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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 10  |  Issue : 4  |  Page : 124-130

Green Coconut Water Supplementation Attenuates Flutamide-induced Testicular Damage in Male Prepubertal Wistar Rats


1 Department of Anatomy, Faculty of Basic Medical Sciences, Kampala International University, Ishaka, Uganda
2 Department of Anatomy, Faculty of Basic Medical Sciences, University of Lagos, Lagos, Nigeria

Date of Submission31-Dec-2022
Date of Decision01-Apr-2023
Date of Acceptance01-May-2023
Date of Web Publication22-Feb-2023

Correspondence Address:
Mr. Abdulkareem Temitayo Olayinka
Ph.D in-view, Department of Anatomy, Faculty of Basic Medical Sciences, Kampala International University, Western Campus, Ishaka
Uganda
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_23_22

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  Abstract 


Background: The enhancement of male reproductive health function remains a paramount desire of every infertile men. Green coconut water (GCW) is the liquid in the inner cavity of an immature coconut fruit. Studies have demonstrated that GCW has endocrine property that regulates the reproductive system. Aim and Objectives: This study was designed to investigates the effects of GCW on the cardinal sperm function parameters such as; sperm count, sperm motility and sperm morphology, and the histo-architecture of the testis in flutamide-treated pre-pubertal wistar rats. Materials and Methods: Thirty-six male prepubertal wistar rats, weighing between 40-70g were divided into six study groups A-F. Group A, B and C received distilled water, flutamide at 25mg/kg and GCW at 20ml/kg respectively for 6 weeks. Group D and E received 25mg/kg flutamide for 2 weeks then GCW at 10ml/kg and 20ml/kg respectively 4 weeks, while Group F received 25mg/kg flutamide and GCW at 20ml/kg concomitantly for 6 weeks. At the end of the experiment, the animals were euthanized; caudal epididymis and the testis were collected for semen analysis and histological evaluation. Results: GCW supplementation was showed to significantly increased sperm count, sperm motility and also brings about percentage decrease in sperm morphology, as well as ameliorates histo-pathological degenerations caused by flutamide on the testis of the experimental rats. Conclusion: The findings from this study suggest that GCW supplementation may effectively enhance male fertility.

Keywords: Flutamide, green coconut water, male factor infertility, sperm count, testis


How to cite this article:
Olayinka AT, Bakare AA, Oremosu AA. Green Coconut Water Supplementation Attenuates Flutamide-induced Testicular Damage in Male Prepubertal Wistar Rats. Niger J Exp Clin Biosci 2022;10:124-30

How to cite this URL:
Olayinka AT, Bakare AA, Oremosu AA. Green Coconut Water Supplementation Attenuates Flutamide-induced Testicular Damage in Male Prepubertal Wistar Rats. Niger J Exp Clin Biosci [serial online] 2022 [cited 2023 Mar 30];10:124-30. Available from: https://www.njecbonline.org/text.asp?2022/10/4/124/370249




  Introduction Top


Globally, and more, especially in Africa continent, the inability of couples within their reproductive ages to conceive is seen to be female partner dependent. However, the body of evidence from recent research revealed that 40%–50% of the infertility cases seen in the 15% of global couples living with infertility challenges are male factor associated.[1],[2] A male is declared to be infertile or subfertile following his inability to impregnate a female after 1 year of regular unprotected sexual intercourse.[3] The causes of male infertility are reported to be multifactorial, with infections and hormonal imbalance being its major causatives.[4] Sperm count and sperm motility are the two principal diagnostic parameters implored for the assessment of male fertility potentials.[5] Decline in either the sperm quantity or sperm quality or both accounts for more than 90% of male infertility.[6] The pathological cause of the decline in sperm function is, however, evidence to stem from either distortion in the control mechanism of sperm production at the pretesticular, testicular, or posttesticular center.[7]

Infertile men suffer huge psychological and emotional stigma from their inability to achieve pregnancy duly with their partners. So much that in their quest for remedy, many seek refuge in the regular use of drugs acclaimed to enhance reproductive function. However, the abuse of drugs of this nature that is either prescribed or nonprescribed was one of the major causes of male infertility as their prolong usage leads to impairment of hypothalamic–pituitary–gonadal functions, increased sperm DNA fragmentation, and reduced sperm quality.[8] It is upon this rationale, this study is geared to hypothesize whether natural herbal supplement as green coconut water (GCW) commonly consumed as refreshing drink, can revamp sperm function with minimal or no side effects or not.

