To investigate the effect of the aqueous extract of the root bark of Morinda morindoides (Rubiaceae) on the reproductive activity of male Wistar rats, three groups (A, B and C) of six rats each were treated with 400,800 and 1600 mg/kg/day of Morinda morindoides root bark extract respectively for 28 days while 5mls/kg of distilled water was given to the control group D. At the end of the experimental period, animals were sacrificed and sperm characteristics, histology of the testes and epididymis were assessed. Morinda morindoides root bark extract caused a significant reduction (p&lt;0.05) in sperm motility, a significant dose dependent reduction in the sperm count and a significant (p&lt;0.05) dose dependent increase in morphological abnormalities of the spermatozoa of the treated rats. Histopathological evaluation of the testis and epididymis revealed varying degrees of degeneration and necrosis of the germinal epithelia cell of the seminiferous tubules and spermiostasis. Morinda morindoides root bark extract has significant anti-spermatogenic effects on adult male Wistar rats which could impair reproductive activities in these male Wistar rats. &nbsp

Abatan, M. O.

Adeleye, O. E.

Adenubi, O. T.

Ajayi, O. L.

Kehinde, O. O.

Olukunle, J. O.

English

Federal University of Agriculture, Abeokuta: FUNAAB Journal

ANTISPERMATOGENIC ACTIVITY OF Morinda morin-doides ROOT BARK EXTRACT IN MALE  WISTAR RATS  O.T. ADENUBI1, J.O. OLUKUNLE1*, M.O ABATAN2, O.L. AJAYI3, O.E. ADELEYE1 AND O.O. KEHINDE4                     1Department of Veterinary Physiology and Pharmacology, College of Veterinary         Medicine, University of Agriculture, PMB. 2240, Abeokuta, Ogun State.            2Department of Veterinary Physiology, Biochemistry and Pharmacology,         University of Ibadan, Ibadan, Oyo State.            3Department of Veterinary  Pathology, College of Veterinary Medicine, University of         Agriculture, PMB 2240, Abeokuta, Ogun State.           4Department of Veterinary  Public Health and Reproduction, College of Veterinary         Medicine, University of Agriculture, PMB 2240, Abeokuta, Ogun State.       *Corresponding author: bukioguns@yahoo.com on the leaves of this plant led to the isolation of nine flavonoids and flavonoid O-glycosides (Cimanga et al., 1995). The struc-ture determination of eight iridoid glycosides was also reported (Cimanga et al., 2003).  There have been various reports on the an-tispermatogenic and antifertility effect of antimalarial agents. Chloroquine, an antima-ABSTRACT To investigate the effect of the aqueous extract of the root bark of Morinda morindoides (Rubiaceae) on the reproductive activity of male Wistar rats, three groups (A, B and C) of six rats each were treated with 400,800 and 1600 mg/kg/day of Morinda morindoides root bark extract respectively for 28 days while 5mls/kg of distilled water was given to the control group D. At the end of the experimental period, animals were sacrificed and sperm characteristics, histology of the testes and epididymis were as-sessed. Morinda morindoides root bark extract caused a significant reduction (p<0.05) in sperm motil-ity, a significant dose dependent reduction in the sperm count and a significant (p<0.05) dose depend-ent increase in morphological abnormalities of the spermatozoa of the treated rats. Histopathological evaluation of the testis and epididymis revealed varying degrees of degeneration and necrosis of the germinal epithelia cell of the seminiferous tubules and spermiostasis. Morinda morindoides root bark extract has significant anti-spermatogenic effects on adult male Wistar rats which could impair repro-ductive activities in these male Wistar rats.  