Effects Of Epimedium And Its Active Ingredients On Female Mouse Reproductive Development

Traditional medicine(TM)is an important and often underestimated part of health services including animal health services.In veterinary practice,the use of TM has a long history in health promotion,maintenance and in disease prevention and treatment,particularly for chronic disease.In Traditional Chinese Medicine(TCM),epimedium(a traditional herbal medicine)and its ingredients have been used over a long period of time in the treatment of several medical conditions such as cardiovascular diseases and estrous enhancement but there is no definite record of how epimedium and its active ingredients affects reproductive development in female mouse.The mouse ovary is composed of its female’s lifetime supply of oocytes,each contained within an ovarian follicle.The onset of follicular growth is an important control point in follicular development.The follicles are formed prior to birth at the resting,primordial stage:once the follicle pool is established,there is a continual and gradual movement of follicles from the primordial into the growing follicle pool.As follicles start to grow,they develop through the primary,secondary,preantral and then antral stages,until they reach the pre-ovulatory,or Graafian,stage.Only oocytes from pre-ovulatory follicles have full developmental competence,able to support embryonic development to term,if fertilized.Therefore,this study was conducted to investigate how Epimedium(EP)and its active ingredients(Epimedium polysaccharide,EPS;icariin,ICA)affect mouse growth and reproductive development through assessment of the effects of these 3 medicines on mouse body weight,assessment of in vitro maturation(IVM)rate of oocytes,reproductive index calculation,and histological evaluation of the ovaries and uterus.In all,one hundred and eighty(180)healthy Kunming female mice were randomly divided into 30 groups(n=6)and orally administered epimedium(EP)at 2,6,10,and 14 mg/0.1 mL,epimedium polysaccharide(EPS)at 2,4,6,and 8 mg/0.1 mL,and icariin(ICA)at 0.1,0.3,0.5,and 0.7 mg/0.1 mL for 7 and 14 days.The control groups were given 0.1 mL of 0.9%physiological saline solution.All mice were routinely raised in a clean area with normal room temperature and fed with standard mice feed and water ad libitum.After 7 and 14 days,they were all sacrificed and their reproductive tracts(uterus,ovi-duct,and ovary)extirpated.The reproductive tracts were weighed to calculate the reproductive index(RI)of the mice;some ovaries were punctured to harvest oocytes for in vitro maturation(IVM)culture and the others were used for histological analysis.This experiment provides the basis for understanding how Epimedium and its active ingredients affect mouse oocyte growth and development and to ascertain which of the 3 medicines works best.The experimental results are briefly outlined as follows:Experiment 1.Effects of EP on ovary and uterus developmentDuring reproductive index analysis,the RI value of 10 mg/0.1 mL group for 7 days and 6 mg/0.1 mL for 14 days were significantly higher than other treatment groups and the control.Also,the RI values for all treatment groups were significantly higher than the control groups for either 7 or 14 day.After culturing the EP treated oocytes,the percentage of oocytes that extruded first polar body(pbI)was more in the treated groups as compared to the control(63.30%).However,there were differences between individual experimental groups and the control(p<0.05).6 mg/0.1 mL had the highest pbI rates in the 14 d group.When the ovaries were observed histologically under the microscope,there seemed to be no significant differences for the number and size of antral follicles among groups.There were lots of secondary follicles in the experimental group(2,6,10,and 14 mg/0.1 mL)than the control group,an indication that EP enhances follicular development.Experiment 2.Effects of EPS on ovary and uterus developmentWhen the female mice were administered with different doses of EPS continuously for 7 and 14 d,there were no negative effects in body weight of the mice.There was also marginal increase in the body weight of the mice.The RI value of 4 mg/0.1 mL group for 14 d was significantly higher than other treatment groups(2,6,8 mg/0.1 mL)and the control.At the end of the IVM experiment for the EPS treatment groups,the pbI extrusion rates of the oocytes were as follows:the percentage of oocytes that extruded first polar body(pbI)after culture was also more in the treated groups as compared to the control.However,there are no differences between experimental groups(except 8 mg/0.1 mL for 7 d group)and the control(p<0.05).The 8 mg/0.1 mL group had the highest pbI rates in the 7 d group and 4 mg/0.1 mL had the highest pbI rate in the 14 d group.During histological evaluation of EPS on ovary development for 7 and 14 d,we also observed no histopathological alterations in the ovaries in the control or EPS-treated mice.There were lots of secondary follicles in the experimental groups(2,4,6,and 8 mg/0.1 mL)than the control group,an indication that EPS like EP enhances follicular development.Experiment 3.Effects of ICA on ovary and uterus developmentThe findings were that ICA also had no negative effects on the body weight of the mice.Again,like EP and EPS experiments,there was marginal increase in the body weight of the mice.The RI value of 0.1,0.3,0.5,and 0.7 mg/0.1 mL groups for 14 d was higher than the control.During oocyte culture evaluation of ICA on the pbI extrusion rates of the oocytes,it was realized that the percentage of oocytes that extruded first polar body(pbI)after culture was also more in the treated groups as compared to the control.However,there were differences between experimental groups(except 0.1 mg/0.1 mL for 14 d group)and the control(p<0.05).The 0.5 mg/0.1 mL group had the highest pbI rates in the 7 d group whilst 0.3 mg/0.1 mL had the highest pbI rate in the 14 d group.In our final experiment,ICA was given orally to adult female mice for 7 and 14 d and then animals were sacrificed respectively.Again,our histological findings suggested that ovaries treated with ICA had no histopathological alterations.There were also lots of secondary follicles in the experimental group(0.1,0.3,0.5,and 0.7 mg/0.1 mL)than the control group,an indication that ICA enhances follicular development just like EP and EPS.These data suggest that epimedium,epimedium polysaccharide,and icariin at an appreciable high dosage have a positive effect on mouse ovary development and among the 3 medicines;icariin seems to work best on mouse ovaries and uterus development based on the IVM and RI results.