Toxicity of Aqueous Wood-Ash Extract of Parkia biglobosa on Male Swiss Albino Mice Timothy Auta Department of Biological Sciences, Federal University Dutsinma, Katsina State, Nigeria E-mail: autatimz@gmail.com; tauta@fudutsinma.edu.ng Phone no:+234(0)8039213141 Presented at the 2nd World Bio Summit & Expo, October 10-12, 2016, Dubai, UAE

INTRODUCTION The use of wood-ash extracts, including that of Parkia biglobosa as food additives and for medicinal purposes by different ethnic groups in Nigeria, without knowledge of its possible reproductive toxicity has been an age long practice (Auta et al., 2015). Recently, there has been growing concern about the deleterious effects of chemicals on male reproductive system (Sharma and Garu 2011). In the male reproductive system, weight loss of the gonads as well as reduced sperm count and epididymal sperm motility are considered standard criteria for the characterization of toxic agents that may cause fertility problems in the treated subject (Ban et al., 1995; Queiroz-Neto et al., 1997). Though there are several reports in literatures on P. biglobosa, there is dearth of information on reproductive toxicity of its aqueous wood-ash extract. Hence, this study reports the damaging effects of aqueous wood ash extract of P. biglobosa on sperms and testicular tissues.

METHODOLOGY Aqueous extraction of the wood ash of P. biglobosa was performed using method described by Auta et al., 2015. Ethical approval was obtained from the University of Ibadan Animal Care and Use for Research Ethical Committee (ACUREC). Four different dose levels of 0, 5, 50 and 100 mg/kg body weight were administered to 20 male mice (five per group) for seven days, which were sacrificed 35 days thereafter. Gonadosomatic index, sperm motility, sperm count, sperm morphology, serum follicle stimulating hormone (FSH), leuitinizing hormone (LH) and testosterone assay, and histopathology of testes were carried out using methods described by Bakare et al., (2009). Data were analysed using descriptive statistics and ANOVA, considered significant at P < 0.05.

RESULTS No significant toxic effect on testicular weight, FSH, LH and testosterone was recorded (Table1). Percentage of abnormal sperm cells increased significantly (Table2). Sperm motility, live/dead sperm and sperm count decreased significantly (Table3), when compared to control. Dose dependent depletion of spermatogenic cells were recorded in the testes (Figure1).

Table 1: Result of Sperm Count and Motility Assay of Mice exposed to aqueous wood ash extract of P. biglobosa Groups Motility (%) Live/Dead (%) Volume(μl) Count (x106/ml) Control 91.5 ± 1.19 a 98 ± 0 a 5.1 ± 0 137.75 ± 1.84 a 5 PB 73.25 ± 2.36 b 97 ± 0.70 ab 116 ± 2.94 b 50 PB 66.75 ± 2.36 b 96 ± 0.71 ab 106 ± 3.49 bc 100 PB 94.25 ± 1.65 b 102.25 ± 5.11 c Values are expressed as Means ± SEM (n = 4 per group). Means in same columns with different superscript letters are significantly different; p<0.05.