GCW is a sterile, nutritious, refreshing, and delicious drink of an average volume of 400 ml, found within the endosperm of a 6–7-month-old green coconut fruit.[9],[10],[11] It is believed to originate from the Southeast Asia region, from where it became widely distributed across other regions in the world.[12] Its phytochemical analysis shows that it is composed of large and traced quantities of vitamins, sugars, minerals, proteins, free amino acids, organic acids, inorganic ions, and growth-promoting factors.[13],[14] Since its introduction into the scientific community in the 1940s, lot of research had since then been conducted to understand better the mechanism of its biochemical activities in the treatment of different disease conditions. GCW supplementation had been reported to significantly reversed increased cholesterol levels in induced myocardial infarction rats fed with a high diet.[15] GCW had been shown to exhibit antidiabetic effects in alloxan-induced rats.[16] GCW was also shown to possess fertility-enhancing potentials as it regulates estrous cyclicity in female rats-induced hyperprolactinemia.[10] The present study was carried out to investigate the effects of GCW on the sperm function parameters and the cytoarchitecture of the testis in flutamide (FLT)-induced prepubertal Wistar rats.


  Materials and Methods Top


Source of green coconut fruit

The immature (green) coconut fruits were harvested from coconut trees grown within the University of Lagos College of Medicine Campus, Lagos. The fruits were authenticated in the Department of Botany, University of Lagos. The green coconut fruits were washed, dehusked, and cracked. The extraction of the GCW was done through the germinal pore, poured directly into an air-tight sterile container, kept in the refrigerator, and replaced every 3 weeks.[17]

Ethical approval

Ethical approval for this study was obtained from the health research ethics committee on animal use of the College of Medicine, University of Lagos Nigeria with clearance reference: CMUL/HREC/04/19/533.

Animals

Thirty-six prepubertal male Wistar rats with an average weight of 40–70 g were used for this study. The animals were kept in standard plastic cages in the animal house of the department of anatomy and were acclimatized for 2 weeks under standard laboratory conditions of room temperature 24°C ± 2°C with a photoperiodicity of 12 h of light alternating with 12 h of darkness. The animals were fed with pelletized feeds and water ad libitum for 6 weeks.

Dosage and administration of green coconut water

The GCW was administered orally at a dose of 10 ml/kg and 20 mL/kg body weight per day for 6 weeks.[18]

Experimental design

The animals were randomly divided into six experimental groups and treated as shown in [Table 1].
Table 1: Description of experimental groups and dose administration

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At the end of the experiment, the epididymis and the testis were obtained for semen analysis and histological evaluation, respectively.

Animal sacrifice and sample collection

On the last day of the experiment, the animals were fasted overnight and then sacrificed by cervical dislocation. After the sacrifice, the abdomen and scrotal compartments of each animal were opened; the testis and the caudal epididymis were excised, trimmed of fat, and immediately weighed before rinsing in cold normal saline. The testis of each rat was fixed in freshly prepared Bouin's fluid for histological processing.

Epididymal sperm count

Epididymal sperm count of the control and treated animals was determined by the method as described in the WHO manual.[20] An incision was made through the cauda of the right epididymis, light pressure was applied to this region, and sperm was extruded. To 1 ml of Ham's F-10 medium, 10 μl sperm of the control and experimental group were suspended. After centrifugation, 5 μL epididymal sperm was diluted with 95 μL of diluent (2.5 g of sodium bicarbonate, 500 μL of 35% formalin, and 12.5 mg of trypan blue in 50 mL of distilled water) and counted using Neubauer hemocytometer. Live sperms appeared bright and colorless, whereas dead sperms showed blue heads.

Sperm morphology

Assessment for the percentage of morphologically abnormal sperm was carried out using a dry preparation technique.[21] The cauda of the right epididymis was rinsed with 0.5 mL of normal saline (0.9% NaCl) to obtain a sperm suspension. Aliquots of sperm suspension were stained with 2% eosin. Hundred spermatozoa per animal were analyzed microscopically at × 400 magnification and counted spermatozoa with abnormal traits as follows twisted body, detached head, round tails, and abnormal neck.

Tissue processing

Harvested testes were fixed in Bouin's fluid, dehydrated stepwisely in graded ethanol, cleared in xylene, and then infiltrated and embedded in paraffin wax. Five micrometer thickness of the paraffin section was taken from the midportion of each testis and stained with hematoxylin and eosin (H and E), as described by Alturkistani et al.[22] Tissues from which slides were taken were randomly selected and examined under a light microscope at × 100. Photomicrograph of the slides was captured by a camera attached to the microscope and then analyzed for histopathology.

