EVALUATION OF EFFICACY OF HERBAL FORMULATIONS IN A MOUSE MODEL OF CADMIUM CHLORIDE-INDUCED TESTICULAR TOXICITY AND INFERTILITY

EVALUATION OF EFFICACY OF HERBAL FORMULATIONS IN A MOUSE MODEL OF CADMIUM CHLORIDE-INDUCED TESTICULAR TOXICITY AND INFERTILITY

1.0 TEST SYSTEM DETAILS:

Species : Mus musculus (Mouse)

Strain : Swiss Albino or BALB/c

Age : 6-7 weeks

Sex : Male and Female

No. of animals : 8 /Group

Total animals : 112 (56 Males + 56 Females)


2.0 TEST ARTICLES DETAILS

HB1: Tribulus terrestris, Dactylorhiza hatagirea and Ayurvedic mineral-based herbal formulation.

 
3.0 ALLOCATION OF GROUPS:

Group No.	Group Description	Disease disease-inducing agent administered	Treatment administered	Dose Volume and Route
G1	Normal Control	PBS was administered by oral route for 35 consecutive days	0.5% MC, p.o., b.i.d.	10 ml/kg, p.o.
G2	Disease Control	Cadmium chloride -2 mg/kg (dissolved in PBS), by oral route for 35 consecutive days	0.5% MC, p.o., b.i.d.
G3	Reference Control		Rapamycin 8 mg/kg, p.o., q.d. + 0.5 % MC, p.o.
G4	Treated with a low dose of HB1		HB1: 10-30 mg/kg, b.i.d.  in 0.5% MC
G5	Treated with intermediate dose 1 of HB1		HB1: 30-100 mg/kg, mg/kg, b.i.d. in 0.5% MC
G6	Treated with dose 2 of HB1		HB1: 100-300 mg/kg,   b.i.d. in 0.5% MC
G7	Treated with a low dose of HB1		HB1: 300-1000 mg/kg, b.i.d.  in 0.5% MC

Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d.: quaque die; bid: bis in die; PBS: Phosphate Buffered Saline

1.0 METHOD:

• Healthy animals will be selected for the study, randomized based on body weight, and assigned to 7 groups of 8 animals each.

• Animals of Group G1 will be designated as normal-control and administered 0.5% MC, p.o., b.i.d.

• Disease control animals (assigned to group G2) will receive 0.5% MC, p.o., b.i.d.

• Animals of group G3 will be treated with reference drug Rapamycin 8 mg/kg, once daily. + 0.5% MC at the dose of 8 mg/kg, i.p., (in the morning) and will be additionally administered 0.5% MC in the evening.

• Animals of group G4-G7 will be treated with HB1, at different dose levels ranging from 10-1000 mg/kg, b.i.d.

• The normal control group (G1) will be administered an injection of Normal Saline oral route for 35 consecutive days. In contrast, animals allocated to groups G2 – G11 will be administered cadmium chloride solution (2 mg/kg, p.o.) dissolved in PBS, for a total duration of 35 days.

• Compound administration will be initiated 14 days before cadmium injection, concurrent with CdCl2 administration, and for 35 days after stopping CdCl2 administration.

• On Day 36 after stopping CdCl2, a fertility test will be performed, wherein males of all the groups will be allowed to mate with proestrus females (1:1) overnight to estimate the index of libido and male fertility index.

• The mated females were allowed to complete the term for litters.

• On Day 37 after stopping CdCl2, animals will be sacrificed under an overdose of thiopentone anesthesia. After suitable anesthesia but before the animal dies, blood will be collected from the retro-orbital plexus to estimate biochemical parameters. Immediately after the animal dies, the testicles, kidneys, and liver will be weighed. Subsequently, semen will be collected for sperm analysis. Thereafter, half a portion of the tested will be fixed in Modified Davidson’s fixative for histopathology. In contrast, the other half will be stored at -80°C for the various biochemical and molecular evaluations.

2.0 PARAMETERS TO BE EVALUATED:

• Body weight: Twice a week.

• Index of libido

• Male fertility index

• Testicle weight and its relative organ weight.

• Serum cadmium and reproductive hormone (Testosterone, LH, and FSH) concentrations

• Testicular glucose, lactate, and LDH activity and antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase in the testis. Non-enzymatic antioxidants in the testis include reduced glutathione. oxidative stress markers in the testis Malondialdehyde and H2O2.

• Sperm analysis

• Gene expression analysis by Real-Time PCR: Bax and Bcl-2 proteins

• Histopathological analysis of Testicle (Hematoxylin & Eosin-stained).

• Immunofluorescence staining of the testicle tissue

3.0 REFERENCE(S):

1. Mishra, R. K., Jain, A. & Singh, S. K. Profertility effects of Shilajit on cadmium-induced infertility in male mice. Andrologia 50, 1–9 (2018).

2. Mouro, V. G. S. et al. Cadmium-Induced Testicular Toxicity in Mice: Subacute and Subchronic Route-Dependent Effects. Biol. Trace Elem. Res. 193, 466–482 (2020).

3. Zhang, L. et al. Protective effect of Lycium barbarum polysaccharides against cadmium-induced testicular toxicity in male mice. Food Funct. 8, 2322–2330 (2017).

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