Experimental Study On Spermicidal Effect In Vitro And The Mechanism Of N-butanol Extract Of Rhynchosia Volubilis Lour

Objective: To study on the spermicidal effects of BERVL in vitro.Methods: The change of sperm motility was determined under a microscope in2minutesafter50μl semen was mixed with200μl BERVL at room temperature. At least200sperm was counted for each sample.Results: No motile sperm was observed in the group mixed with250mg/ml BERVL.And in groups of125mg/ml and100mg/ml as well as90mg/ml, we could see(2.340±1.379)%and (2.412±1.193)%and (4.730±3.231)%of motile sperm wasobserved, respectively.Conclusions: BERVL has spermicidal function. In dose of90mg/ml, we couldn’t see anysperm moving forward, and we presume90mg/ml is minimum spermicidalconcentration in vitro. Objective: To study BERVL’S impact on lactobacillus acidophilus in vitro, and evaluatingthe security of BERVL as spermicides.Methods: AGAR dilution method: To inoculate100μl prepared Lactobacillus acidophilusCGMCC1.1854with200μl of BERVL in the diameter of60mm Petri dishes, thenadded some MRS agar. After mixing together, MRS agar plates were incubated18-24h at37℃to observe results. Tubes dilution method: Marked in15ml tubesand added4800μl MRS, then100μl of the prepared Lactobacillus acidophilusCGMCC1.1854and200μl of BERVL were metastasized to tubes, incubated18-24h at37℃to observe results after mixing well.Results: AGAR dilution method: In groups of500,250,125,100,90,62.5mg/ml,theinhibitory rate to lactobacillus acidophilus CGMCC1.1854was100%,89.19%47.99%,40.97%,32.96%and20.56%; Tubes dilution method: In groups of500mg/ml and250mg/ml,100%of lactobacillus acidophilus CGMCC1.1854hasbeen inhibited in growing. And in group of125mg/ml，100mg/ml,90mg/ml and62.5mg/ml, we found the inhibitory rate separately was50%,45.54%,35.7%and21.4%.Conclusions: Both in AGAR and tubes dilution methods, we found BERVL had inhibitoryeffect on lactobacillus acidophilus. And in different concentrations ofBERVL, the inhibitory rate to lactobacillus acidophilus was different. Many plant’s extract spermicidal agents induce disruption of the plasma membranethrough inhibiting the specific enzymes in sperm membrane. sperm apoptosis is animportant feature during sperm happen process，and the auto-apoptosis is very important tosustain the normal population. Induce too much apoptosis is a kind of abnormalpathological phenomena, which is close to male infertility. So in the present study, wedetected sperm membrane capacity by Eosin Y Staining Experiment and useImmunohistochemical test to evaluate sperm apoptosis and necrosis, as to explore themechanism of BERVL spermicidal activity.Experiment1: Eosin Y Staining ExperimentObjective: Studying on the impact on sperm membrane of BERVL by Eosin Y Stainingexperiment. To explore its mechanism of spermicidal effects in vitro.Methods: First，10of differences semen50μl were mixed with200μl BERVL in dose of125,100and90mg/ml. Then Eosin Y mixed with the sperm suspension at ratioof1:1. The last, we inspected five at high magnification vision continuouslyand counted at least200sperm. To observe the changing of the sperm headwithout shading and tail swelling, as well as calculated the rate of sperm headwithout stain and tail swelling.Results: In the groups of the control,125,100and90mg/ml, the rate of sperm headwithout dying and tail swelling was (81.44±5.71)%,(6.24±3.31)%，(5.21±3.91)%and (7.85±4.71)%；And the sperm tail swelling rate was(97.50±10.13)%,(83.49±7.05)%,(93.96±6.62)%and (92.738±9.428)%.Conclusions: After BERVL mixed with sperm, we found both the rate of sperm headwithout dying and tail swelling descent significant(p＜0.05), whichsuggested BERVL disrupted sperm membrane obviously. The declineamplitude of the sperm head without shading is more obvious than the tailswelling after test(p＜0.05), that told us BERVL through damaged sperm head membrane to caused sperm dead. Experiment2: Sperm apoptosis in Hoechst33342/PI double stainObjective: To study the relationship between sperm apoptosis and BERVL, as to explorethe mechanism of spermicidal effects.Methods: Firstly,10differences semen50μl were separate mixed with200μl BERVL indose of125,100and90mg/ml. Secondly, the sperm suspension was stainingwith Hoechst33342/PI at the ration of1:1in dark3-5min. The last, at highmagnification vision continuously and counted at least200sperm inultraviolet stimulating under fluorescence to observe and calculate the rate ofnormal, early or middle apoptosis and late apoptosis as well as necrosis sperm.The normal sperm is light blue, its nuclear is ground. The early or middleapoptosis sperm is bright blue, its nuclear is fragments. The late apoptosis aswell as necrosis sperm is red.Result: In the groups of the control,125,100and90mg/ml, the nor mal sperm rate was(94.46±5.11)%,(3.29±2.00)%,(3.53±3.35)%and (5.01±2.71)%; The early andmiddle sperm apoptosis rate was (2.64±1.31)%,(6.09±2.08)%,(5.65±3.71)%and (4.98±2.27)%; The late apoptosis and necrosis sperm rate was(2.89±2.54)%,(90.12±3.54)%，(90.91±4.40)%and (90.11±4.51)%。Conclusions: Compared with the control group, the normal sperm rate descendsnoteworthy (p＜0.05), and the sperm apoptosis and necrosis rate wassignificant high after experiment (p＜0.05). We recognized BERVLaccelerated sperm sperm necrosis very fast.