| Artificial breeding was considered as the most effective way to protect the turtles from extinct by the international turtle conservation organizations(IUCN/SSC, TFTSG and TCF), but the artificial breeding problems of most turtles were still unresolved. To improve the ability of turtle reproduction, a new way of gonad development and egg laying amounts through the way of nutrition to solve these problems was made. The study of the reproductive nutrition mainly concentrated in fish and shrimp, hardly any in turtles. PUFAs was one of the most important components of vitellogenin and biological membrane of embryonic cells, significantly influenced animal reproductive performance(gonad development, egg laying, egg quality, etc.), and was one of the indispensable nutritional factors during life history. So far, there were few studies about the mechanism of PUFAs on ovarian development of turtle.The relationship between lipid metabolism and vitellogenesis was studied, so that the suitable lipid nutrition could be added into the dietary of Chinese Strip-necked Turtle during the vitellogenic stage, to lay a foundation for solving the reproductive problems under artificial condition. The female adult turtle which weighted 1557±307 g was selected, and were domestication in the breeding room of hainan normal university for two weeks. After that, the healthy ones were divided randomly into four groups named 3S∶0F group(soybean oil, fish oil, 1∶0), 2S∶1F group(soybean oil, fish oil, 2∶1), 1S∶2F group(soybean oil, fish oil, 1∶2), and 0S∶3F group(soybean oil, fish oil, 0∶1) according to the proportion of soybean oil and fish oil added into the dietary. Fed twice a week, lasted for a year. Every two months six turtles were selected randomly from each group, their blood was used to measure the content of vitellogenin, low density lipoprotein cholesterol, high density lipoprotein cholesterol, triglyceride and total cholesterol. The liver was used to analysis the fatty acid composition, the expression of L- FABP gene and the activity of the lipases including: hepatic lipase, lipoprotein lipase, fatty acid synthase and hormone sensitive lipase. The muscle was used to measure fatty acid composition. The activity of pancreatic lipase and histological structure of small intestine was studied. The results are as follows:1. The effects of dietary PUFAs on the accumulation of the yolk protein were studied by measuring the contents of VTG in the serum of M. sinensis.The interaction effects of VTG between the vitellogenic stage and the dietary processed significantly(F=1.969, df=25, P=0.019<0.05). The different dietary PUFAs had no significant effects on VTG in the early vitellogenic stage(P>0.05). In the mid-late stage, PUFAs especially n-3 PUFAs could increase the content of VTG in serum significantly(P<0.05), and the content of VTG of 0S∶3F was almost 2 times more than 3S∶0F. The VTG of 0S∶3F was more than 1.5 times of the mixed oil groups, almost 3 times of 3S∶0F(P < 0.05). During the spawning season, the content of VTG generally decreased significantly, and the VTG of 0S∶3F were 3 times more than 3S∶0F(P < 0.05). In conclusion, the content of VTG was significant effected by dietary PUFAs especially n-3 PUFAs. VTG was regulated by the vitellogenic stages. Exogenous vitellogenesis was given the priority in the mid-last vitellogenic stage, greatly influenced by the dietary.2. In order to figure out the mechanism of how PUFAs influenced the accumulation of the yolk protein, a further study to explore the influences of dietary PUFAs on the entire process of lipid metabolism during the vitellogenic stages:(1) The influence of the lipid absorption processLipids were mainly absorbed in the small intestine. The effects of the dietary PUFAs on the lipids absorption process could be evaluated by measuring the activity of PL of the different treatment groups of the small intestine as the physiological aspect and the changes of the histological structure of the small intestine as the histological aspect during the different vitellogenic stage. The exogenous lipid absorption was less in the early vitellogenic stage, and the activity of PL was low. The activity of PL increased significantly in the mid vitellogenic stage, and up to the peak in November, almost 3 times over than the early stage, indicated that the exogenous lipid absorption also reached a peak in this period. There was no significant difference between the activity of PL of the different treatment groups(P>0.05), the activity of PL generally down to a minimum to response to the limitation of the excessive lipid intake. PL activity gradually increased in the spawning season to replenish the consumption of lipids. The activity of 3S∶0F and 1S∶2F were all higher than the rest groups during the different vitellogenic stages, and the PL activity of 3S∶0F was 2.2 times over than 0S∶3F in the early, middle vitellogenic stages and spawning season, and even reached 4.4 times of 0S∶3F in the early stage(P<0.01), indicated that n-3 PUFAs had a significant inhibition effect on PL. In addition, the dietary could also influenced the lipid absorption affecting the histological structure of the small intestine: the villus length of 3S∶0F was only about 7/9 of the rest groups(P<0.05). The V/C of 1S∶2F was almost 1.4 times of the 3S∶0F(P<0.05), indicated that the dietary PUFAs could increase the maturity of the enterocyte by increasing the length of the small intestinal villus and V/C value, to gain the ability of lipid absorption.