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: 30-100 mg/kg, mg/kg, b.i.d. in 0.5% MC |
||
|
G6 |
Treated with dose 2 of |
HB1: 100-300 mg/kg, b.i.d. in 0.5% MC |
||
|
G7 |
Treated with a low dose of |
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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