| Heshouwuyin is a complex preparation which is from six kinds of Chinese herbs(Heshouwu, Roucongrong, Huainiuxi, Yinyanghuo, Danshen，Fuling).Objective: To investigate the effects of Heshouwuyin on sperm quality of aging rats byobserving the changes of morphology of testis and function of sperm in Natural aging rats.Methods:1. Groups of experimental animals: Wistar rats which were12months old wererandomly divided into five groups: young control group (YCG); natural aging group (NAG,intragastric administration of normal saline);Heshouwuyin group1(SWY1G, after16monthsof birth, Heshouwuyin treatment was intragastrically administered for60days);Heshouwuyin group2(SWY2G, after17months of birth, Heshouwuyin treatment wasintragastrically administered for30days); Shouwu pill control group (SWPG, after16months of birth, intragastric administration of Heshouwupill).2. Collection of samples: The rats were anesthetized using chloral hydrate afterweighing. Blood sample(2Ml) was collected from post-inner canthus venous plexus using acapillary pipet. The supernatant was extracted after centrifugation at1000r/min for10minutes to determinate testosterone. The epididymis was taken out at room temperature.Sperm cells were collected from cauda of epididymis for observing sperm quality. Thebilateral testicular tissues were taken quickly, weighted using an electronic analytical balancefor calculating testicles index.3. Items of observing3.1Determining the testosterone contents in SerumBiological chemiluminescence method was used to determine testosterone contents inserum from each group of rat.3.2Testicles index calculationTesticles index=weight of testis/body weight×100%.3.3The morphology of testicular tissue Morphological changes were observed by HE staining of testis.3.4Integrity of the plasma membraneSperms were stained by the acetoacetate and propidium iodide method.200sperms werecounted (Green fluorescence indicated the sperm with intact plasma membrane, and redfluorescence referred to the sperm with damaged plasma membrane)under the fluorescencemicroscopy. To calculate the percentage of sperms with green fluorescence.3.5Sperm mitochondrial functionUsing rhodamine123, sperms were stained. Observing by the fluorescence microscopy,200sperms were counted to calculate the percentage of green fluorescent sperms (greenfluorescent sperms had function with normal mitochondrial, and weakened greenfluorescence or sub-section phenomenon indicated decreased mitochondrial function).3.6Sperm DNA integritySperm DNA integrity were detected by Sperm DNA acridine orange assay kit underfluorescence microscopy. A total of200sperms were counted to calculate the percentage ofgreen fluorescent sperms.（(green fluorescent sperms are sperms with nomal DNA, red oryellow fluorescence are sperms with abnomal DNA).Results:1. The testosterone levels of nature aging group was obviously lower than that of youngcontrol group (P <0.01); The concentration of testosterone in Heshouwuyin and Heshouwupill group were improved significantly compared with nature aging group (P <0.05). Thebetter curative effect of Heshouwuyin group was obvious than that of Heshouwuyin pillgroup(P <0.05).2. Testicles index: Compared with the young control group,the testicles indexsignificantly decreased in the natural aging group (P <0.01); The testicles index obviouslyincreased in Heshouwuyin groups1, Heshouwuyin groups2and the Shouwu pill groupcompared with the natural aging group. The efficacy of Heshouwuyin group1was thebest（P<0.05）. 3. Morphological observation of testicular tissues: In the young control group,seminiferous tubules had no atrophy and collapse, showed intact limiting membrane with nothickening and fibrosis, all kinds of spermatogenic cells and supporting cells were visible inthe lumen, While in the natural aging group, the basement membrane of seminiferoustubules was partially thickened, and seminiferous tubules have atrophy and collapse, germlayers were reduced in number and cells were sparse; In all three drug treatment groups, theseminiferous tubule showed a significant improvement in the morphology and structure,basement membrane tended to be complete, and germ cell layers of the seminiferousepithelium were increased in number. Heshouwuyin group1had a better effect than othergroups.4. Plasma membrane integrity observations: Compared with the young control group,the number of sperm cells with green fluorescence obviously decreased in the natural aginggroup (P <0.01); but the number of sperm cells with green fluorescence markedly increasedin Heshouwuyin groups and Heshouwu pill group compared with the natural aging group,and the efficacy of Heshouwuyin group1was the best (P <0.05).5. Sperm mitochondrial function: Compared with the young control group, thenumber of sperm cells with green fluorescence obviously decreased in the natural aginggroup (P <0.01); But the number of sperm cells with green fluorescence significantlyincreased in the Heshouwu pill group and the two Heshouwuyin groups compared with thenatural aging group, and the efficacy of Heshouwuyin group1was the best（P<0.05）.6. Sperm DNA integrity assessment: Compared with the young control group, thenumber of sperm cells with green fluorescence in the natural aging group significantlydecreased, but the number of sperm cells with red or yellow fluorescence increaseddramatically (P <0.01). Compared with the natural aging group, the number of sperm cellswith green fluorescence was more, but the number of sperm cells with red or yellowfluorescence was significantly less in the Heshouwuyin and Shuwu pill groups. Moreover, themost number of sperm cells with normal DNA was Heshouwuyin group1(P <0.05).Conclusion: Heshouwuyin will improve the testis morphology, increase testicles index andtestosterone level, regulate sperm function. |
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