Histomorphometric studies of the effects of Telfairia occidentalis on alcohol-induced gonado-toxicity in male rats.

BACKGROUND: Available evidence suggests that 50% of couples with infertility are male related. Over 40% of these males consume alcohol which has been reported to be a reproductive toxicant causing depletions in the epithelium of seminiferous tubules hence reducing sperm counts and sperm morphology.
OBJECTIVE: To determine the effects of aqueous leaf extract of Telfairia occidentalis on alcohol-induced cyto-architectural changes in the testis.
METHODS: Aqueous leaf extract of Telfairia occidentalis (T. occidentalis) was administered by gastric gavage at a dose of 250 mg/kg and 500 mg/kg body weight daily, while 2 g/kg body weight of ethanol at 30% v/v was administered daily to mature male Sprague-Dawley rats. The experiment was in 2 phases. Phase 1 had groups A1-F1 and lasted for 4 weeks while phase 2 had groups A2-F2 and lasted 8 weeks. Parameters tested include: testicular histology, relative volume density, sperm parameters, malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione.
RESULTS: In both phases, there were depletions in the seminiferous epithelium, decreased sperm quality and increased MDA and SOD in animals that received alcohol only compared to control. Likewise, a significant increase of seminiferous epithelium of animals that received respective doses of 250 mg/kg and 500 mg/kg of T. occidentalis only compared to control. Animals that received T. occidentalis and alcohol simultaneously had a significant increase in seminiferous epithelium and sperm quality with decreased MDA level.
CONCLUSION: T. occidentalis attenuated the deleterious effects of alcohol to the cyto-architecture of the testis, protected the seminiferous epithelium, reduced oxidative stress and promoted spermatogenesis.

Introduction

To most couples the desire to have one’s own biological children is strong and compelling. The effects of infertility on these couples can be devastating. Infertility leads to psychological stress, anxiety and depression Centers for Disease Control and Prevention [CDC], 2012 [11]. There are over 186 million couples in developing countries alone (excluding China) who are affected by infertility [51]. Rates of infertility vary considerably from country to country; in the worst-affected areas, over 25% of couples may be unable to have children [39].

On a practical level, many families in developing countries depend on children for economic survival. So, while many people would not consider infertility a disease in itself, it is certainly a social and public health issue as well as an individual problem [51].

In Nigeria, the data on infertility indicates that disorders in males and females account for an equal proportion of infertility with the male factor being associated with a greater percentage of primary infertility [33]. Available evidence reveal that male factor infertility has not been given due prominence in issues of reproductive health [21], [38], [41].

[38], had attributed aspects of male infertility to alcohol consumption. Records have it that, over 42% of males with infertility are alcohol consumers [12], [38]. Several studies have shown that alcohol impairs sexual performance and desire. It also decreases testosterone levels, sperm count and sperm motility contributing to fertility problems [50], [6]. Studies on humans have even shown that heavy consumption of alcohol adversely affects sperm count [20], [25]. In addition, there is evidence suggesting that long term use of alcohol will lead to permanent damage of the male gonad, including a permanent reduction in the growth and maturation of male gametes [50], [52]. This may be due to the fact that alcohol atrophies the cytoplasmic mass of Leydig cells responsible for the secretion of the male sex hormone – testosterone [50]. Any decrease in testosterone will adversely affect spermatogenesis [47].

In spite of these deleterious effects, alcoholic beverages are still widely consumed domestically and socially. In our previous study, animals administered alcohol showed a correlation between decreased testosterone levels and increased oxidative stress [17]. This suggests that alcohol may be acting through oxidative stress-inducing mechanisms. Therefore, our hypothesis is that reduction in alcohol-induced oxidative stress will enhance fertility.

Antioxidants such as flavonoids, ascorbic acid and tocopherol have been reported to significantly reduce oxidative stress and improve sperm parameters [15], [34]. These antioxidants and other phytochemicals have been reported present in Telfairia occidentalis (T. occidentalis) [23].