Statistical analysis

The results were analyzed using GraphPad Prism 9.3.0 (GraphPad Software, La Jolla, USA). Data were collected and expressed as mean ± standard deviation statistical analysis between the mean of the control group and treatment group was determined using a one-way analysis of variance. Multiple comparisons were done using Tukey's post hoc test. The mean difference is statistically significant at P < 0.05.


  Results Top


Sperm indices

There was a significant decrease in sperm motility in the FLT-treated group in comparison with the control and GCW-only group [Figure 1]. There was also a significant increase in sperm motility in FLT + GCW Low dose (LD), coadmin, and FLT + GCW High dose (HD) groups (P < 0.05) compared to the FLT-treated group (P < 0.05). Sperm count analysis in [Figure 2] showed a significant decrease in the FLT group in comparison with control and GCW groups. However, there was also a significant increase in sperm count in the FLT + GCW LD, coadmin, and FLT + GCW HD compared to the FLT-treated group. Sperm morphology assessment in [Figure 3] reveals a significant percentage increase in abnormal sperm in the FLT-treated group when compared to the control group. Whereas, in GCW only, FLT + GCW LD, coadmin, and FLT + GCW HD groups, the percentage of abnormal sperm was shown to be significantly reduced compared to the FLT-only-treated group.
Figure 1: Effects of GCW on sperm motility in flutamide-induced prepubertal Wistar rats (a) Significantly different from the control. (P ≤ igni) (b) Significantly different from the Flutamide group. GCW: Green coconut water

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Figure 2: Effects of GCW on sperm count in flutamide-induced pre-pubertal Wistar rats. (a) Significantly different from the control. (P ignifi) (b) Significantly different from the Flutamide group. GCW: Green coconut water

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Figure 3: Effects of GCW on sperm morphology in flutamide-induced prepubertal Wistar rats. (a) Significantly different from the control. (P ignifi) (b) Significantly different from the flutamide group. GCW: Green coconut water

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Testicular histology

Photomicrograph of the testicular section of rats in the Control group, [Figure 4] showed a normal testicular profile, with intact basement membrane cells, distinct seminiferous tubule lumen containing spermatozoa and well compacted normal interstitial recess. However, testicular section of rats treated with FLT only in [Figure 5], showed an abnormal testicular profile, with broaden interstitial recess containing diminished Leydig cells, depleted seminiferous tubule lumen containing scanty sperm cells and densely degraded basement membrane cells, on comparison with the Control, GCW only and other GCW post-treated groups. Testicular photomicrograph of rats treated with GCW only in [Figure 6] also reveals a normal testicular profile comparable to control group. [Figure 7] showed a remarkable recovery in the histoarchitecture of the testis in rats post treated with GCW LD when compared to the FLT only group. Likewise, in [Figure 8], GCW HD was revealed to restore the testicular integrity with evidence of spermatogenic cell series recovery on comparison with the FLT group. [Figure 9] also indicates a restored testicular profile when compared to the photomicrograph of the FLT group.
Figure 4: Photomicrograph of the control group. HE, ×100; Normal histoarchitecture. L- Lumen, IS- Interstitial space with Leydig cells, SS- spermatogenic series and BM- Basement membrane

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Figure 5: Photomicrograph of the FLT group. HE, ×100; L- Lumen with scanty sperm cells, SS- Depleted spermatogenic cells series, SE- Degenerated seminiferous epithelium. Arrow indicating interstitial space with damaged Leydig cells. FLT: Flutamide

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Figure 6: Photomicrograph of the GCW group. HE, ×100; SS- progressive spermatogenic cell series, L- sperm cells present in the lumen, BM- Basement membrane with seminiferous epithelium. Arrow indicating interstitial space with abundant Leydig cells. GCW: Green coconut water

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Figure 7: Photomicrograph of the GCW LD group. HE, ×100; L- lumen containing sperm cells, SS- Spermatogenic cells series. Regenerating seminiferous epithelium (Arrowed). GCW: Green coconut water, LD: Low dose

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Figure 8: Photomicrograph of the GCW HD group. HE, ×100; L- sperm cells in lumen, LC- Leydig cells. Arrow showing spermatogenic cell series recovery. GCW: Green coconut water, HD: High dose

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Figure 9: Photomicrograph of the CO-admin group. HE, ×100. SS- spermatogenic cell series, L- seminiferous tubule lumen with spermatozoa. Intact basement membrane (Arrowed)

