSH) were determined from one part of the homogenate. Another part of the homogenate was centrifuged at 9000 RPM to obtain the supernatant (S9) fraction. The S9 fraction was used to calculate total protein, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione-S-transferase (GST). The LPO and GSH level in the testis were estimated by the previously used methods (27,28). The SOD and CAT level were measured using earlier described procedures (29,30). GPx, GST, and GR were assessed as per the previous protocols (30,31). Lowry method was used for total protein estimation (32).

StAR gene expression study
Total RNA from the part of the testis was extracted using the Aurum TM Total RNA Mini kit (Bio-Rad, USA) according to the manufacturer's instructions. The RNA purity and concentration were measured by spectrophotometric analysis at A 260 /A 280 nm absorbance ratio. The cDNA was prepared from extracted mRNA using the iScripts TM cDNA synthesis kit (Bio-Rad, USA). cDNA was amplified by quantitative real-time PCR (Bio-Rad, USA) using SsoFast TM EvaGreen® Supermix (Bio-Rad, USA). Rat-specific primers were designed for the genes of interest: StAR (Steroidogenic acute regulatory protein), and RPL-19 (Ribosomal protein L-19); details of the primers used are listed in (Table II). Housekeeping gene RPL-19 was used as a reference gene. Cycling stages were followed as: -Step 1-3 min at 95°C; step 2-40 cycles at 95°C for 10 sec, 60°C for 30 sec, and 30 sec at 72°C; step 3-dissociation stage. The threshold cycle values of each sample were used for PCR data analysis. Relative quantitative RT-PCR gene expression was analyzed using the fold change method by calculating 2 −▵▵ .

Histopathological examination
The testes were stored in 10% formalin. A transverse section (5-µm thick) of testis was cut with a semiautomatic rotary microtome (Yarco YSI 060, US) and stained with hematoxylin-eosin (H&E). The section was examined under a microscope at 400x magnification.

Ethical considerations
The present research was approved by the Institutional Animal Ethical Committee of Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, India with approval number IGNTU/IAEC/2018/10. The animals were cared for and treated as per the rules prescribed by The Committee for Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India.

Statistical analysis
Statistical analysis was performed by ANOVA and post hoc least significant difference tests using GraphPad Prism software version 8 (GraphPad Software, USA). Data were expressed as mean ± standard error (SE). The results were expressed as statistically significant at a probability level of p < 0.05.

Results
In context to all the parameters, no significant difference was observed between the healthy control group and the vehicle control groups (VC-I, VC-II). Hence, healthy control was used for all comparisons.

Body and organ weight
The Bwt and reproductive organs of animals of the D-gal group were significantly decreased when compared to the control group. The treatment of TP and SB substantially increased the Bwt and reproductive organs weights decreased by Dgal. The results of Bwt and reproductive organs weight has been summarized in the (Table  III).

International Journal of Reproductive BioMedicine
Netam et al.

Sperm parameters
The results showed a significant (p < 0.001) decrease in the epididymal sperm counts in the D-gal administered groups as compared to the control groups. The injection of TP and oral administration of SB significantly elevated the reduced sperm count in the Post-TP (p = 0.73), Comb-TP (p = 0.12), Post-SB (p = 0.39), and Comb-SB (p < 0.01) groups when compared to the exposure group (D-gal).
A significant (p < 0.001) decrease in the mean sperm motility was observed in the D-gal group when compared to the control group. On TP and SB administration, a non-significant (p > 0.05) increase in sperm motility was seen in Post-TP, Comb-TP, Post-SB, and Comb-SB groups when compared to the exposure group (D-gal) ( Table IV).
The results of sperm abnormalities are summarized in (Tables V and VI)

Serum T level
The D-gal exposed group exhibited a significant (p < 0.001) reduction in serum T level compared to the control group. On the other hand, treatment with TP caused a significant elevation in serum T levels in the treated groups Post-TP (p < 0.001) and Comb-TP (p < 0.001) as compared to the exposure group D-gal. Further, a significant increase in T level was also observed after the administration of SB in both Post-SB (p < 0.001) and Comb-SB (p < 0.001), suggesting that it has a strong influence on testicular steroidogenesis ( Figure 2).

LH and FSH
In the D-gal group, the LH and FSH levels in the serum increased significantly (p < 0.001) as compared to the control group. The administration of TP significantly (p < 0.001) reduced the elevated LH and FSH levels in Post-TP and Comb-TP when compared to the exposure group (D-gal). SB-administered groups showed nonsignificant (p > 0.05) decreased LH levels when compared to (D-gal). FSH level was also decreased in Post-SB (p = 0.59) and Comb-SB (p = 0.03) groups as compared to D-gal ( Figure 2).