T. occidentalis is an important staple vegetable consumed in Nigeria and several other countries. The leaves are rich in minerals (such as calcium, potassium, sodium, phosphorus and magnesium), iron, antioxidants, vitamins (such as thiamine, riboflavin, nicotinamide, ascorbic acid, retinol and tocopherol). and phytochemicals such as phenols, Tannins, and Flavonoids [23], [28], [30]. It has a high profile of aminoacids which include; alanine, aspartate, glycine, glutamine, histidine, lysine, methionine, tryptophan, cystine, leucine, arginine, serine, threonine, phenylalanine, valine, tyrosine and isoleucine [49], [23]. In view of these profertility components of T. occidentalis, this study is aimed at determining the effects of T. occidentalis on alcohol-induced cyto-architectural changes in the testis.

Materials and methods

Extract preparation

Healthy T. occidentalis plant was obtained from a local market in Lagos, Nigeria. It was authenticated in the Department of Botany of the University of Lagos and a specimen was deposited in the herbarium with a voucher number – LUH 2763. One kilogram (1 kg) of freshly cut T. occidentalis leaves with the stalk was separated from the stem, washed with clean water to remove dirt and sand. The water was drained and the leaves chopped. This was then squeezed and filtered with sieve to obtain an aqueous extract of the T. occidentalis leaves. The aqueous leaf extract was prepared at intervals of 3 days, refrigerated and served to the animals fresh [40].

Administration of extract and Alcohol

Aqueous leaf extract was administered orally by gastric gavage at a dose of 250 mg/kg and 500 mg/kg body weight daily Saalu et al. [44], while 2 g/kg body weight of ethanol was administered orally daily at a concentration of 30% v/v [17].

Experimental animals

A total of 60 adult male Sprague–Dawley rats weighing between 170 and 200 g were randomly selected from the Nigerian Institute of Medical Research (NIMR) located in Yaba, Lagos. The animals were housed in the Laboratory Animal Center, College of Medicine, University of Lagos. The animal house was well ventilated with a temperature range of 28–32 °C under day/night 12–12 h photoperiodicity. The rats were fed with standard rat chow (Pfizer Nig Ltd). They had access to water ad libitum. The experiment was in 2 phases. Each phase had 6 groups (A- distilled water, B- 30%v/v alcohol, C- 250 mg/kg of T. occidentalis D-500 mg/kg of T. occidentalis, E-250 mg/kg of T. occidentalis + Alcohol, F-500 mg/kg of T. occidentalis + Alcohol) with 5 rats per group. The first phase was for 4 weeks and had groups ranging from A1 to F1 while the second phase was for 8 weeks and had groups ranging from A2 to F2. All experimental protocols followed the guidelines approved by the Ethics Committee of the College of Medicine, University of Lagos, Nigeria.

Sperm count

This was done using the new improved Neubauer‘s counting chamber (Hematocytometer). The epididymal fluid was diluted with normal saline solution by adding 0.9 ml to 0.1 ml of the crushed epididymis. This chamber was then filled with sperm fluid and placed under a binocular light microscope using an adjustable light source. The ruled part was then focused and the number of spermatozoa counted in five 16-celled squares Keel and Webster, [32].

Sperm motility

The sperm motility analysis was carried out at room temperature using one epididymis of each rat. The percentage of sperm motility was calculated using the number of live sperm cells divided by the total number of sperm cells (motile and non-motile), from two samples per epididymis of each rat. All sperm cells that were not moving at all were considered to be non - motile, while the rest, which displayed some movement, were considered to be motile [53].

Testicular malondialdehyde (MDA) and testicular antioxidant enzymes

The lipid peroxidation products were estimated by measuring TBARS and were determined by modifying the method of Niehaus and Samuelson [36]. Antioxidants such as reduced glutathione (GSH) and Superoxide dismutase (SOD) were estimated by employing modified methods of [18] and [43], respectively.