(2) The effects on lipid transfer processLipids were broke down by lipolytic enzymes after absorbed into the blood, and were transferred to the liver by carrier proteins to affect the levels of serum lipids.The activity of LPL and HL in the liver all reached to the peak in the early vitellogenic stage. The activity of LPL and HL changed with n-6 PUFAs in the dietary, and the highest activity occurred in 2S∶1F, almost 3 times of 0S∶3F(P<0.05), even 4 times in LPL(P<0.01). The activity of HSL down to a minimum at the same time, and the HSL activity of 0S∶3F was significantly higher than the rest groups(P<0.05), even achieved about 1.4 times of 3S∶0F, indicated an improving effect of PUFAs on HSL. Considered of the changes of the lipolytic enzymes, explained this time was mainly composed of endogenous lipid mobilization. The HDL-C fell to the minimum, while the LDL-C increased to the peak, indicated that a large number of lipids were transferred to the ovaries from the tissues such as the liver, resulting in decreased serum lipid levels, especially TG, down to a minimum. But PUFAs had no significant effects on HDL-C, LDL-C and TG, the content of TC of 0S∶3F was significantly lower than 3S∶0F and 1S∶2F(P<0.05), as a response to reduce the demanding for TC.In the mid-late vitellogenic stage, the activity of HL and LPL kept around two-thirds of the early stage, and the September in the middle stage were less than a half of the early stage, and the HSL activity reached to the peak at this time, it was a mark that the exogenous decomposition of lipid increased while the endogenous decomposition of lipid decreased. HL and LPL were significantly affected by dietary PUFAs in September of the middle stage and the late stage, and HSL activity was significantly affected by dietary PUFAs in the mid-late vitellogenic stage, the exogenous lipids were the main lipid nutrition source at this time. HDL-C content gradually increased to the peak in the late vitellogenic stage, but not significantly influenced by dietary PUFAs(P>0.05). LDL-C content fell to the minimum in September of the middle vitellogenic stage, less than one-sixth of the early stage. In September of the middle vitellogenic stage, dietary PUFAs significantly increased the expression of L-FABP, and 0S∶3F was 2 times over than the rest groups(P<0.05). Dietary PUFAs directly inhibited LDL-C to transfer the lipids significantly increased the expression of L-FABP and indirectly increased the content of HDL-C, accumulated more lipids for exogenous vitellogenisis. LDL-C restored and maintained at a high level in November of the middle stage and late stage to prevent the excessive accumulation of lipids on the liver. A significant increase of serum lipids in the middle stage, and reached to the peak in November, especially TG, almost 5.5 times more than the early stage, which were the results of exogenous lipids intake. Exogenous vitellogenisis reduced in the late vitellogenic stage, less lipids intake and slightly lower serum lipids.During the spawning season, there was no significant influence of the dietary PUFAs on the expression of L-FABP and the activity of HL and LPL of the liver. The activity of an HSL gradually reduced, and was influenced by the dietary PUFAs significantly. The content of HDL-C and LDL-C was gradually reduced, and the serum lipids down to a minimum in the late spawning season. With the ending of the vitellogenisis in the spawning season, the energy demand of the body was greatly reduced.(3) Effects on lipid utilization processIn the early stage, the activity of hepatic FAS fell to a minimum, fish oil added groups were significantly higher than 3S∶0F. In the mid-late vitellogenic stage, FAS activity gradually increased, and the peak appeared in September of the middle stage, about 1.5 times more than the early stage, suggested that the lipid synthesis metabolism in the liver was active. The FAS activity of 0S∶3F was significantly higher than 1S∶2F(P<0.05)in November of the middle stage. In the late vitellogenic stage, the activity of FAS of 0S ∶ 3F was significantly higher than the rest groups(P<0.05), indicated that the ability of de novo synthesis of the liver was regulated directly by dietary PUFAs in the mid-late stage. FAS activity fell significantly in the late spawning season, obviously affected by dietary PUFAs. After the “De novo synthesisâ€, parts of the lipids were transferred to the muscle to store, and the rest were deposited in the liver. So the influence of dietary PUFAs on the composition of fatty acid in muscle and liver could be used as an important index to evaluate the process of the utilization of lipids. Dietary PUFAs had a more significant influence on the fatty acid composition of the muscle than the liver, in the liver only C12∶0, C18∶2n6t, C22∶1n9, EPA and n-3/n-6 PUFAs were significantly affected by the dietary PUFAs, while in the muscle the fatty acids were all significantly affected by the dietary PUFAs(P<0.05), except C18∶2n6c,C18∶3n3,C23∶0 and n-3/n-6 PUFAs. The liver accumulated lots of MUFAs(mainly C18∶1n9c) and SFAs(mainly C16∶0), the liver also had the most kinds of fatty acids than the dietary and the muscle. |
