| Zebrafish(Danio rerio) is a kind of typical small tropical fish,because of its small individual, fast development, eggs embryos, invitro fertilization, embryos transparent, easy to observe and feed, etc. Its widely applied in developmental biology, genetics, environment toxicology, immunology etc. Zebrafish thus becomes one of the most extensive research background mode vertebrate.We taked zebrafish as the research object, using histology and toxicology and reproductive biology research methods to compare the effece of different concentration of ethanol and methyltestosterone to the survival rate,growth vitality and gonads structure of Zebrafish.We observe the structure and developmental capacity of ovaries by fixing with Bouin's and paraffin section.The result showed that zebrafish ovaries is close type, roughly symmetrical couple,grows to sexual maturation about 90 days after born.. Mature ovaries were milk-white,full of II-V phases oocytes,more than 80% volume was occupied by V phase oocytes,its fit for zebrafish was typical asynchronic ovarian. II phase oocytes dyeing were modena,phase III oocyte began to appear zona radiata, phase IV oocyte yolk produce quickly, phase V oocyte was maturation in the free state, nuclei disappeaered.Using the concentration gradient of 0 ml/L (control group), 0.50 ml/L, 1.00 ml/L, 2.00 ml/L, 4.00 ml/L, 8.00 ml/L ethanol feeding fertilized eggs and breeding juvenile fishes 5 d. Got fertilized eggs hatch rates was 61%, 59.5%, 55%, 40%, 17.5%, 10%, juvenile fishes'survival rate were 67.78%, 66.84% ,58.8%, 47.68 %, 20.2%, 12.3%. In 4ml/L above groups observed one-eyed deformity, spinal bending and die. Results showed that 0.50 mg/L group of fertilized eggs'hatch rate and the survival rate of juvenile fishes with no significant difference, not deformities. So less than 0.50ml/L ethanol solution of zebrafish was safe doses. Above 0.50 mg/L could obviously effect to hatch rate and surviva rate.Using the concentration gradient of 0mg/L (control group), 1.00 mg/L, 2.50 mg/L, 5.00 mg/L, 10 .00mg/L, 20.00 mg/L of methyltestosterone (MT) hatch fertilized eggs and breeding fishes after hatched 10 d for 40 d, and fixed juvenile after hatched above 12 d observation development of gonad per 3 d. Experimental results showed that the group can distinguish sex latest in 35d. The survival rate respectively fertilized group were 34.75%,33%,31%,23%,15.75%,9.75%, Male rate was50.35%, 56.89%, 74.94%, 93.61%, 95.3%, 0%. Survival rate of juvenile fishes were 74.92%,66.60%,62.15%,41.74%,24.39%,12.48%;male rate were 49.60%,58.17%,78.23%,92.89%,98.75%,0% in each group.And with the increase of the concentration of MT, fishes'length and weight were reduced. Results show that the MT has obvious inhibition to growth vitality and survival of Zebrafish.While the methyltestosterone dose reached 10mg/L , cased a few infertility fish, 20mg/L of MT could induced group infertility, soaking method of MT in the male control dose of zebrafish should be in 5~10mg/L.Using involved MT 0mg/kg (control group), 30 mg/kg, 60 mg/kg, 120 mg/kg, 240 mg/kg, 400 mg/kg of hormone feed fed 60 day-old female and sexual maturation female zebrafish 30d,then testcrossing. Results showed that in 60 day-old group, the survivl rate were 95%, 90%, 75%, 55%, 35%, 70% .GSI respectively were 0.1074, 0.0998, 0.0756, 0.0582, 0.0386, 0.0278. The survivl rate of sexual maturation group were 100%, 95%, 80%, 55%,40%, 65%;GSI respectively were 0.2247,0.1905,0.1531,0.1210,0.0275,0.0407。With the MT dosage increased, the growth vitality of zebrafish were obviously inhibition, but in 400 mg/kg group were obviously improved. MT has obvious inhibition to spawning quantity and hatch rate of Zebrafish. Results showed that MT has obvious inhibition in female zebrafish's spawning, hatch , survival rate , growth vitality, and GSI. 400 mg/kg MT feed fed female zebrafish 30d can induction hormone neutered, thus showing higher growth vitality.Mature female zebrafish gonad observation results showed. With the MT dosage increased,each group's ovarian development has inhibition deeper and ovarian structure changed more obviously. 30 mg/kg group phase V maturation oocytes obviously reduction,60 mg/kg group phase II above oocytes occupied area reduced to 60%,120 mg/kg group began to observed oocytes apoptosis process. 240 mg/kg group phase II above oocytes within the gonads accounted for only about 10%.400 mg/kg group could hardly identify gonad organization, except a few degradation phase II oocytes. MT made oocytes apoptosis process : inhibit oocytes'development-- nucleus structure changed-- organelles structure changed-- membrane structure damaged and apoptosis body formed-- apoptosis body diffusion and oocytes shrinkaged--disintegration. |
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