Testicular histology preparation

The histology of the testes was done by modification of method reported by Kayode et al. [31]. The organs were harvested and fixed in Bouin‘s fluid for 24 h after which it was transferred to 70% alcohol for dehydration. The tissues were passed through 90% and absolute alcohol and xylene for different durations before they were transferred into two changes of molten paraffin wax for 1 hour each in an oven at 65 °C for infiltration. They were subsequently embedded and serial sections cut using rotary microtome at 5 microns. The tissues were picked up with albumenized slides and allowed to dry on hot plate for 2 min. The slides were dewaxed with xylene and passed through absolute alcohol (2 changes); 70% alcohol, 50% alcohol and then to water for 5 min. The slides were then stained with haematoxylin and eosin. The slides were mounted in DPX. Photomicrographs were taken at a magnification of x400.

Phytochemical screening

Qualitative and Quantitative phytochemical analysis of the aqueous leaf extract of T. occidentalis was done in accordance with Soni and Sosa [48]. While modifications on the report by Grindberg and Williams [27] on high performance liquid chromatography was adopted to quantify the vitamins.

Morphometry

The primary aim was to estimate the volumes of seminiferous tubule epithelium (seminiferous epithelium) and interstitium in the testis. This was done in accordance with Howard and Reed [29] and Baines et al. [8]. Four sections per testis, and six microscopical fields per section, were randomly chosen for analysis. Fields were sampled as images captured on a Leica DM750 brightfield microscope (Germany) via LAZ software. Volume densities of testicular ingredients were determined by randomly superimposing a transparent grid comprising 35 test points arranged in a quadratic array. Test points falling on a given testis and its ingredients were summed over all fields from all sections. The total number of points hitting on a given ingredient (lumen (EL), epithelium (EE), interstitium (EI)), divided by the total number of points hitting on the testis sections (ET) multiplied by 100, provided an unbiased estimate of its%volume density/volume fraction.

Statistics

The sperm parameters were log transformed to improve normality of data. Differences between groups were compared using one way ANOVA and LSD post hoc test. This was done using the SPSS. The data was expressed as mean ± standard deviation [4].

Results

Sperm motility and sperm count analysis

After the first phase of the study, there was a significant decrease in sperm motility of group B1 when compared to groups A1, C1, D1, E1 and F1 (p < 0.05). There was also significant decrease in groups E1 and F1 compared to group A1. The sperm count of group B1 decreased significantly compared to groups A1, C1, D1, E1 and F1 (p < 0.05; Table 1).

Table 1

Sperm motility and sperm count after 4 weeks.

Groups	Description	Motility (%)	Count(106/ml)
A1	Control (distilled water)	98.5 ± 0.71^	88.3 ± 9.90^
B1	Alcohol (30%v/v)	42.0 ± 1.41*	24.7 ± 1.27*
C1	250 mg/kg of T. occidentalis	93.5 ± 4.95^	95.9 ± 3.82^
D1	500 mg/kg of T. occidentalis	96.5 ± 2.12^	91.1 ± 1.70^
E1	250 mg/kg of T. occidentalis + Alc	61.5 ± 9.19*^	69.8 ± 13.4^
F1	500 mg/kg of T. occidentalis + Alc	75.5 ± 6.36*^	86.4 ± 8.70^

v: volume; T. occidentalis: Telfairia occidentalis

Values are expressed as mean ± Standard deviation.

*p < 0.05 compared with A1; ^p < 0.05 compared with B1. One-Way ANOVA.

After the second phase of the study, the sperm motility showed a significant decrease in group B2 compared to group A2. There was also a significant increase in groups C2, D2, E2 and F2 compared to group B2. In the sperm count, there was a significant decrease in sperm count of group B2 compared to groups A2, C2, D2, E2 and F2. There was also a significant increase in groups C2 and F2 compared to group A2 (p < 0.05; Table 2).

Table 2

Sperm motility and sperm count after 8 weeks.

