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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 70  |  Issue : 2  |  Page : 106-112

Biotherapeutic sufficiency of virgin coconut oil on paraquat-mediated reproductive dysfunction


1 Department of Anatomy, Faculty of Basic Medical Sciences, College of Medicine, Alex Ekwueme Federal University Ndufu-Alike Ikwo, Abakaliki, Ebonyi State, Nigeria
2 Department of Medical Physiology, Faculty of Basic Medical Sciences, College of Medicine, Alex Ekwueme Federal University Ndufu-Alike Ikwo, Abakaliki, Ebonyi State, Nigeria

Date of Submission25-Dec-2019
Date of Acceptance29-Apr-2021
Date of Web Publication30-Jun-2021

Correspondence Address:
Dr. Joseph Alo Nwafor
Department of Anatomy, College of Medicine, Alex Ekwueme-Federal University Ndufu-Alike, Ikwo (AE-FUNAI), PMB 1010, Abakaliki, Ebonyi State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JASI.JASI_243_19

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  Abstract 


Introduction: Paraquat (PQ) affects male reproductive health with the risk of infertility. Virgin coconut oil (VCO) has antioxidative and fertility properties. Thus, the histology of the testis and seminal vesicles, spermatic indices, testosterone, malondialdehyde (MDA), and superoxide dismutase level in Wistar rats exposed to PQ and treated with VCO was investigated. Material and Methods: Twenty-four rats (150–200 g) were divided into groupings (A-D) with six rats per group. Control (Group A) had a daily dose of 5 ml/kg of normal saline (NS) throughout the experimental period of 24 days. Group B received NS and oral dose of 12.75 mg/kg of PQ at the last 3 days of the experiment. Groups C and D were treated with 5 ml/kg and 10 ml/kg of VCO, respectively, for 21 days before PQ. The rats were then sacrificed. Blood samples were collected for biochemical analysis and the organs were harvested for histology. Results: There was no significant rise in MDA except in Group B. Groups C and D had significant decreases in sperm cells with unconventional pinhead, contrasting difference in active motile cells and testosterone when compared to Group B (P < 0.05). Normalcy in sperm count and a decrease in sperm cells with headless tails were only seen in Group C (P < 0.05). Histological evaluations showed minimal damage in VCO-treated groups. Discussion and Conclusion: VCO has a promising effect against PQ by modulating the activities of its free radicals in a dose-dependent manner.

Keywords: Fertility, oxidative stress, paraquat, reproductive health, virgin coconut oil


How to cite this article:
Nwafor JA, Uduma-Ukpai C, Okechukwu J, Uchewa O, Ekaikite O, Ikpeze S. Biotherapeutic sufficiency of virgin coconut oil on paraquat-mediated reproductive dysfunction. J Anat Soc India 2021;70:106-12

How to cite this URL:
Nwafor JA, Uduma-Ukpai C, Okechukwu J, Uchewa O, Ekaikite O, Ikpeze S. Biotherapeutic sufficiency of virgin coconut oil on paraquat-mediated reproductive dysfunction. J Anat Soc India [serial online] 2021 [cited 2021 Sep 26];70:106-12. Available from: https://www.jasi.org.in/text.asp?2021/70/2/106/320280