Keywords: extract, Morinda morindoides, rats, sperm. INTRODUCTION Morinda morindoides (Rubiaceae) is a plant with a high reputation in the traditional management of malaria, diarrhoea, amoebi-asis, haemorrhoids, gonorrhoea and rheu-matic pains (Kambu, 1990). Biological stud-ies have supported some of these traditional uses (Tona et al., 1999, 2001; Cimanga et al., 2003, 2006). Previous phytochemical studies J. Nat. Sci. Engr. Tech. 2010, 9(1):99-105 Journal of Natural  Sciences, Engineer-ing and Technology  99 ISSN - 2277 - 0593 © UNAAB 2010 larial drug, has been discovered to have negative effects on sperm motility and fer-tility as a whole (Okanlawon et al., 1993); quinine is also known to inhibit spermato-genesis (Osinubi et al., 2004). Furthermore, pyrimethamine was observed to arrest sper-matogenesis and cause infertility in a dose-dependent manner. Cessation of admini-stration of the aforementioned drugs re-sulted in full restoration to normal fertility status (Awoniyi et al., 1993; Consentino et al., 1990).  Many medicinal plants have also been re-ported to have antispermatogenic effects. Alstonia boonei, a tropical plant, reputed in traditional medicine to have anti-malarial, antipyretic, analgesic and anti-inflammatory properties (Ojewole, 1984, Olajide et al., 2000) was reported to cause dose depend-ent changes in the body weight, organ weights and sperm characteristics in male rats. Azadirachta indica, another medicinal plant with very potent antiplasmodial activi-ties in mice, has also been reported to cause mass atrophy of spermatogenic elements and Leydig cells (Gbile, 1986).  There has not been any documented report on the effect of the root bark extract of Morinda morindoides on sperm parameters. This study was therefore carried out to in-vestigate the effects of the administration of aqueous extract of M. morindoides root bark on the spermiogram of male Wistar rats.  MATERIALS AND METHODS Animals Twenty four adult male Wistar rats (Mean body weight- 180.0±3.33g) were used for this study. The rats were housed in the Ex-perimental Animal Unit of the College of Veterinary Medicine, University of Agricul-ture, Abeokuta, Ogun State, Nigeria. They were kept in well-ventilated metal cages at ambient temperature and a period of 12 hour light and 12 hour darkness was maintained. The rats were fed standard ration (Vital Feeds Limited, Ibadan) and clean water ad-libitum.  Plant material The root bark of Morinda morindoides was pur-chased from Kuto Market in Abeokuta, Ogun State. Identification and authentication was done at the Forestry Research Institute of Nigeria (FRIN), Ibadan, Nigeria.  Plant extraction The root bark of Morinda morindoides was air dried, pulverized, finely sieved and 500g of the plant was soaked in 1 litre of distilled water for 24 hours after which it was filtered. Thereafter, the filtrate was evaporated to dryness and 1g of it was dissolved in 20mls of distilled water to give a concentration of 50mg/ml.  Experimental procedure The rats were randomly divided into four groups (A-D) of 6 rats each. The rats in groups A, B and C were given aqueous ex-tract of Morinda morindoides root bark at 400mg/kg, 800mg/kg and 1600mg/kg body weight respectively once daily orally for 28 days. Group D rats were the control to which 5mls/kg distilled water was adminis-tered orally once daily for 28 days. Thereaf-ter, the rats were euthanized by placing them in a glass chamber containing cotton wool soaked in diethyl ether till they lost con-sciousness followed by cervical dislocation. A ventral midline abdominal incision was then made using a scalpel blade size 14 to expose the abdominal organs. The testis and epididymis of each rat were identified, care-fully removed and a small incision was made on the caudal epididymis to squeeze out the O.T. ADENUBI1, J.O. OLUKUNLE1, M.O ABATAN2, O.L. AJAYI3, O.E. ADELEYE1 AND O.O. KEHINDE4 J. Nat. Sci. Engr. Tech. 2010, 9(1):99-105 100 semen content on a glass slide for semen evaluation. The testis and epididymis were thereafter preserved in Bouins fluid for histopathological evaluation.  