Groups	Description	Motility (%)	Count (106/ml)
A2	Control (Distilled water)	67.5 ± 3.54	48.95 ± 1.06
B2	Alcohol (30%v/v)	32.5 ± 3.54*	31.2 ± 1.27*
C2	250 mg/kg of T. occidentalis	71.0 ± 1.41^	79.25 ± 1.48*^
D2	500 mg/kg of T. occidentalis	57.5 ± 3.54^	56.05 ± 2.62^
E2	250 mg/kg of T. occidentalis + Alc	62.5 ± 3.54^	57.55 ± 3.61^
F2	500 mg/kg of T. occidentalis + Alc	67.5 ± 2.12^	77.4 ± 3.39*^

v: volume; T. occidentalis: Telfairia occidentalis.

Values are expressed as mean ± Standard deviation.

* p <0.05 compared with A2; ^p < 0.05 compared with B2. One-Way ANOVA.

MDA and antioxidant enzymes

MDA levels increased significantly in group B1 when compared to groups A1, C1, D1, E1 and F1 in the first phase of the study. The SOD levels of group D1 decreased significantly when compared to group B1. There was no significant difference in GSH when compared to groups A1 or B1 (Table 3).

Table 3

MDA and antioxidant enzymes analysis after 4 weeks.

Groups	Description	MDA (nmol/ml)	SOD (min/m/protein)	GSH (μmol/ml)
A1	Control (distilled water)	6.87 ± 0.19	107.71 ± 5.20	0.063 ± 0.03
B1	Alcohol (30%v/v)	12.10 ± 0.52*	120.66 ± 3.92	0.042 ± 0.03
C1	250 mg/kg ofT. occidentalis	6.34 ± 0.29^	113.01 ± 15.80	0.101 ± 0.09
D1	500 mg/kg ofT. occidentalis	5.92 ± 0.76^	82.23 ± 23.84^	0.021 ± 0.01
E1	250 mg/kg ofT. occidentalis + Alc	7.54 ± 0.38^	125.31 ± 10.49	0.031 ± 0.03
F1	500 mg/kg ofT. occidentalis + Alc	6.21 ± 0.47^	114.64 ± 4.74	0.018 ± 0.01

v: volume; T. occidentalis: Telfairia occidentalis; MDA: Malondialdehyde; SOD: Superoxide dismutase; GSH: Glutathione reductase

Values are expressed as mean ± Standard deviation.

*p < 0.05 compared with A1; ^p < 0.05 compared with B1. One-Way ANOVA.

In the second phase of the study, there was a significant increase in MDA level of group B2 when compared to groups A2, C2, D2, E2 and F2. SOD levels in groups B2 and C2 increased significantly compared to group A2. The GSH levels of groups E2 and F2 increased significantly compared to group B2. Also, there was a significant increase in GSH of group F2 compared to group A2 (Table 4).

Table 4

MDA and testicular antioxidant enzymes analysis after 8 weeks.

Groups	Description	MDA (nmol/ml)	SOD (min/mg/protein)	GSH (μmol/ml)
A2	Control (distilled water)	9.42 ± 0.95	32.27 ± 0.91	0.154 ± 0.01
B2	Alcohol (30%v/v)	16.75 ± 2.90*	54.07 ± 15.3*	0.136 ± 0.06
C2	250 mg/kg ofT. occidentalis	8.68 ± 0.48^	58.93 ± 14.16*	0.195 ± 0.03
D2	500 mg/kg ofT. occidentalis	8.75 ± 0.22^	39.58 ± 5.87^	0.229 ± 0.13
E2	250 mg/kg ofT. occidentalis + Alc	9.70 ± 1.90^	39.56 ± 4.43^	0.275 ± 0.04^
F2	500 mg/kg ofT. occidentalis + Alc	9.63 ± 0.86^	35.4 ± 2.36^	0.308 ± 0.00*^

v: volume; T. occidentalis: Telfairia occidentalis; MDA: Malondialdehyde; SOD: Superoxide dismutase; GSH: Glutathione reductase

Values are expressed as mean ± Standard deviation.