  Introduction Top


Adverse male reproductive health from varying toxicities has been intimately related to infertility.[1] The testis, seminal vesicle, and epididymis are affected sites that are prone to pathological changes since they contribute to mammalian reproducibility.[2] It is believed that fertility challenges are apparently worrisome, especially in societies where birth expectancies are high. Certain agrochemicals like paraquat (PQ) under different trade names are used as a better alternative to manual weeding for their efficacy.[3] However, their usages posit dangers to the reproductive function as reported elsewhere.[4] Redox cycling and intracellular oxidative stress has been implicated in the mechanism of PQ-mediated toxicity.[5],[6] Oxidative stress is an important factor that affects the development of male fertility because of very high rate of cell division and mitochondrial oxygen consumption in testicular tissues.[7] The associated health risks from PQ toxicities result from accidental ingestion and dermal exposures with the intensity of toxicities being dependent on dose.[8],[9] Currently, there are no antidotes for the treatment of PQ poisoning and therapeutic management is mostly supportive and directed toward changing the disposition of the poison.[10] Development of an efficient antioxidant therapy from novel compounds of plant origin may provide the necessary antidote that could attenuate the toxic effects of PQ on reproductive function due to their presumptive adequacies. Virgin coconut oil (VCO) is a plant product consumed as a dietary food. Unlike ordinary coconut oil, VCO is enriched with more polyphenols, tocopherols, sterols, and squalene.[11] Perhaps, this suggests why it has continued to capture recent interest as an antioxidant.[12] From the foregoing premise, it became germane to explore the efficacies of dietary supplementations of VCO on the male reproductive organs in circumstances of PQ toxicity.


  Material and Methods Top


Fresh mature coconuts numbering thirty (30) were gotten from a coconut farm situated in Ikwo Local Government Area of Ebonyi State, Nigeria.

Plant preparation and extraction

The coconut shells were removed and the fleshy endosperms were crushed into a viscous slurry. Five hundred ml of water was added to the slurry obtained and squeezed through a fine sieve to obtain coconut milk. The resultant coconut milk was left for about 24 h to facilitate the gravitational separation of the emulsion.[13],[14] Extraction of VCO was done by adopting a modified wet extraction method described in a previous study.[15] The oil on the top layer was scooped, de-moisturized, and filtered through a fine sieve. It was then stored at room temperature for the experiment.

Preparation of the reproductive toxin

PQ dichloride used as the toxicant was purchased under the trade name Paraeforce, a product of Nanjing Redsun Biochemical Company Ltd. The working solution was constituted within a fume cupboard. 2.5 ml of the stock solution (concentration of 200 g/L) was diluted in 400 ml of distilled water, shaken, and left to stand for some minutes before use.

Experimental animals

Twenty-four (24) Wistar rats (150 g–200 g) were purchased and housed in the Animal Facility of Alex Ekwueme Federal University Ndufu Alike Ikwo, Ebonyi State (AE-FUNAI), Nigeria. The animals were kept in well-ventilated cages under standard conditions. They were fed with rat pellet and drinking water ad libitum. Their weights were taken by the aid of an electronic weighing balance during acclimatization and for the duration of the experiment. Acclimatization period was for 1 week and the experiment lasted for 24 days.

Induction of toxicity

Target organs were induced with toxicity through oral administration of 12.75 mg/kg bwt of PQ chloride. This dose represents 11.20% of the reported oral LD50 of PQ in male Wistar rats.[16]

Dose determination of virgin coconut oil and administration route

An oral dose of 5 ml/kg of VCO with reported health benefit served as the low dose for this present study. A high oral dose of 10 ml/kg of VCO being twice the low dose was equally used in this study. These administered doses were considered safe as the calculated percentages lie below the limit of the established oral LD50 of VCO.[17]

Experimental design

The animals were randomly assigned into six groups of four rats each. Group A served as control that received a daily dose of 5 ml/kg bwt of normal saline (NS) throughout the experimental duration. Group B served as the positive control that received NS as vehicular medium and 12.75 mg/kg bwt of PQ on the last 3 days of the experiment. The animals in Group C were pretreated with 5 ml/kg bwt of VCO for 21 days followed with a toxic dose of PQ. Group D animals were equally pretreated with a high dose of VCO for 21 days before toxic administration of PQ.

Animal sacrifice and sample collection for biochemical evaluations

At the end of the experiment, the animals were fasted overnight, anesthetized with diethyl ether. Following a simple cardiac puncture, blood samples were collected into plain tubes and centrifuged at 3000 rpm for 10 min using a tabletop centrifuge (P/C 03). The sera were separated and stored in aliquots at −25°C for biochemical assays of testosterone, superoxide dismutase (SOD), and malondialdehyde (MDA) using commercially available Analyse Gold Kits. The pelvic area was quickly and carefully incised. The testis and seminal vesicles were harvested, blotted on filter paper, and fixed in formal saline for routine histology.