Steroidogenic enzymes
The testicular 3β-HSD and 17β-HSD enzyme levels were significantly lowered (p < 0.001) in the exposed group (D-gal) than in the control group. In both the TP-treated groups (Post-TP, Comb-TP) the administration of TP significantly (p < 0.001) increased the 3β-HSD level relative to the exposure group (D-gal); however, this increase was nonsignificant (p > 0.05) in case of 17β-HSD. The administration of SB significantly (p < 0.001) increased the 3β-HSD level and 17β-HSD enzyme levels in both Post-SB and Comb-SB groups when compared to D-gal ( Figure 3). The results showed the protective effect of SB and its influence on gonadal steroidogenesis.
The current study's findings reveal a significant  (Table VII).

Antioxidant enzymes
In testis, the LPO level was significantly increased (p < 0.001) in the D-gal exposed group when compared with the control group.  (Table VIII).

StAR mRNA expression
The RT-PCR results showed that the StAR mRNA expression in the D-gal exposed group was significantly (p < 0.001) downregulated as

D-gal
In the present investigation, D-gal exposure resulted in decreased Bwt, sex organs weight, sperm count, and motility with increased sperm abnormalities. The decline in Bwt in D-gal-exposed

Treatment with TP and SB
In the current study, TP treatment increased Histopathological analysis in previously conducted study exhibited a normal histoarchitecture of testis after TP treatment through amelioration of testicular oxidative stress in rat and mice model (38,39).
The results suggest that exogenous T treatment may be responsible for improving steroidogenesis and spermatogenesis in aging rat testis. However, long-term TP treatment has been linked with prostate cancer and a potential risk to cardiac health, obstructive sleep apnea, and erythrocytosis (14). A decrease in intratesticular T production has also been reported in long-term therapy of TP, indicating its dependency. Hence, we studied the steroidogenic effect of SB, a natural substance with the capability of enhancing endogenous T production.
SB is an Ayurvedic preparation used as sexual function enhancer in traditional Indian Ayurvedic medicine (20). In the current investigation, the oral administration of SB significantly increased the mean Bwt, testis weight, and other accessory sex organs weights. Further, treatment of SB also considerably improved sperm counts and motility with a reduction in sperm abnormalities in Dgal exposed rats. The increased spermatogenic activity is related to SB's anti-oxidative and ROS scavenging properties, with greater androgen availability in treated animals. It has been reported that the SB (gold ash) treatment increased the total sperm count and motility percentage in healthy adults and infertile patients (41).
In the present study, we found that SB significantly increased the serum T level and reduced LH and FSH hormones in D-gal exposed rats. The SB treatment also increased the SB is considered as herbo metallic formulation where herbal extracts used in the preparation of SB weren't used for their medicinal benefits but rather for their capacity to chemically reduce the gold metal and mechanically simplify the grinding process (42). In order to understand the composition, the physico-chemical characterization of SB used in the study has been previously characterized by various modern methods like XRD and EDX, which revealed  (14).
According to Ayurveda, the ayurvedic metallic formulation is free from any toxic effects. Besides its traditional belief, various in-vitro and in-vivo toxicological studies conducted previously concluded that SB is nontoxic (19). Toxicity study of 90 days was conducted in Wistar rats where a high dose of SB (13.5 mg/kg Bwt) did not cause any alteration in weight and histopathology of organs (liver, kidney) in treated rats (24).

Conclusion
The study concluded that the D-gal caused several reproductive impairments through increased oxidative stress and decreased T synthesis, affecting sperm count, motility, and morphology. TP and SB reduced oxidative stress, increased sperm count, increased steroidogenesis, and improved testicular histopathology in the D-gal aging model. The findings suggested that maintaining androgen levels through exogenous T treatment may help in sustaining gonadal activity. But the exogenous T treatment uses have increased cause for concern because of the various associated reported toxicities. According to this study, SB maintained the T level endogenously via increased antioxidant activity and restored normal spermatogenesis, and steroidogenesis was altered by D-gal. Further study is needed to find out the various target protein of SB to elucidate its exact mechanism of action. The study concludes that SB may prefer over TP as a therapeutic agent against D-gal-induced testicular toxicity, as it has a long history of usage in India, and a number of studies have established its safety in both humans as well as animal models.