Testosterone, Progesterone, And Flutamide Affect The Behavior, Fertilization Rate, And Longevity Of The Male Rotifers

In this research,Brachionus calyciflorus has been adopted as the research object. The effect of testosterone, progesterone and flutamide on swimming behavior,mating behavior,fertilization rate,life and size of male B. calyciflorus were investigated. The relationship between the male swimming speed and fertilization rate was analyzed. And the effect of any two of the testosterone, progesterone and flutamide on swimming speed alteration and fertilization rate. The research provides reference for male rotifer as male invertebrate reproductive toxicology test animals.The results of research are as the follows:1. The method of the induction of Brachionus calyclflorus sexual reproductionAt 25 ? under the laboratory culture, take the parthenogenesis population of Brachionus calyciflorus through 300 cells mesh sieve to enrichment. Rinse it with double distilled water several times and take out protozoa and tiny impurities while cultivation process. After getting high density population, feed the population 1×106 cells/mL chlorella to test the induced effect on sexual reproduction with different initial density. The results showed that the groups of 50, 100, 500, 1000 ind/mL were stable in population density at 4 days and there was no significant difference. The population density was decreased significantly at 2000 ind/mL in the second day.After the second day, in groups of 500, 1000, 2000 ind/mL, a large amount of males appeared and some resting eggs came out. When the initial population density was at 500 ind/mL, the induced effect of sexual reproduction accessed to optimum in the respect of resource utilization and population stability.2. The swimming behavior of male Brachionus calyciflorus under the different environmental hormonePick out the male rotifer which could swim quickly and born within 4h from the population after sexual reproduction. Take use of Olympus BX41 microscope, DP70 image capture system and Cellsens Standard software to film and capture the motion state of male rotifers, then make the male rotifers be exposed to testosterone (0.5-6 mg/L), flutamide (0.5-6.0 mg/L) and progesterone (0.5-6.0 mg/L). After 2h later,capture the motion state again. The results showed that there was no significant influence (P>O.05) on swimming sinuosity of male rotifers exposed to testosterone,flutamide and progesterone. Testosterone had significantly increased the swimming speed alteration of males. The regression equation is Y=-3.9597X2+28.4066X-6.5309,R2=0.89 P=0.02<0.05. Progesterone had significantly decreased the swimming speed alteration of males. The regression equation is Y=-0.1692X2-7.7092X +0.4543,R2=0.98 P=0.00<0.01. Flutamide had significantly decreased the swimming speed alteration of males too. The regression equation is Y=-0.1667X2-8.9862X+1.3892,R2=0.99 P=0.00<0.01. That is to say, swimming speed alteration of male B.calyciflorus is sensitive to environmental hormone.Pick out male and female individuals which bor within 4h from the rotifer population after sexual reproduction, then make the male and female rotifer be exposed respectively to three kinds of hormone in proportion of 10:3 to conduct the mating behavior observation experiments. Compared with the control group, the recognition ability of males was all improved by testosterone in 1h, 2h and 3h(P<0.05). In 4h, 5h and 6h, the recognition ability of males all descended, the progesterone results in substantial suppression of males mating recognition ability;At 6h, the density of each group all comes to extremely low degree. The result of flutamide is the same as progesterone. In general, male of B. calyciflorus mating recognition rate index is susceptible to environmental hormone; however,it also has much to do with male's life and individual discrepancy.3. The reproductive ability of male Brachonus calyciflorus under the different environmental hormoneIn Brachionus calyciflorus' male fertilization rate experiment through exposing male rotifer to experiment exposed to testosterone (0.5-6.0 mg/L), flutamide (0.5-6.0 mg/L) and progesterone (0.5-6.0 mg/L) with 24h, the male fertilization rate increased significantly under testosterone with different concentration (P<0.05), and reached the highest at 2 mg/L which the males fertility rate was 48.61±3.18%; the fertilization rate of male rotifer were suppressed by progesterone and flutamide(P<0.05), furthermore, the higher the dug concentration was the stronger the suppression effect became.In a single individual life experiment, the individual life of males was reduced by testosterone (0.5-6.0 mg/L). However with the rise of the concentrations of testosterone, the descent range of life length varies slightly. The reason may be that the testosterone may be contributed to the male body of swimming velocity so that more energy was consumed. As a result of the males have no consumption system,where energy comes from is embryo. Thus, all reasons lead to a decrease in life length. The individual life length of males wasn't influenced significantly by flutamide (0.5-6.0 mg/L) and progesterone (0.5-6.0 mg/L).By combining what effect different hormones in different concentration have on the males' swimming speed alteration and fertilization rate, a comprehensive analysis can be obtained. Nevertheless, it is easy to find that the males swimming speed index and fertility rate index matched the S curve. The regression equation is Y=42.17/(1+(9.51-X)0.0525)+4.29, R2=0.9456 P=1.82× 10-11<<0.01. This S curve can expound the relationship between the movement and reproductive capacity of male B.calyciflorus.4. Hormone combination experiment of testosterone-flutamide ,testosterone-progesterone and progesterone-flutamideAccording to two-way ANOVA, the affection of the male's swimming speed alteration and mating rate was analyzed under the two hormones combined effect(testosterone-flutamide, testosterone-progesterone and progesterone-flutamide). The hormone antagonism was appeared on mating rate under testosterone-flutamide and testosterone-progesterone. But there is no significant interaction on swimming speed alteration under three kinds of hormone combination and mating rate under progesterone-flutamide. There was strongest inhibitory effect under P1F1: at this time,the swimming speed alteration is 10.5% and the fertilization rate was 3.1%.