High Efficient,Non-invasive And Repeated Collection Of Ejaculates From Rat Using Penile Vibratory Stimulation And Analysis Of Its Semen Quality

Rat is one of the most important experimental animal which played an indispensable role in biomedical research,particularly in reproduction.However,according to our best knowledge,there is no easiest and efficient method available for semen collecting in rat.In this study,we successfully collected semen through penile vibratory stimulation ejaculation(PVSE)from laboratory rat which is an easiest and efficient method as compared with rectal probe electro-ejaculation(RPE).We found that the ejaculation rate,semen weight,concentration and motility of semen collected with PVSE were substantially better than that of RPE(P<0.05),and the malformation is lower.Although PVSE was a time-consuming as compared to RPE,but quality of semen was improved.Additionally,the ejaculation rate,sperm concentration and motility were significantly higher in two-day internal than that of five-day interval.In two-day internal,the rats semen were collected using the PVSE method for three months,we had harvested the high quality semen continuously and steadily.We found that the electrical stimulation and the use of anesthetics can significantly reduce the motility of rat sperm.Ten old rats(age of 60 weeks)were treated with PVSE method to collect semen,but there was no success.Four sexually fertile female rats were artificially inseminated with semen from PVSE and 12 healthy offspring were born successfully.The rat semen were diluted with six different sperm extenders containing HTF,SR,mKRB+egg,egg,DM and TTE,respectively,the sperm motility survival rate with mKRB+egg was significantly higher(P<0.05)compared to other five extenders at 4℃,while in SR dilution rat sperm survival rate was the lowest.Rat semen were incubated at 25℃ and 37℃ for 2 hours,respectively,but there is nosignificantly difference on the decline of sperm motility.The diluted sperm were frozen and thawed,the most notable finding was that sperm frozen in mKRB+egg had the greatest post~thaw motility but no more than 10%.We also added different concentration of glycerol,ethylene glycol,propylene glycol,sorbitol,acetamide,dimethylacetamide,DMSO and trehalose based on mKRB+egg extender,but did not achieve the desired results.Here,for the first time,we successfully set up PVSE for rat,a repeated collection of semen with high efficient and non-invasive method.