*p < 0.05 compared with A2; ^p < 0.05 compared with B2. One-Way ANOVA.

Testicular histology

Cross section of the testis of animals after the first phase of the study showed that groups A1, C1, D1, E1 and F1 had a normal cellular composition in their germinal epithelium with sperm cells in the lumen and a normal interstitium. However, animals in group B1 that received alcohol only, showed a marked depletion of spermatogenic cells as depicted in Fig. 1.

Fig. 1

Photomicrographs of testis (×400) after 4 weeks of administration, showing normal cellularity in germinal epithelium (GE), lumen (L) filled with sperm cells and interstitial cells of Leydig in the interstitium (I) in groups A1 (control), C1 (250 mg/kg body weight of T. occidentalis), D1 (500 mg/kg body weight of T. occidentalis) and E1 (250 mg/kg body weight of T. occidentalis + Alc) and F1 (500 mg/kg body weight of T. occidentalis + Alc). Group B1 (alcohol 30%v/v) showing depleted spermatogenic cells of the GE.

Cross section of the testis of animals after the second phase of study showed that groups A2, C2 and D2 showed normal cellular composition with no obvious aberrations compared to group B2 that showed marked depletion of spermatogenic cells with a widened and a hypo-cellular interstitium (Fig. 2). Groups E2 and F2 showed normal cellularity in the germinal epithelium but a widened and hypocellular interstitium (Fig. 2).

Fig. 2

Photomicrographs of testis of second phase (×400). A2 (control), C2 (250 mg/kg body weight of T. occidentalis), D2 (500 mg/kg body weight of T. occidentalis) showing normal GE. Groups E2 (250 mg/kg body weight of T. occidentalis + Alcohol), F2 (500 mg/kg body weight of T. occidentalis + Alcohol). Group B2 (alcohol 30%v/v) showing depleted spermatogenic cells in the germinal epithelium (GE) after 8 weeks of administration.

Morphometric (stereological) analysis

After the first phase of the study, the volume density of the germinal epithelium of group A1 had no significant difference with group B1. However, there was a significant increase in groups C1, D1, E1 and F1 when compared to groups A1 and B1. The lumen density significantly decreased in group B1 compared to group A1. The interstitium had a significant increase in group B1 compared to A1. There was also a significant decrease in the interstitium of groups C1, D1, E1 and F1 compared to group B1 as reported in Table 5.

Table 5

Morphometric analysis of testis after 4 weeks administration.

Groups	Description	Germinal epithelium (%)	Lumen (%)	Interstitium (%)
A1	Control (distilled water)	63.70 ± 0.99	15.00 ± 0.71	21.25 ± 0.35
B1	Alcohol (30%v/v)	62.60 ± 0.14	10.24 ± 1.08*	27.10 ± 1.27*
C1	250 mg/kg of T. occidentalis	69.20 ± 2.12*^	11.89 ± 1.54	18.90 ± 0.56^
D1	500 mg/kg of T. occidentalis	69.55 ± 1.06*^	12.83 ± 2.93	17.35 ± 3.75^
E1	250 mg/kg of T. occidentalis + Alcohol	71.98 ± 0.46*^	11.95 ± 1.34	16.05 ± 1.77*^
F1	500 mg/kg of T. occidentalis + Alcohol	68.05 ± 0.92*^	11.40 ± 0.99	20.50 ± 1.84^

v: volume; T. occidentalis: Telfairia occidentalis

Values are expressed as mean ± Standard deviation.

*p < 0.05 compared with A1; ^p < 0.05 compared with B1. One-Way ANOVA.

