Comparative Study On Gonad And Gamete Development And Haemocytes Of Different Ploid Rainbow Trout (Oncorhynchus Mykiss)

Rainbow trout (Onchorynchus mykiss Walbaum) was one of important cultivated cold water species. In order to improve quality of the products and output of the stock, cultivating artificial-inducing sterile triploid rainbow trout has come into practice in recent years. The tetraploid rainbow trout has already been inducted and cultivated successfully as the parent of breeding of triploid rainbow trout. Due to the polyploidisation of chromosome, the reproductive mechanism of the polyploid rainbow trout has changed and resulted in triploid female rainbow trout sterile. Since the gonad was the foundation of fishes for breeding, comprehensively understanding the regularity of gonadal development and gametogenesis can benefit controlling the process of breeding. Although there were some studies on the gonadal development of triploid rainbow trout, the systemic studies on histology, cytology, reproductive endocrinology have not finished in the whole developmental process, especially on gonadal development at the early stage. Up to now, there were no systemic reports about triploid ovaries from normal to diapause.This experiment was done with diploid and triploid rainbow trout of 4~35 months old and with tetraploid rainbow trout of 26 and 35 months old. The comparative studies on gonad of diploid, triploid and tetraploid rainbow trout were done in several aspects such as anatomic, histological ones and the changes of reproductive hormone. This study also made location of diploid and triploid female rainbow trout's vitellogenin by immune histochemistry method. In addition, the blood cell characteristics of polyploid rainbow trout were researched. The conclusions were showed as follow:1. 35 month-old diploid female rainbow trout was near sexual maturity, the most of the ovary developed to stage IV+++ and ovulated. 26 month-old diploid male is up to maturity, the most of them developed to stage V (spermiation stage). The GSI of female and male rainbow trout before sexual mature was increasing constantly and reached the peak near the time of reproduction. The HSI of female rainbow trout was descending obviously in the midphase and anaphase of ovary development, and was negatively correlated to GSI.2. The ovaries of triploid rainbow trout developed abnormally and immaturely. The ovaries of triploid and diploid rainbow trout have no differences between stage I. After stage II (8 month old), ovaries diapause, the front parts of them were intumescentia slightly and the posterior parts linear. Some individuals have small number of normal developmental oocytes. The GSI was keeps in low level and lower than one of diploid individuals of the same stage. The HSI was lower than one of diploid rainbow trout and there were no obvious correlation compared with GSI. Triploid rainbow trout had morphological normal development spermary which developed slower than diploid spermary. Only a few individuals maturited and came into stage V at 35 month-old. The GSI was lower than that of diploid individuals.3. The gonadal morphology of tetraploid was similar to that of diploid, but the sexual maturation of teraploid was later than that of diploid. At 35 months old, a few ovaries developed to the stage IV+++, and mostly samples were still at the stage IV++. At 26 months old, a few spermaries developed to stage V. The GSI of tetraploid was not significantly different from that of diploid at same stage.4. The oocyte's yolk nucleus of diploid rainbow trout looked like lump in the early egg yolk genesis, and transferred and formed outer ring at the cortex. It was the source of endogenous egg yolk, composing by Gaogi apparatus and mitochondria. Soon after the growing links were disappearing, a basophilla silkiness link formed in the oocyte's cytoplasm and disappeared in stage III.5. The triploid rainbow trout's ovary diapaused and underwent the process: ovogonium→oocyte in the earlier of yolk development→ovogonium cluster→grp-androgone cytocyst. The influence of chromosome triplication on female triploid fish mainly took place at the early ovary development. There was no difference between female triploid rainbow trout and diploid one in ovaries development at the gonad differentiated stages. Oocyte was abortion at the diplotene stage of meiosisⅠ(advanced stages of nucleolus periphery, protophase of Vitellogenesis) rather than at amphitene and pachynema of meiosisⅠin other reports. The ovaries had the tendency of masculinization at the midanaphase of gonadal development. The triploid female sterility resulted from comprehensive factor: the abortion of oocyte at the diplotene stage of meiosisⅠwas due to the disorder separation of the paired chromosome. After abortion of oocyte, oogonium can not further development in ovaries, this resulted from separating block of the matrix cell in the wall of ovogonium cluster. Duing to the separation, oogonium lost the interaction with the follicular cells and could not go on further differentiation. Oogonium which was liberated from outside of oogonium cluster could continue developing and maturate.6. The triploid male trout could finish spermatogenesis, but their spermary developed slower than the spermary development of the diploid in the same stage. More dead cells appeared, the sperm of triploid trout was large with the inequality of size. The impact of chromosome triplication on male triploid mainly happened in the advanced stage (stage of metamorphosis) of spermatogenesis.7. The abortive generative cells in the spermary and ovaries of triploid rainbow trout was phagocytized by sertoli's cells (male), grp- sertoli's cells (female) and fibroblast.8. Tetraploid female rainbow trout's