Experimental Groups (mg/kg bw) Table 2: Sperm morphology parameters of mice exposed to aqueous wood ash extract of P. biglobosa Parameters Experimental Groups (mg/kg bw) 0 (DW) 5 PB 50 PB 100 PB Tailless head 4.25 ± 0.63a 4.75 ± 0.48 a 4.25 ± 0.63 a 5.0 ± 0.41 a Headless tail 4.25 ± 0.25 a 4.25 ± 0.48 a 4.75 ± 0.63 a 4.5 ± 0.65 a Rudimentary tail 1.75 ± 0.48 a 2.75 ± 0.25 a 2.0 ± 0.41 a 2.5 ± 0.29 a Curved tail 7.0 ± 0.41 a 8.75 ± 0.25 bc 8.0 ± 0.41 ab 9.25 ± 0.25 c Curved midpiece 8.0 ± 0.41 a 8.75 ± 0.63 ab 9.75 ± 0.63 b 10.0 ± 0.41 b Bent midpiece 7.75 ± 0.63 a 9.0 ± 0 b 9.25 ± 0.25 b Looped-tail 1.25 ± 0.25 a 2.0 ± 0.41 ab 2.25 ± 0.25 b 1.75 ± 0.25 ab Bent tail 7.25 ± 0.25 a 9.0 ± 0.41 b 10.0 ± 1.41 b Total number of abnormal sperm 41.5 ± 0.5 49.25 ± 2.17 49.5 ± 1.04 53.0 ± 1.08 Total number of normal sperm 367.25 ± 3.61 355.75± 4.15 350.5 ± 1.04 348.25 ± 1.65 Total number of sperms 408.75 ± 3.15 405.0 ± 2.04 400.0 ± 0 401.25 ± 1.25 % of Abnormal cells 10.16 ± 0.19 a 12.17 ± 0.59 b 12.38 ± 0.26 b 13.21 ± 0.27 b Values are expressed as Means ± SEM (n = 4 per group). Means in same row with different superscript letters are significantly different; p<0.05.

Table 3: Sperm Count and Motility of Mice exposed to aqueous wood ash extract of P. biglobosa Groups Motility (%) Live/Dead (%) Volume(μl) Count (x106/ml) Control 91.5 ± 1.19 a 98 ± 0 a 5.1 ± 0 137.75 ± 1.84 a 5 PB 73.25 ± 2.36 b 97 ± 0.70 ab 116 ± 2.94 b 50 PB 66.75 ± 2.36 b 96 ± 0.71 ab 106 ± 3.49 bc 100 PB 94.25 ± 1.65 b 102.25 ± 5.11 c Values are expressed as Means ± SEM (n = 4 per group). Means in same columns with different superscript letters are significantly different; p<0.05.

A B C D Figure 1: Photomicrograph of mice testes exposed to aqueous wood ash extract of P. biglobosa. Control (A) with numerous regular variably sized ST packed full with normal spermatogenic cells. B (5 mg/kg) shows closely packed, numerous and large STs with moderate amounts of spermatogenic cells. However, there are a few STs (arrows) which are depleted of spermatogenic cells. C (50 mg/kg) showing variably-sized STs (arrows) with irregular outline and contain moderately depleted amounts of spermatogenic cells. The usual gradation from basal to apical/lamina compartment is generally absent. D (100 mg/kg) shows tunica albuginea (star) is thickened. There are variably-sized STs with irregular outlines and severely depleted (arrows) amounts of spermatogenic cells suggestive of testicular atrophy. H&E. 100X (B-D) 400X (A). ST: seminiferous tubules

DISCUSSION In general, damage to the sperm cell is said to occur either by physiological, cytotoxic or genetic mechanism (Otitoloju et al., 2010). Exposure to the extract could have produced pituitaryhypothalamic or sex hormonal effects which in turn affected spermatogenesis; it might have resulted to abnormalities in seminal fluid leading to functional or structural impairment of sperm (Odeigah, 1997). The decrease in sperm viability (live/dead), progressive sperm motility and sperm count could be due to the effect of this extract on the epididymis by acting as a spermatoxic agent on maturing or matured spermatozoa (Pacific et al., 1995). The dose-dependent increase in percentage of sperm head abnormalities suggests aqueous wood ash extract of P. biglobosa have caused damage to the premeiotic stages of spermatogenesis; during spermatogenesis, DNA synthesis occurs before pre-meiotic phase and no further DNA synthesis occurs throughout spermatogenesis in the cell cycle (Odeigah, 1997).

CONCLUSION Parkia biglobosa extract depletes sperm motility, live/dead sperms and causes increase in the number of abnormal sperm cells, increasing possibilities of infertility. Aqueous wood-ash extract of Parkia biglobosa had damaging effects on sperm cells and testicular tissues, which could compromise reproductive potentials.

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