Statistical Analysis The mean and standard error of mean were calculated for all the sperm parameters. Analysis of variance (ANOVA) was used to establish any significant difference in all the stated parameters. p<0.05 was considered significant in all cases.  RESULTS There was a significant decrease (p<0.05) in the progressive sperm motility and percent-age sperm live/dead ratio of the male Wis-tar rats treated with the aqueous extract Morinda morindoides root bark when com-pared with the control (Table 1). In addi-tion, sperm count of the Wistar rats follow-ing oral administration of 400mg/kg, 800mg/kg and 1600mg/kg doses of the aqueous extract of M. morindoides root bark was significantly reduced in a dose-dependent manner when compared with the control (Table 1).  At lower doses (400mg/kg and 800mg/kg), there was no significant change in the total number of abnormal spermatozoa in the treated groups compared with the control. However, at 1600mg/kg, there was a sig-nificant increase (p<0.05) in abnormal sper-matozoa such as headless tail, curved tail and curved mid-piece when compared with the control group (Table 2).  Histopathology of the testis and epididymis of rats administered different doses of aque-ous extract of Morinda morindoides root bark revealed varying degrees of degeneration and necrosis of the germinal epithelia cell of the seminiferous tubules and spermiostasis (Fig I & II).  DISCUSSION  Administration of the aqueous extract of the root bark of M. morindoides caused a reduc-tion in progressive sperm motility, percent-age sperm live/dead ratio and sperm count. Some secondary morphological sperm ab-normalities such as headless tail, bent tail and curved mid-piece were higher in the treated rats.  M. morindoides has been reported to have a wide safety margin. It was observed that oral administration of the extract of M. morin-doides to rats up to 6000mg/kg neither showed mortality nor any apparent signs of weakness in the animals (Shoba & Thomas, 2001). Doses ranging from 100mg/kg to 6000mg/kg had been used in clinical trials and in this study, three doses; 400, 800 and 1600mg/kg were tested 400mg/kg is mostly used by herbal practitioners and this dose was used and its multiples.  Sperm motility depends on the coordinated propagated flagella wave under acetylcholi-nesterase control (Nelson, 1972). Fructose utilization and glucose oxidation are impor-tant means by which spermatozoa derive energy for their motility. The reduction in the progressive sperm motility of the treated rats seen in this study could be due to the acetylcholinesterase inhibition and glucose lowering properties of the specie of this plant (Olajide et al., 1999).  The significant decrease in the sperm count supported by the various degrees of degen-eration in the histologic sections of the testis and epididymis, suggests that M. morindoides root bark extract is capable of permeating the blood-testis barrier (Baldessarini, 1980).  ANTISPERMATOGENIC ACTIVITY OF MORINDA MORINDOIDES ROOT ... J. Nat. Sci. Engr. Tech. 2010, 9(1):99-105 101 Since several studies have reported the anti-fertility effects of anti-malarial agents, this result is in consonance with previous stud-ies. Chloroquine, quinine and quinacrine have been reported to inhibit Leydig cell steroidogenesis and fertility in the male (Adeeko & Dada, 1998).  