Semen collection and evaluation of sperm parameters

Prior to fixing the testis, the caudal tip of the epididymis was excised and placed into a Ringer's physiological saline solution. The tissue was minced and left for a few minutes to liberate the spermatozoa into the solution. Sperm count procedure and determination of morphological defect in sperm were done according to an earlier descriptive method.[18] Sperm motility was evaluated following a small drop of semen on a warmed slide, mixed with one drop of warm sodium citrate, and covered with a glass slip. The percentage of sperm cells in a unidirectional progressive movement over a field on a slide was observed, using a research light microscope as previously described.[19] Sperm motility was classified as highly motile sperm (sperms that could be seen to cross the field of view very quickly), slightly motile sperms, and nonmotile sperms. Morphological defects in sperms were classified accordingly.[20],[21] In general, all values were expressed as percentages of normal sperm.

Estimation of testosterone level

This was done using the Testosterone AccuBind Microplate EIA test system following a descriptive procedure.[22]

Estimation of malondialdehyde and superoxide dismutase

The amount of lipid peroxidation using MDA was estimated following an earlier descriptive method.[23] Quantification of SOD was determined in line with known descriptive techniques.[24]

Ethical approval

Guidelines relating to National Institutes of Health Guide for the Care and Use of Laboratory animals (NIH Publications No. 8023, revised 1978) were adhered to.[25]

Data analysis

The data obtained in this present study were subjected to statistical analysis using the Statistical Package for Social Sciences (SPSS) software, manufactured by IBM, Chicago, USA version 23. Values were expressed as mean ± standard deviation for the groupings. Differences in the mean between the groups were analyzed using one-way ANOVA. Statistical significance was considered at P ≤ 0.05.


  Results Top


Comparing the groups [Table 1], there was a significant difference in MDA level between groups that received only PQ (Group B) and control group (Group A) (P ≤ 0.05). SOD was significantly increased in high-dose VCO pretreatment group when the mean value was compared with Group B (P ≤ 0.05). Testosterone level decreased significantly in Group B when the mean value was compared with control (P ≤ 0.05). However, there was a significant increase in testosterone hormone in high-dose VCO pretreatment group when compared to Group B (P ≤ 0.05).
Table 1: Testosterone, SOD and MDA level.

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Sperm morphology showed that among all the abnormalities observed, there was a significant difference in sperm cells presented with pinheads and headless tails between control and Group B (P ≤ 0.05). Although there was also a decrease in pinhead sperm cells of Group D, only low-dose VCO pretreatment group (Group C) had significantly decreased number of sperms with headless tail and pinheads when values were compared with Group B (P ≤ 0.05). A detailed comparison is given in [Table 2].
Table 2: Sperm Morphology

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Differences in the sperm motility and sperm count were observed between the respective groups [Table 3]. Group B had a significantly lowered sperm count when compared with control (P ≤ 0.05). Contrastingly, the sperm count was significantly preserved when a comparison was made between Groups B and C (P ≤ 0.05). The result further showed that sperm motility was not significantly affected in both Groups C and D as otherwise seen in Group B when their mean values were compared.
Table 3: Sperm Motility and Count.