After the second phase of the study, there was no significant difference in the volume density of the germinal epithelium of groups A2 and B2. However, the volume density of the germinal epithelium of group B2 decreased compared to group A2 (p > 0.05). There was a significant increase in groups C2, D2 and E2 when compared with group B2. The volume density of the lumen also showed no significant difference between groups A2 and B2 but there was a significant decrease in groups D2 and E2 compared with group B2. The interstitium of group B2 increased compared to A2. There was a significant decrease in groups C2, D2 and E2 when compared to group B2 (Table 6).

Table 6

Morphometric analysis of testis after 8 weeks.

Groups	Description	Germinal epithelium(%)	Lumen (%)	Interstitium (%)
A2	Control (distilled water)	61.09 ± 3.41	14.60 ± 0.41	24.33 ± 3.78
B2	Alcohol (30%v/v)	50.50 ± 0.71	16.50 ± 0.14	37.05 ± 6.43
C2	250 mg/kg of T. occidentalis	64.25 ± 1.06^	13.95 ± 0.78	21.80 ± 0.28^
D2	500 mg/kg of T. occidentalis	64.90 ± 0.14^	13.30 ± 0.14^	21.80 ± 0.28^
E2	250 mg/kg of T. occidentalis + Alcohol	65.15 ± 0.21^	13.70 ± 0.42^	21.10 ± 0.71^
F2	500 mg/kg of T. occidentalis + Alcohol	50.80 ± 4.53	14.20 ± 1.70	35.00 ± 2.83

v: volume; T. occidentalis: Telfairia occidentalis.

Values are expressed as mean ± Standard deviation.

*p < 0.05 compared with A2; ^p < 0.05 compared with B2. One-Way ANOVA.

Qualitative analysis of T. occidentalis leaves shows the presence of flavonoids, tannins, phlobatannins, terpenoids, cardiac glycoside, saponins, alkaloids and steroids (Table 7). After the quantitative analysis as depicted in Table 8, total saponins, total phenol and total flavonoids had higher values compared to total taninins and the total alkaloids present. There were also high values of vitamins A, C and E (Table 8).

Table 7

Qualitative Phytochemical Analysis of Aqueous Extract of Telfairia occidentalis.

S/No	Phytochemicals	Status
1	Flavonoids	+
2	Tannins	+
3	Phlobatannins	+
4	Terpenoids	+
5	Cardiac glycosides	+
6	Alkaloids	+
7	Steroids	+
8	Saponins	+
9	Coumerins	–
10	Quinones	–

Table 8

Quantitative phytochemical analysis of aqueous extract of Telfairia occidentalis.

S/No	Phytochemicals	Quantity
1	Total Phenols (%)	15.36
2	Total Alkaloids (%)	5.62
3	Total Saponins (%)	16.2
4	Total Flavonoids (%)	12.36
5	Total Tannins (%)	7.33
6	Vitamin C (mg/g)	3.06
7	Vitamin A (mg/g)	3.28
8	Vitamin E (mg/g)	2.46

Discussion

At the end of both phases of this study, alcohol significantly reduced sperm count and sperm motility by subjecting the spermatozoa to increased oxidative stress-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids (PUFAs) [5], [9] and their cytoplasm contains low concentrations of scavenging enzymes [45], [46]. Increased formation of ROS has been correlated with a reduction of sperm motility [2], [7]. The link between ROS and reduced motility may be due to a cascade of events that result in a rapid lose of intracellular ATP leading to axonemal damage and sperm immobilization [9], [16].

It was also observed that malondialdehyde (MDA) a by-product of lipid peroxidation [17], [45] was increased in the animals that received alcohol only. This depicts an increase in lipid peroxidation. This study further showed that an increased lipid peroxidation elicited an increased production of superoxide dismutase to neutralize the effects of the free radicals. This is in consonant with the findings by [17] but contradicts that of [37] and [19] who reported that acetaldehyde a product of the biotransformation of alcohol in situ might harm testicular function indirectly by decreasing antioxidant defenses. The observed increase in MDA with animals that consumed alcohol alone may also infer that an increase in SOD alone may not suffice for effective testicular antioxidant defense against lipid peroxidation.

