1. The effects of aqueous leave extract of Basella alba on reproductive parameters of streptozotocin-induced diabetic male wistar rats
Dennis S. Arokoyo a, Ibukun P. Oyeyipo b, Stefan S. Du Plessis b , Yapo G. Aboua a
aFaculty of Health and Wellness Sciences, Cape Peninsula University of Technology, RSA
bDivision of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, RSA

2. Background
Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia and due to defects in insulin secretion, insulin action, or both (Diabetes care 2012)
Management with medicinal plant is a major research focus in developing countries
Basella alba is one of such plants reported to have anti-DM effects (Bamidele et al., 2014) and increase libido (Kuete and Efferth., 2010)

3. Background Basella alba (a vegetable) Malabar spinach Indian spinach
Common names;
Malabar spinach
Indian spinach
Ceylon spinach

4. Aim and Objectives
Aim: To investigate effect & mechanism underlying the effect of Basella alba (B.A) aqueous leave extract on sperm parameters in diabetic Wistar rat.
Objectives:
To assess the effect of DM on;
Sperm parameters
Gonadal hormones.
Weight and relative weight of testes and epididymis
And a possible ameliorative role of Basella alba on such effects.

5. Materials and Methods Plant extract preparation
Fresh leaves of B.A was washed, air-dried and grinded into fine powder.
100 g of the fine power was poured into 1000 ml of boiling distilled water
Allowed to boil for another 5 min. then kept off heat for 30 min. for adequate infusion.
This was filtered and the filtrate freeze-dried to powdery extract.
Extract dissolved in normal saline and administered orally by gavage.

6. Materials and Methods HT = Healthy treatment(B.A , 200mg/kg)
Animals & study design
Male Wistar rats
Age 8-10 weeks
(n=10/group)
HC = Healthy control (N/S, 0.5ML/100g BW)
Diabetic Control (N/S, 0.5ML/100g BW)
DC =
DT = Diabetic treatment (B.A, 200mg/kg)
HT = Healthy treatment(B.A , 200mg/kg)

7. Materials and Methods Induction of DM and blood sugar measurement
Diabetes was induced in groups DC & DT with a single intraperitoneal injection of streptozotocin (55mg/kg).
DM was confirmed 72 h after by testing the fasting blood sugar (FBS) using glucometer and glucose test strip.
FBS and weight was then recorded weekly in all four groups throughout the study.

8. Materials and Methods Collection of samples & analysis
All animals were euthanized after four weeks of treatment.
Blood was collected into serum clot activator tubes, centrifuged at 4000rpm for 10min at 4⁰C and serum stored at -80⁰C.
Right testes & epididymis were removed, weighed and caudal epididymal spermatozoa analysed immediately.

9. Materials and Methods 𝑶𝒓𝒈𝒂𝒏 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈) 𝑩𝒐𝒅𝒚 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈)
Relative organ weights was calculated using the formula;
Sperm motility and concentration was performed with computer-aided sperm analysis (CASA).
(SCA®; Microptic, Barcelona, Spain).
Sperm viability was determined by the Eosin (EO) dye exclusion test;
10µl sperm suspension + 20µl eosin + 30µl nigrosin
A thin smear was prepared & air-dried for 24hr
% viability determined by counting of 100 spermatozoa at x100 magnification (Eliasson, 1977).
Live spermatozoa = unstained, Dead sperm = red stained
𝑶𝒓𝒈𝒂𝒏 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈) 𝑩𝒐𝒅𝒚 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈)

10. Materials and Methods % Normal sperm morphology; Hormonal assays;
SpermBlue (SB) fixative and stain was used
Sperm Class Analyzer® with a blue filter, at x60 magnification was used to analyse the slides.
All spermatozoa which do not overlap with each other or with background staining were considered.
Hormonal assays;
Testosterone, LH and FSH were analysed in serum samples using radioimmunoassay technique

11. Statistical analysis Done using Graphpad prism version 5
Values were expressed as mean ± SEM
Bonferroni post-test: Comparison of all four groups.
Differences between group means: One-way AN0VA
Statistical significance at p ˂ 0.05.

12. Results
Effect of Basella alba on FBS (Fasting blood sugar)

13. Results
Effect on organ weight & relative organ weight

14. Results

15. Results . Effect on sperm parameters Healthy control Diabetic control
Healthy treatment
Diabetic treatment
Concentration
(million/ml)
4.50 ± 0.49
2.23 ± 0.57 *
3.88 ± 0.88
4.52 ± 0.63 a
% Motile
74.30 ± 7.23
53.30 ± 10.37
70.90 ± 6.48
69.50 ± 6.88
% Viability
70.13 ± 2.0
31.67 ± 1.63 **
70.43 ± 1.73
50.50 ± 1.73 *a
% Normal Morphology
62.96 ± 2.71
41.67 ± 2.33 **
68.00 ± 2.80 b
53.67 ± 2.39 a
*P<0.05, **P<0.001 between groups and healthy control.
aP<0.05, bP<0.001 between groups and diabetic control.

16. Results .
Levels of gonadal hormones in serum

17. Discussion
The ↑Testosterone & ↑LH in DC group is consistent with previous reports of higher & normal serum testosterone in T1DM compared to healthy controls.
(Meyer et al., 2000 & Dandona et al., 2009)
T2DM usually causes hypogonadotrophic hypogonadism where serum testosterone is low
(Amaral et al., 2008; Pontes et al., 2011)

18. Discussion B.A STZ 55mg/Kg T1DM AGEs Testosterone Insensitivity
(Busineni et al., 2015)
T1DM
↓fat = ↑testosterone
(Tomar et al., 2006)
(Mallidis et al.,2011)
DM Neuropathy
↑Testosterone
AGEs
(Sexton et al., 1997)
Testosterone Insensitivity

19. Conclusion
Basella alba ameliorates most of the harmful effect of DM on sperm parameters by regulating the secretion and actions of gonadal hormones among other mechanisms.

20. References
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American Diabetic Association. Diabetes care 2012; volume 35: supplement 1, s64-s75.
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