ovary and oocytes could develop normally and be fertile. The spermatogenesis was quite similar to one in diploid trout and slightly lagged behind. The diameter of sperm head was 2.82±0.33μm, and the volume was obviously bigger than triploid (2.37±0.12μm ) and diploid (1.86±0.12μm ).9. The levels of GtH-Ⅰand 17β-E2 in serum of diploid female rainbow trout increased constantly during immaturity, and decreased near maturity. The contents of GtH-Ⅱand T were low at the protophase of development, and increased slowly and constantly, but increased quickly near sexual maturity. It indicated that 17β-E2 was produced by GtH-Ⅰthrough the stimulation to gonad, and promoted the development of oocytes. GTH-II promoted the ovum to maturity. The changes of reproductive hormone in triploid female rainbow trout were obviously different from diploid, the levels of GtH-Ⅰ, GtH-Ⅱand 17β-E2 in the serum fluctuate within a low range, and the regularity was not obvious. There was a constant increasing in the levels of T in triploid female rainbow trout after 21 months and was higher than the diploid at the same stage, which provides a physiological evidence for the masculinization-like of ovaries in triploid rainbow trout. The changes of GtH-Ⅰ, GtH-Ⅱ, 17β-E2 and T in tetraploid female rainbow trout was as the same as diploid.10. The levels of GtH-Ⅰand 17β-E2 in immature diploid male rainbow trout increased constantly and became lower near sexual maturation. The contents of GtH-Ⅱand T was low at the protophase of development, increased slowly and constantly, and shew a quickly increase near sexual maturity. The male individuals had two periodic changes during 11-35months. The change s of GtH-Ⅰ, GtH-Ⅱ, 17β-E2 and T in male triploid rainbow trout were identical to ones in the diploid, but lagged behind diploid at the same stage, and existed only in one variation cycle. The contents of GtH-Ⅰ, GtH-Ⅱ, 17β-E2 and T in male tetraploid rainbow trout had the same change regularity with the diploid.11. Vitellogenin of rainbow trout's ovum were extracted and purified by saturated ammonium sulfate fractional precipitation and gel chromatography. Lipovitellenin contained sugar, phosphorus and adipose with 123.2 kDa molecular weight. Its antiserum reacted only with the vitellogenin (Vg) in the normal female fish plasma without exogenous inducer, which indicated that it was a female peculiar protein.12. Immuno-histochemistry allocation showed that the hepatocyte and blood had Vg in diploid rainbow trout's ovaries whichdeveloped to stage III~V, positive reaction of its ovaries was hysteresis and the oocyte had Vg during stage IV~V. In other developmentive periods, the Vg in diploid female rainbow trout's hepatocyte and blood presented the negative reaction. The Vg in liver, blood and gonad of triploid female rainbow trout also shew the negative reaction, and the Vg in liver, blood and gonad of diploid and triploid male rainbow trout in every developmental stage had similar negative reaction, which indicated that there no inducing factor in surrounding to induce the unusual expression of vitellogenin. The Vg negative reaction appeared in intestine tissue of both male and female diploid and triploid rainbow trout in every developmental stage, illustrating the formation of Vg had not any relation with intestines.13. The red blood corpuscles were bigger, longer in triploid and tetraploid rainbow trout than the diploids. The size of triploid and tetraploid rainbow trout's erythrocyte were 17.35±1.59×10.52±0.70μm and 18.66±2.28×11.08±1.47μm respectively, greater than the diploid (13.30±1.31×8.34±0.63μm ) , and they were all larger than diploid(13.30±1.31×8.34±0.63μm)and had no significant deviation from expectant theoretical value (1.5︰1, 2.0︰1) . The rate of short diameter to long diameter for erythrocyte was 0.60 and 0.59 respectively, and was smaller than diploid (0.63). Because of the number of erythrocyte per unit in triploid rainbow trout was smaller than in diploid, the volume of packed red cells was obviously smaller than diploid. Erythrocyte in triploid rainbow trout was less fragile than in diploid, there were no significant differences in levels of hemoglobin per unit volume blood between triploid and diploid rainbow trout.14. Some of red cell nucleus looks like dumbbell or dikaryon. Hypotonic test showed that the erythrocytes were not terminal differentiated cells in the peripheral blood of polyploid rainbow trout and had ability to division. The dikaryotic erythrocyte's karyodieresis was earlier ones of nuclus than cytokinesis and the dumbbell-like red blood corpuscle was middle course of synchronized division in the karyoplasm and cytoplasm.15. The development of erythrocyte in triploid rainbow trout passed through primitive, inmature and ripe stages. The proportion of immature blood cell was13.8%±1.24%, 4.14%±0.99%, 5.52%±0.58% and 3.96%±0.47% respectively in the triploid rainbow trout's erythrocyte series, monocyte series, lymphocyte series and granulocyte series, and was higher than the corresponding index of the hematogenic organ. The abnormal division of the erythrocyte in the peripheral blood was the main causes of inmature red blood corpuscles increase.16. The proportion of erythrocyte was 66.90%±3.85% in the blood cell from head kidney, which was higher than corresponding index from the spleen and liver (56.90%±1.77%, 47.20%±2.14% ) .The proportions of monocyte and granulocyte were 21.80%±1.58% and 4.92%±0.01% in the blood cells from liver, higher than corresponding index from the spleen and head kidney (17.80%±2.34%, 4.87%±0.99%;6.82%±1.24%, 3.11%±1.10%). The proportion of lymphocyte was 14.90%±1.29% in the blood cell from liver, higher than corresponding index from the head kidney and spleen(13.20±2.86%, 12.60%±2.74%).