Study On Triploid Induction And Sperm Morphology Of Chlamys Farreri

Triploid breeding experiment of Chlamys farreri was conduted in this paper.Hyperhaline treatment and hypotonic treatment methods were used for triploidinduction by inhibiting polar bodyⅡ(PB2)releasing．Hyperhaline treatment methodwas a new method of triploid induction. Five different triploid induction methodswere compared containing cold shock, heat shock, CB,6-DMAP and hypotonictreatment Chlamys farreri oocytes were first stripped, promoted and then induced byhypotonic treatment method. Then the suitable induction parameters of triploidChlamys farreri oocytes with hypotonic treatment were studied. The sperm ofChlamys farreri was smeared and stained by two dyes Eosin and Crystal Violet forcomparison of the staining effect and to evaluate the sperm quality in order to find outthe suitable dying methods for the analysis of sperm morphology of Chlamysfarreri．All the relevant suitable induction parameters were obtained in this research.This research tried to find a best triploid induction method to support shellfish geneticbreeding research and improve the development of the aquiculture.The main contents consist of the following three parts.1. Research of the suitable induction parameters of triploid Chlamys farreri withhypotonic treatment which oocytes were first stripped and then promoted to be morematuration in vitro.In vitro ammonia seawater and5-HT were proved can improve the Chlamys farrerioocytes to more maturation.The suitable improving maturation parameters were0.005%ammonia seawater and0.002M5-HT. In this research Chlamys farreri oocytes werefirst stripped and then treated by0.005%ammonia seawater and0.002M5-HT invitro and then fertilized artificially.Different salinity(S=10、12、14、16),initialtreatment time(at the time when the first PB1,40%-50%percents of PB1and the firstPB2were observed)and duration time(15,20and25min),were tested out at19°C water temperature in this research. Results indicated that ammonia seawater canobviously induce the Germinal Vesicle Breakdown and5-HT can induce the alreadyGVBD oocytes more maturation. The feitilization rate was up to41.28%after theammonia seawater and5-HT treated. Results indicated that the suitable inductionparameters were: the fertilized eggs were treated20min with hypotonic a solution of14salinity at the time when40%-50%percents of PB1were released, can get thehighest triploidy rate which was up to (54.28±8.48)%．2. Triploid induction in Chlamys farreri by hyperhaline treatment and comparisonwith other treatment methods.Different salinity(S=40、42.5、45、47.5、50、52.5),initial processing time(at the timewhen the first PB1,40%-50%percents of PB1and the first PB2were observed) andduration time(10,15,20and25min)were tested at19℃water temperature in thisresearch. Results indicated that the suitable induction parameters were: the fertilizedeggs were treated20min with hyperhaline a solution of47.5salinity at the time when40%-50%percents of PB1were released, can get the highest triploidy rate which wasup to (95.56±2.87)%.Then five different triploid induced methods were comparedincluding cold shock (1℃), heat shock (30℃), CB (0.005ppm),6-DMAP(60mg/L)and hypotonic treatment. The triploids induced by hyperhaline treatment were higherthan all the five methods. The hyperhaline treatment method results were higher thanhypotonic treatment (the difference was not significant), higher than other fourmethods (the difference was very significant).The hyperhaline treatment was the besttriptoid induction methods. Hyperhaline treatment method was a very safe andnon-toxic method for the experimenters and larvae. And in addition, it was simple andlow cost so very suitable for industrialization promotion.3. Different dyeing methods on evaluate Chlamys farreri sperm qualityThe sperm of Chlamys farreri was smeared at the glass and then stained by twodyes Eosin and Crystal Violet to compare the staining effect and to evaluate the spermquality so that find out the suitable staining methods for the analysis of spermmorphology of Chlamys farreri. After20mg/ml Eosin was mixed with the spermsolution, the sperm head、neck and tail were obviously clear. The parts of the sperm structure and the background were also clean. Therefore, this method was fit for thesperm characteristic observation. Penetration staining method was the best methodsfor the5mg/ml Crystal Violet. Obvious sperm characteristics containing the brunetsperm head and the light tail with very clear background were all observed in theglass after5mg/ml of Crystal Violet stain the sperm. The main malformation wasfound in tail of the sperm, few in the head according to the sperm features shown bystaining. The results indicated that there were no significant differences in theevaluation of the normal sperm by the5mg/ml of Crystal Violet and by20mg/mlEosin (P＞0.05), so either could be used to evaluate the Chlamys farreri sperm quality.The ratio of curved tail sperm of Eosin is higher than Crystal Violet, but will notaffect the overall evaluation standard. So Eosin and Crystal Violet methods could bothbe used to evaluate the Chlamys farreri sperm characteristics accurately andproduction practice.