THE EFFECT OF COGON GRASS ROOT ETHANOL EXTRACT ON REPRODUCTIVE ORGAN AND EPIDIDYMAL SPERM QUALITY OF MOUSE WITH DIFFERENT AGES

This study aims to determine changes related to epididymal sperm parameters in mice given cogon grass root ethanol extract (CGREE). The experiment was carried out by dividing three groups of mice aged 4-week old (early pubertal), 8-week old (adult), and 24-week old (old) as control and treatment groups. The treatment groups were given 115 mg/kg body weight of CGREE for 14 days. The mice were sacrificed on day 15 to asses reproductive organ indexes and quality of the epididymal sperm. Index of epididymis and ductus deferens indices significantly reduced in the 4-week old treatment group compared to control (0.23 vs 0.33; 0.08 vs 0.16, respectively), and vesical seminal significantly reduced in 8week old treatment group compared to control (0.76 vs 0.68). The epididymal sperm quality significantly decreased in all treatment groups, but the sperm concentration in early pubertal group showed an increase. These results indicate that CGREE can enhance several semen parameters in prepubertal and adult mice but this effect is not apparent in old mice. ____________________________________________________________________________________________________________________


INTRODUCTION
In this modern era, almost all big pharmaceutical companies take part in the mass production of chemically synthesized medicine. However, in some parts of the world especially in developing countries, access to traditional practicing and utilization of herbal medicine is very popular. World Health Organization (WHO) estimates that 80% population of developing countries are using herbs for health care and therapy (De, 1994;WHO, 2002). African, Indian, and Chinese count on traditional medicine up to 90%, 70%, and 40% of their population, respectively (Benzie and Watchel-Galor, 2011). This phenomenon could be due to the affordable price, ease of access, and side effect, and lower toxicity compared to synthetic chemical medicine (Koger and Bryson, 2004;Parkavi et al., 2012). The use of traditional medicine is not limited only to developing countries. In the United States, almost 40% of the adult population and 11.8% of children use traditional medicine as a remedy (Benzie and Watchel-Galor, 2011;Yakubu et al., 2008a).
Many common traditional herbs such as turmeric (Curcuma longa), papaya (Carica papaya), mondia (Mondia whitei), Chinese hibiscus (Hibiscus rosa Sinensis), neem (Azadirachta indica), black pepper (Piper nigrum), and celery (Apium graveolen) are used as supplementary medicine and also have antispermatogenic and anti-fertility for men. Cogon grass (CG) is a herbal medicine that has been traditionally used widely to relieve symptoms occurred in children and adults such as urinary tract infection and intestinal disorders including dysentery and diarrhea (Okwu and Josiah, 2006) myalgia, night sweats, piles, and common cold (Koger and Bryson, 2004) and antibacterial activity (Parkavi et al., 2012).
The previous research reported that CG exhibits various results in infertility (Zhou et al., 2013;Lubis et al., 2018). The difference in results encourages us to evaluate the effect of cogon grass root ethanol extract (CGREE) administration on reproductive organs and epididymal sperm quality of mice at different ages. Finally, the result could enhance the general understanding of whether CG could only affect mice fertility in certain age or all level of ages. This issue may become a consideration as preventive medicine to solve infertility problems.

MATERIALS AND METHODS
Cogon grass was purchased from Solo, Central Java, Indonesia. Cogon grass roots was macerated with 95% ethanol for 72 hours, filtrated with a vacuum filter, and concentrated in a vacuum evaporator. The concentrated extracts was then suspended with 0.5% carboxymethyl cellulose (CMC) and separated to a concentration of 115 mg/kg body weight. The extract was subjected to preliminary phytochemical screening and the results were presented in Table 1.
Thirty six male mice of DDY with different ages (4week old, early pubertal), 8-week old, adult, and 24week old, (old) purchased from PT Bio Farma, Indonesia were used in this experiment. Animals were housed in cages equipped with tap water under 24 hours light and fed with standard feed. The study was approved by the Ethics Review Committee of the Faculty of Medicine, Universitas Padjadjaran (1263/UN6.C10/PN/2017). This study was performed in the Mouse Animal Laboratory, Faculty of Medicine and Laboratory of Animal Reproduction and Artificial Insemination Faculty of Husbandry, Universitas Padjadjaran.
Animals were divided into three groups according to animal ages. Animals in group I, II, and III aged 4week-old (12 mice), 8-week-old (12 mice) and 24week-old (12 mice) respectively. Six of 12 animals in each group were treated orally with CGREE of 115 mg/kg body weight for 14 days, while the rest (six mice) was not fed by CGREE (control). The animal was weighed and sacrificed using anesthetic isoflurane on the day 15. The reproductive organ body weight ratio was calculated according to the previous research (Yakubu et al., 2008b). The cauda epididymis was used to evaluated sperm characteristics.