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Histological micrographs

The result of the histology showed that sections of the testis from the Control had normal tissue architecture with numerous and well-organized seminiferous tubules lined spermatogenic cells with normal progression of spermatogenesis [Figure 1]. PQ administration resulted in loss of seminiferous tubules, resorption in the mass of sertoli cells, and spermatogenic cell series with possibilities of an arrest of spermatogenesis [Figure 2]. However, pretreatment with VCO showed an appreciable protection against the adverse effect of PQ in a dose-dependent fashion [Figure 3] and [Figure 4]. Micrographs of the seminal vesicle of control (Group A) showed normal convoluted mucosal folds with mucosal crypts and secretory cells, interstitial spaces, and smooth muscles [Figure 5]. In contrast, micrographs of Group B [Figure 6] administered with PQ only showed severe fragmentations of mucosal folds and other deleterious effects on the seminal vesicle. Mild-to-moderate damage was observed in the histoarchitecture of Group C [Figure 7] and Group D [Figure 8] pretreated with 5 ml and 10 ml/kg VCO, respectively.
Figure 1: Micrograph of control section of the testis show normal architecture with well clustered and numerous convoluted seminiferous tubule (ST) lined by sertoli cell (SC), well-nourished spermatogenic cells (SPC). H & E x400

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Figure 2: Micrograph of section of the testis administered with paraquat only (Group B) shows Apoptotic Seminiferous Tubules (ApST), Shrinked and Unevenly spaced Seminiferous Tubules (marked by yellow star), Interstitial clogs (Icgs) H & E x400

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Figure 3: Micrograph of section of the testis pre-treated with low dose VCO for 21days before paraquat administration (Group C) shows nourished, large and well clustered Seminiferous tubules (L & WCs ST) with preserved spermatogenic appearance. H & E x400

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Figure 4: Micrograph of section of the testis pre-treated with high dose VCO for 21days against paraquat toxicity (Group D) shows Nourished, larger sized and Well clustered seminiferous tubules with preserved spermatogenesis except for mild presence of vascular and interstitial clogs (Icgs) H & E x400

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Figure 5 : Micrograph of Control section of Seminal vessicle (Group A) shows healthy appearance such as numerous convolutional Mucosal folds (MF) with mucosal crypts (MC) and secretory activity, Interstitial spaces (IS), presence of well dispersed smooth muscles (SM) of (x400)(H/E)

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Figure 6 : Micrograph of Seminal vesicle (Group B), show severe alteration of the mucosal foldings (marked by arrows), disoriented and Atrophied Smooth Muscles (ASM), Interstitial Erosion with associated lymphocytic infiltration. H & E x400

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Figure 7 : Micrograph of Seminal vesicle pre-treated with 5ml/kg VCO before paraquat (Group C) show reduced and distorted folding (RDMF) of the mucosal gland, a moderately preserved smooth muscle mass (MPSM) around the gland as well as mild damage to the Interstitium. H & E x400

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Figure 8 : Micrograph of Seminal vesicle pre-treated with 10ml/kg VCO before paraquat (Group D) show improved architecture with welldefined and finely dispersed smooth muscle (WDSM), enveloping the abundant and better organized mucosal folding and cryptic formation. H & E x400

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  Discussion Top


PQ is a toxic agent that affects bodily organs on environmental and occupational exposures.[26] Establishing its reproductive toxicity and providing a potential biotherapy are central to the priority setting of this study. According to the WHO (2010), the preliminary basis for ascertaining male infertility is through clinical evaluation of sperm concentration, morphology, and motility.[27] It is believed that the uptake of reactive metabolites of PQ via processes of ionic mimicry into the tissues critically interferes with the molecular and genetic transcription factors like the HSF2 and OVOL1 expressed in germ cells as well as WT1, RHOX5, and SOX8 expressed in sertoli cells for regulating the development and maturation of sperm cells. The sluggish and nonmotility of the sperms could be due to impairment in the production of sufficient Adenosine triphosphate (ATPs) hat would have otherwise facilitated propulsive movements. Although sperm count and sperm motility are regarded as the first and most important predictors of fertility than sperm morphology, primary morphological defects like acephalic sperms are associated with decreased fertility.[28],[29] Therefore, these sperm cells cannot initiate the acrosome reaction step of fertilization process. This impairment has earlier been associated with disorders in the spermiogenic epithelium which occur in testicular degeneration.[30] The low sperm count, vast depth of nonmotile sperm, and morphological abnormalities mediated by sublethal exposure to PQ without treatment are clear evidence that it could cause a rapid progression to infertility. This finding agrees with an earlier report on the hazards of PQ to sperm.[31]