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  Discussion Top


The findings from the present study showed that GCW supplementation at 10 ml/kg and 20 ml/kg significantly improved sperm quantity (count) and sperm quality (motility and morphology), and as well ameliorates testicular damage caused by FLT administration on the cytoarchitecture of the testis in prepubertal Wistar rats. Furthermore, the deleterious effects of FLT treatment on sperm function and the histology of the testis demonstrated in this study agree with the report of Oremosu et al.[23]

Semen analysis is a cardinal measure considered for the assessment of male fertility.[24] Results of sperm parameters evaluated in this present study following FLT treatment showed a significant decline in sperm count and sperm motility in FLT-treated rats compared to the control and GCW-treated groups. The observed decrease in sperm count may be attributed to the deleterious effect of FLT on the Leydig cells which produces testosterone needed for spermatogenesis. Furthermore, the decline in sperm motility noticed in this study suggests the antiandrogenic action of FLT on the development and functional growth of the epididymis where the sperm acquires its functional maturation traits. The progressive movement and fertilizing capability are the two maturation traits the sperm acquires within the epididymis.[25] According to Perobelli et al.,[26] FLT was reported to compromise the functional maturation role of the epididymis on the sperm through the accelerated movement of sperm through the epididymis in FLT-treated rats. Sperm morphology has been shown to correlate with an increase in sperm DNA fragmentation.[27] Therefore, the percentage increase in abnormal sperm structure demonstrated in this study may be suggestive of FLT alteration effects on the ultrastructure structures of the sperm as previously reported in the work of Lydka et al.[28]

The ameliorative activities of GCW to improve sperm function in FLT-treated rats in this study may be attributed to the actions of its active phytochemical constituents. Phytochemical screening of GCW for its active constituents shows that GCW is abundant in free amino acids, vitamins, organic acids, inorganic ions, and plant hormones.[29] Its free amino acid and vitamin profiles showed that it is rich in aspartate, arginine, alanine, lysine, and ascorbic acid.[29] Such that isolated supplementation of each of them had been proven by previous studies to remarkably improved sperm quantity and quality. Aspartate is an endogenous amino acid essential for the release of luteinizing hormone and testosterone in human and had been demonstrated to significantly increased sperm concentration and motility in subfertile men.[30] Arginine supplementation had also been reported to significantly improved sperm motility in infertile men without evidence of side effects.[31] L-arginine enhanced male fertility through the synthesis of polyamines in seminal fluid (putrescine, spermidine, and spermine) needed for sperm growth and differentiation, and regulation of nitric oxide essential for sperm motility.[32],[33] Thus, the improvement in sperm counts, sperm motility, and sperm morphology noticed in this study suggests that the ability of GCW to restore spermatogenesis through the revival of testosterone-releasing function of the Leydig cells, as well its impact to reconfer upon the epididymis its functional maturation role of sperm motility. Our findings on this, therefore, lie in consonance with the report of Nair and Rajamohan.[34] on the recovery impact of GCW on sperm parameters in nicotine-intoxicated rats.

Previous studies had reported the role of ascorbic acid dietary supplements in the treatment of male infertility as it significantly increased sperm concentration, improved sperm motility, reduced the number of agglutinated sperm, and protects sperm from endogenous DNA damage that increases the prevalence of abnormal sperm in infertile men.[35],[36] Although this study does not estimate testicular oxidative stress markers, the marked distortion to the histoarchitecture of the testis FLT-treated rats, with obvious depletion of the basement membrane and the spermatogenic series cells may therefore suggest the effect of FLT to induced lipid peroxidation on the basement membrane, thereby giving way for the buildup of free radical species within the seminiferous tubule of the treated rats' testis as posited by Ray et al.[37],[38] Conversely, GCW administration in this study at 10 ml/kg and 20 ml/kg are shown to revive the cytoarchitecture of the FLT-treated rats by enhancing the regeneration of the seminiferous epithelium, proliferation of the spermatogenic cells and cellularity of the interstitial space. GCW is rich in free radical scavengers such as vitamin C and phenolic compounds – catechin and salicylic.[29],[39] Phenolic compounds are phytochemicals well noted as powerful antiperoxidases which counter lipid peroxidation in the biological system by scavenging free radicals and quenching lipid peroxidative side chain through their hydrogen-donating ability.[40] It is upon this thought, we assumed that the testicular cytoarchitectural recovery observed following GCW supplementation in FLT-treated rats may probably be due to this mechanism.


  Conclusion Top


GCW supplementation at a dose of 10 ml/kg and 20 ml/kg significantly improved sperm function parameters and restored testicular damage in FLT-induced prepubertal Wistar rats. Further study is recommended to investigate the impact of GCW on stereological indices and the actual active constituents which underpins its actions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1]



 

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