Epididymal Sperm Concentration
Epididymal sperm was collected by mincing 100 times of the cauda epididymis in 1 mL of NaCl, The 10 µL of semen sample was then diluted with aqua dest up to 300 µL of final solution (dilution factor = 30). Subsequently, 10 µL of the final solution was loaded into hemocytometer and allowed to stand for 5 minutes.
The number of existed sperm in 5 random squares of hemocytometer (SN) was counted on 200x magnification using phase-contrast microscope. Finally, the sperm concentration was estimated by multiply sperm number (SN), multiplication factor (50000) and dilution factor (30).

Sperm Motility
The percentage of motile sperm was evaluated visually in each sample by viewing a drop of sperm suspension diluted with Tris buffer solution (3.63 g of Tris-hydroxymethyl aminomethane, 0.50 g glucose, 1.99 g of citric acid, and 100 mL of distilled water) on a pre-warmed (37 C) slide under a pre-warmed coverslip with a phase-contras microscope at 400x magnification. A total of 200 sperm were counted and categorized as motile and immotile, scored between 0-100%.

Sperm Morphology
Sperm with abnormal morphology have head or tail defects such as no hook and double head or no tail, bent, short tail, and double tail. A drop of eosinnigrosin and sperm were smeared in a glass slide and then allowed to dry and flushed with methanol. A total of 200 sperm cells evaluated using a phase-contras microscope at 1000x magnification.

Data Analysis
All the analyses were carried out using Prism software version 8 (GraphPad Inc, San Diego, CA, USA). Data presented as average ± standard deviation and compared using the T-test. Different in mean was considered at P<0.05.