Cimanga, R.K., Kambu, K., Tona, L., Hermans, N., Apers, S., Totte, J., Pieters, Table 1:  Effects of Morinda Morindoides root bark extract on sperm characteristics     of male wistar rats  Groups Sperm motility Sperm live/dead ratio Sperm volume Sperm count Group A 22.50+13.20* 86.3+2.40* 5.15+0.0 45.75+5.10* Group B 47.50+6.34* 90.00+2.0* 5.13+0.0 40.75+1.10* Group C 12.50+7.50* 70.00+5.80* 5.18+0.0 35.50+2.40* Group D (Control) 93.75+1.30 98.00+0.00 5.18.+ 0.0 129.00+ 5.00 Values are mean +standard error of mean *p<0.05. O.T. ADENUBI1, J.O. OLUKUNLE1, M.O ABATAN2, O.L. AJAYI3, O.E. ADELEYE1 AND O.O. KEHINDE4 Table 2: Effects of Morinda Morindoides roots bark extract on sperm morphology      of male wistar rats Groups Headless tail Tailess head Rudi-mentary tail Bent tail Curved tail Bent Mid Piece Curved Mid Piece Looped Tail Coiled tail Total abnor-malities Group A 166.00+ 48.4 249.50+ 27.1 13.50+ 4.8 41017.3+ 23.6.5* 3903.8+ 273.6* 4422.5+ 57.0 3871.5+ 263.6 11.0+ 5.8 13.50 + 4.8 402.5+ 2.5 Group B 239.00+ 27.3 163.75+ 47.6 16.00+ 4.9 4629.00+ 235.5 3822.3+288.5* 4544.8+ 226.9 4567.00+220.3* 13.50+ 4.8 11.00+ 4.1 400.00+ 0.0 Group C 340.0+ 27.0* 239.25+ 27.3* 16.25+ 4.7 5822.5+ 255.5* 4433.00+57.7* 4827.5+ 286.7 5347.3+ 25.3* 18.50+ 2.5 8.50+ 2.5 402.50+ 1.4 Group D (Control) 116.25+ 54.9 68.75+ 47.5 8.50+ 4.8 2492.00+ 279.0 2767.00 + 29.0 3494.2+ 240.0 2696.25+300.66 13.50+ 4.8 6.00+ 2.9 403.75+ 2.4 Values are mean ± standard error of mean *p<0.05. J. Nat. Sci. Engr. Tech. 2010, 9(1):99-105 102 Figure 1: Section of testis with degeneration and necrosis of the germinal epithelial     cells of the seminiferous tubules with spermiostasis. X250 ANTISPERMATOGENIC ACTIVITY OF MORINDA MORINDOIDES ROOT ... Figure 2: Section of testis with degeneration and necrosis of the germinal epithelial                 cells of the seminiferous tubules with spermiostasis. H&EX300 J. Nat. Sci. Engr. Tech. 2010, 9(1):99-105 103 Figure 3: Section of the epididymis that revealed that the epididymis is empty and contains               few spermatogonia cells (severe spermiostasis) with no sperm produced at all. X250 O.T. ADENUBI1, J.O. OLUKUNLE1, M.O ABATAN2, O.L. AJAYI3, O.E. ADELEYE1 AND O.O. KEHINDE4 CONCLUSION In conclusion, this study shows that daily oral administration of 400-1600mg/kg body weight of Morinda morindoides root bark ex-tract for a period of 28 days has significant anti-spermatogenic effects on adult male Wistar rats as seen with most other anti-malarial herbs. Further studies aimed at elu-cidating the activities of Morinda morindoides root bark extract would be worthwhile.  REFERENCES Adeeko, A.O., Dada, O.A. 1998. Chloro-quine reduces the fertilizing capacity of epididymal sperm in rats. African Journal of Medicine and Medical Sciences, 27:63-68.  Awoniyi, C.A., Chandrashekar, V., Hurst, B.S., Kim, W.K., Schlaff, W.D., 1993. The effects of chronic administration of pyrimethamine on spermatogenesis and fertility in male rats. J. Androl., 14(3): 174-179.  Baldessarini, R.J. 1980. Drugs and the treatment of psychiatric disorders: the phar-macological basis of psychiatric disorders. In: Goodman LS, Limbird LE, Mlilnoff PB, Ruddon RW, Gilman AG, editors. 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(Manuscript received: 15th April, 2010; accepted:, 9th July, 2010). ANTISPERMATOGENIC ACTIVITY OF MORINDA MORINDOIDES ROOT ... J. Nat. Sci. Engr. Tech. 2010, 9(1):99-105 105 

Antispermatogenic Activity Of Morinda Morindoides Root Bark Extract In Male Wistar Rats

https://www.journal.unaab.edu.ng/index.php/JNSET/article/view/1053/1008

Antispermatogenic Activity Of Morinda Morindoides Root Bark Extract In Male Wistar Rats

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Federal University of Agriculture, Abeokuta: FUNAAB Journal