This study also demonstrated that T. occidentalis increased testicular antioxidant enzymes and decreased MDA levels when administered alone. It also prevented the ravaging effects of alcohol on sperm count, sperm motility and testicular antioxidant enzymes when administered with alcohol. The findings from this study has shown that T. occidentalis is rich in antioxidant constituents such as flavonoids, saponins, vitamin E, vitamin C and vitamin A. This is in concordance with reports form Kayode and Kayode [30]. Therefore, it is plausible deducing that these rich antioxidant constituent of T. occidentalis boosted the testicular non-enzymatic and enzymatic antioxidants to effectively scavenge the free radicals preventing lipid peroxidation. The consequence is hereby reflected in the increased sperm count and sperm motility. This finding is in concordance with the reports by [42] and [9]. Moreso, vitamin E, a chain-breaking, non-enzymatic antioxidant also found in T. occidentalis inhibits lipid peroxidation in membranes by scavenging peroxyl (RO•) and alkoxyl (ROO•) radicals [45]. The ability of vitamin E to maintain a steady state rate of peroxyl radical reduction in the plasma membrane depends on the recycling of vitamin E by external reducing agents such as ascorbate (present in T. occidentalis) or thiols Saleh and Agarwal, 2002 [45]. The improved sperm parameters are also attributed to the amino acid content of T. occidentalis [23]. Amino acids such as alanine, glycine, cystine and arginine which are present in T. occidentalis have been reported to preserve sperm cells and improve their motility [10], [3]

In this study, alcohol depleted spermatogenic cells and reduced the volume density of the germinal epithelium. This is in concert with our previous study [3] and the study by [35] who reported that ethanol significantly inhibited the proliferative activity of the spermatogonia in all stages of the seminiferous tubules cycle, it significantly decreased the seminiferous tubules diameter, degenerated germ cells and decreased the number of Leydig cells. For decades, it has been made clear that testosterone which is produced by the interstitial cells of Leydig is a necessary prerequisite for the maintenance of established spermatogenesis [54]. The reduced cellularity of the interstitium in testis of animals that treated with only alcohol would consequently lead to a decrease in testosterone resulting in the poor spermatogenesis observed.

T. occidentalis aqueous leaf extract also maintained the histoachitecture of the testis, increased the proliferative activity of spermatogonia and maintained the volume density of the interstitium compared to the control animals. T. occidentalis has been reported to be rich in carotene which reduces lipid peroxidation [22], [24]. From our observation, when T. occidentalis aqueous leaf extract was administered concomitantly with alcohol; it protected the testis from the pernicious effects of alcohol. This protective nature of T. occidentalis is enhanced by some of its phytochemical constituents: the presence of ascorbic acid which is known for its protection on cell membranes and its scavenging effects on free radicals [22], [9]. In clinical trials, vitamin E supplementation has been found to increase fertilization rates possibly by improving membrane integrity, reducing oxidative damage and lipid peroxidation potential [26], [14]. It is plausible that the antioxidants and micronutrients example zinc and folates in T. occidentalis [24] militated against the ravaging effects of alcohol on the testis. Moreover, Aitken and Roman [1], reported that flavonoids, vitamins A, C and E promote spermatogenesis. Hence, the increase in the volume density of the germinal epithelium of animals administered only T. occidentalis is a consequence of the positive effects of its multivitamins and phytochemicals. Albeit, the reason why T. occidentalis when administered with alcohol was more effective at 250 mg/kg per body weight as compared to 500 mg/kg per body weight remains indefinable although Iron – pro-oxidant may be a plausible culprit [13].

In conclusion, alcohol depleted the germinal epithelium and caused widening and hypocellularity of the interstitium. T. occidentalis however, did not only promote germinal epithelial growth but protected the cyto-architecture of the testis from the damaging effects of alcohol. Hence, T. occidentalis leaves augments spermatogenesis and attenuates alcohol-induced oxidative stress via an antioxidant system of activities.