Introduction Of Meiotic Gynogenesis And Breeding Of Families’ Offspring In Orange-spot Grouper Epinepgelus Coioides

Groupers are the world’s rare fishes. In this study, the orange-spot grouper （Epinephelus coioides） had been studied for the fertilization cytology, meiosis gynogenesis, gynogenesis progeny genetic identification and family breeding research. The specific studies are as follows:1. Crossbreeding between Epinephelus coioides （（?）） and Epinephelus lanceolatus （（?）） was conducted by artificial fertilization. Eggs were collected at Osec、30s、lmin30s、2min、3 min、 4min、5min、6min、7min、8 min、9min、10min、11 min、12min、13min、14min、 15 min after insemination. They were fixed by Simith solution, embedded by paraffins and stained by H.E. Series of processure of histological section, for the differnt characteristics of orange-spot grouper eggs, were modified. The results showed that the sperm of giant grouper entried eggs of orange-spot grouper very quickly. There had been sperm entried egg in 30-60 seconds post insemination.Observation results of tissure section showed that mature eggs of orange-spot grouper remained at metaphase of second maturation division. The eggs was activated as sperm penetration into the egg. With development of second maturation division.At 2 minutes after insemination,the second maturation division has been developed into menalphase. At 3-6minutes after insemination, sperm-aster appeared. At 5minutes after insemination, the second polar-body was taken out. At 7～15minutes after insemination, male pronucleus and female pronucleus got closer to each other. Then they combined together and a clear junction line was observed, at last zygote nucleus formed and later karyotheca became faint.15minutes after insemination, first karyokinetic division was developed.2. The gynogenetic diploids of orange-spot grouper were induced by fertilization of mature eggs of orange-spot grouper with ultraviolet （UV）-irradiated sperm of giant grouper combined with cold shock. The result showed that the fertilization rate and hatchability presented typical Hertwig effect when the giant grouper sperm were treated in the appropriate intensity, in the condition of that, the haploid control had a higher fertilization 83.5%and haploid rate 80%. At the condition of tempeture 4～6℃, the initiation time of 6 min after fertilization, it could get fertilization 40% and hatchability 29.7%.Duncan multiple comparison results indicated that the cold shock treatment water temperature group and duration of group difference in the fertilization rate was not significant. Only the hatchability in duration group differences was extremely significant （p= 0.002）, difference between water temperature treatment group was not significant （p=0.396）.It may be relevant the comprehensive effect of temperature and duration time.3.1n this study,we get 8 microsatellite loci（D316、Mbo061、GAGO1、Mbo066、GAA-1、 RH_GATA₀03、Ec-122、CA-2）,which was different between Epinephelus coioides and Epinephelus lanceolatus, and then using them to indentify the reproduction of Epinephelus coioides which was induced by artificially gynogenesis. The results showed that the induced rate was 94.1% in the offspring.These specific microsatellite markers could distinguish normal diploid、gynogenetic diploid and parents.4. We selected mature individuals from 500 able-bodied male and female broodstock populations grouper in order to foster whole countryman family, using the way of artificial insemination and oxytocin. Based on the characteristics of the family breeding eggs was less, small water seedlings were studied. Selection results showed that a total of 28 tail groupers were selected using to manual operation and manufactured 8 groups of fertilized eggs successfully.Based on males slant grouper had less the amount of sperm,we injected hormones to improve their semen volume, determined female maturity,and grasped the female spawning time were the key to success for eggs.The results showed:with the increase of the breeding time, Nitrate （NO3--N） and nitrite nitrogen （NO2--N） showed increase trend and a significant linear relationship, y= 4.3854x0287,R2= 0.8625. Dissolve oxygen of the water was sustainably over 5.7 mg.L-1, pH ranged from 7.15 to 7.92, the ammonia nitrogen concentration was above 5 mg.L-1in the later stage of breeding, temperature ranged from 27℃ to 29℃, salinity changed from 30 to 34, during breeding average light intensity ranged from 1075 Lx to 2329 Lx. The levels of ammonia nitrogen, Nitrate （NOV-N）, nitrite nitrogen （NO2--N）, dissolve oxygen and pH in pool bottom were significantly different from surface and intermediate water, all the fry lived normally during breeding.