Histological evaluations of the testis and seminal vesicle in the group exclusively exposed to PQ also corroborate its adverse effect. Besides forming the blood–testis barrier, the sertoli cells otherwise called “nurse” cells respond to FSH to promote spermatogenesis. The degenerated mass of the sertoli cells lining the empty seminiferous tubules may point toward the germ cell aplasia or Del Castillo syndrome. The genomic integrity of these spermatogenic cells may be compromised since reactive oxygen species (ROS) have been reported to affect spermatogonia DNAs.[32],[33] These findings agree with a similar report that PQ is genotoxic as well as cytotoxic in germ cells.[34] Furthermore, the severe spermatogenic arrest observed could be linked to the diminished level of testosterone since the primary role of the male steroid hormone is in spermatogenesis. This finding agrees with a previous report that PQ toxicity alters testosterone levels.[35],[36] The mucosal folds and glands of the seminal vesicle are composed of granular epithelia cells that are active in protein synthesis.[37] Impairment of protein synthesis and channel blockage via PQ-mediated damage will affect the general integrity and functionality of the tissue. The atrophied smooth muscle may suggest impairment in the rhythmic contraction of the muscle to release abundant seminal fluid rich in fructose and prostaglandin that nourishes the sperm cells as they move into the ejaculatory duct during ejaculation.[38] By implication, there is a diminished level or absence of factors that increase sperm motility, factors that suppress the immune response in the female reproductive tract against semen, and factors that clot and then liquefy semen in the vagina. These events culminate in the progression of infertility.

Testicular tissues are susceptible to oxidative stress damage because they have comparably higher levels of unsaturated fatty acids than other tissue. Intense oxidation of lipid membrane is due to increased ROS.[39] This alters the structure, function of the cell membrane, and inadvertently disposes cellular organelles to functional impairment.[40] In the present study, the high lipid peroxidation could be attributed to the excessively generated ROS from PQ in oxidative stress. This lends credence to a previous report on the basis of PQ-mediated damage.[41] The surge in the activity of SOD as seen in VCO high-dose pretreatment group is a pointer toward oxidative stress as it suggests the adaptive responses of the antioxidant in mopping up excess free radicals. This finding in tandem with a previous report on intracellular events associated with oxidative stress.[42]

The minimal damage in the histology of the testis, seminal vesicle, sperm, and biochemical parameters after pretreatments with VCO could be attributed to its tocopherols, polyphenols, and phenolic compounds like p-coumaric acid, protocatechuic acid, vanillic acid, ferulic, caffeic acid, and lauric acid.[43],[44] Being quite profound in the VCO low-dose group, it is believed that these biologically active ingredients are present in proportions that do not distort the cellular homeostatic balances. They may aid to considerably forestall rapid depolarization of the seminiferous membrane for intracellular uptake of reactive metabolites of PQ that could affect sperm development. Consequently, the chances of an occurrence of a progressive nonobstructive azoospermia may be halted since the structural organization and functional role of the sertoli cells, Leydig cells, seminiferous tubules, and accessory organs of reproduction were relatively sustained. This finding appears novel as it demonstrates the benefit of VCO on the seminal vesicle. It may also be related to documented evidences on the benefits of VCO on the testis, as well as other reproductive indices in a dose-dependent fashion.[45],[46]


  Conclusion Top


Reproductive toxicity caused by PQ has remained a health risk of major concern in various populations, especially in West Africa. The treatment options when available are usually expensive and few. VCO has shown a more beneficial effect on male fertility and reproduction at low dose by impacting positively on the functionality of the testicular tissue and seminal vesicle in the release of fluid during ejaculation. This could be attributed to its naturally occurring constituents. Thus, harnessing and developing the pharmacologically active ingredients as therapeutic supplements may protect and promote the reproductive health from adverse risks associated with PQ.

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]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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