RESULTS AND DISCUSSION
The extracts obtained by successive solvent subjected to preliminary phytochemical screening, which revealed the presence of alkaloids, flavonoid, steroid, saponin, tannin, and phenol (Table 1). Generally, oral administration of CGREE for 14 days caused a decrease of testis index in all treatment groups ( Figure 1A), although the treatments had no significant effect. The epididymis and ductus deferens indexes was significantly reduced in 4-week old treated animals compared to control (0.23 vs 0.33; 0.08 vs 0.16, respectively, Figure 1C and 1D). On the contrary, the seminal vesicle index showed a different pattern, it tends to increase in all treatment groups and was significantly increased in the 8-week-old treatment group compared to control group (0.76 vs 0.68, Figure 1B). The sperm concentration was significantly increased in the early pubertal treated group compared to control group (0.68±0.25x10 6 /mL vs 7.24±1.92x10 6 /mL, P<0.05, Figure 2A). On the contrary, adult mice treated group showed significant decreased in sperm concentration compared to control (9.02±1.38x10 6 /mL vs 14.57±2.3x10 6 /mL, Figure 2A). Our experiment also indicated a significant decreased in sperm motility of all treatment groups compared to control ( Figure 2B). On the other side, abnormal sperm morphology was increased significantly about two-fold in all treatment groups compared to control ( Figure  2C). Figure 3 presented the detail of abnormal sperm morphology from control and treatment groups. The result showed a decrease in head abnormal morphology in all treated animals compared to control although it was not significant. Interestingly, the neck and tail abnormalitywas increased in all treated animals. The neck abnormality was increased significantly in 4-week old group and the tail abnormality was increased significantly in 8-week old group.
The results also showed that CGREE contained saponin, alkaloid, phenolic, and steroid in their substance. The previous research reported that alkaloids, steroids, phenolic acids, and flavonoids have a noticeable anti-fertility potential (Chakravarty et al., 2003;Russo and Borrelli, 2005;Manthri et al., 2013). Therefore, the presence of saponin, alkaloid, phenolic, and steroid in CGREE, which act either alone or in combination, may play a role as antifertility. The activity of saponin as anti-fertility and abortifacient (Dande and Patil, 2012) as well as sperm immobilization (Dubey and Dubey, 2019) have been sufficiently confirmed in animals model. In the present study, the CGREE was tested for its anti-fertility activities at different age of male mice by observing reproductive organ and epididymal sperm quality.
Moreover, CGREE administration indicated a significant effect on reproductive organ indexes, mainly in early pubertal and adult treatment groups. The phenomena indicated that the administration of CGREE may induce disturbance production and bioavailability of androgen which influences the function and growth of androgen-dependent organs, mainly testes, epididymis, seminal vesicles, and ventral prostate (Klinefelter et al., 2008). There was a pattern that seminal vesicles, testes, and epididymal indexes in a mature animal treatment group was decreased which may correlate with bodyweight gain. The present study showed that the weight of adult and old mice was slightly increased or constant. The seminal vesicle will reach its peak size so the index tends to decline. On the other hand, the early pubertal mice given CGREE had a double-up weight gain compared to control group. The increased in organ weight was followed by an increased in body weight which in turn caused an increased in organ-body weight index.
Generally, the result showed that the epidydimal sperm quality was reduced in all treatment groups compared to control group except for early pubertal and abnormality (C) of control and cogon grass root extract treatment groups after 14 days. * (asterisk) indicates statistically significant differences (P<0.05) between control and treatment groups Figure 3. Sperm abnormalities types of control and cogon grass root extract treatment groups after 14 days. A= Head abnormal morphology, B= Neck abnormal morphology, C= Tail abnormal morphology. * (asterisk) indicates statistically significant differences (P<0.05) between control and treatment groups mice group which showed an increase in sperm concentration. The reduction of sperm concentration may correlate with the decreased of testis index which had detrimental effect on spermatogenesis. The administration of CGREE may induce the disturbance of germ cells in tubules and intra tubules as organs for maintaining sperm production. Reduction in sperm motility in this present study indicated a disturbance in epididymis or disruption during the maturation process. Epididymis damage caused alteration on luminal epididymal fluid containing ion which contributed directly to control cAMP concentration and leads to activation of protein phosphorylation.
The disruption of protein phosphorylation causes the reduction of ATP production that is crucial as energy for sperm movement (Gervasi and Visconti, 2017). The decreased of sperm motility in all treatment groups also strongly correlated to the increased of abnormal sperm morphology, mainly in the neck area where mitochondria are located to provide energy source for the sperm's motility.
The primary defect of sperm such as head abnormal morphology was decreased in all treatment groups compared to control. On the other side, the increased of neck and tail abnormal morphology indicated that the administration of CGREE orally induced secondary sperm abnormality. Secondary sperm abnormality could happen in epididymis where maturation takes place after sperm exit from seminiferous tubules. Based on the results, it might be possible that the disruption of sperm development occured during maturation in the epididymis.
The present research also indicated that the gavage of CGREE in a short time induced alteration of reproductive organs followed by a decreased of epididymal sperm quality. Since CGREE contained alkaloids it had detrimental effect on sperm quality. Yakubu (2012) reported that alkaloid extracts from Chromolaena odorata deteriorate sperm quality and hormones in male mice, decrease sperm count, motility, and density as well as increase abnormal sperm morphology. Kumar and Sachin (2013) also reviewed that plants contained alkaloids have anti-ovulatory, abortifacient, and contraceptive effect. Another study about ginseng (Panax) which contained an abundant of ginsenosides (saponin) and flavonoid caused a significant decrease in total sperm abnormalities, increase in sperm numbers and motility as well as an increase in plasma concentration of testosterone, FSH, and LH (Leung and Wong, 2013). In this present study, combination of these substances in CGREE may deteriorate sperm quality, decrease sperm count and motility, as well as increase abnormal sperm morphology. However, the most affected and accurate change observed in 8-week-old mice (adult). It was predicted that those phenomena may cause by maturation and sufficient function of sexual organs evaluated in this age. A CGREE effect in early mature age (early pubertal group) could be biased because of pubertal changes such as increasing organ weight and hormonal concentration until they physiologically sufficient. On the other side, old mice group organ function was also physiologically deteriorated due to many factors such as external environment exposure, decrease in hormonal production or decrease the sensitivity of hormones receptor thus the CGREE effect may not adequate to revert to its normal value. In conclusion, the most accurate disruption in all parameters observed in 8-week-old mice.

CONCLUSION
The orally administration of CGREE enhance several reproductive organ indexes in prepubertal in adult mice but does not affect the old mice.