| Pelidiscus sinensis is one of the most important aquaculture species in China,especially in the southern region.It is not only delicious but also contains high medicinal value.In recent years,with the development of the national economy and the improvement of the per capita living standard,the demand and purchase of Chinese soft-shell turtle are also increasing,which makes the production scale of Chinese soft-shell turtle growing and the high density intensive culture production becomes normal,but many problems also have been caused at the same time.The continuous deterioration of the aquaculture water environment and the severe degradation of germplasm resources have lead to frequent stress of the Chinese soft-shell turtle,resulting in frequent disease,reduced survival rate,and huge losses in aquaculture production.Stress refers to the situation where the body’s environment changes or other factors cause the body’s own homeostasis imbalance,which is widespread in all kinds of animals and plants..When the body is stimulated by the outside,it will increase the secretion of glucocorticoid(GC)by the adrenal cortex.Glucocorticoids are also called "adrenocorticotropic hormones".GC not only regulates the synthesis of proteins,carbohydrates,and lipids,but also regulates the metabolic processes of these substances.GC also has anti-inflammatory effects and is an important part of the organism’s response to external environmental stress.The glucocorticoids in reptiles such as turtles are mainly corticosterone(CORT).The stress response can regulate the body homeostasis to a certain extent and keep the body healthy.However,living in the long-term stress state will keep the corticosterone level elevated and inhibit the body’s immunity.It will cause great harm to its own growth and immunity.In this experiment,the levels of plasma corticosterone in different ages,genders,and body weights of Chinese soft-shelled turtle under normal growth conditions were studied and analyzed.The effects of temperature changes(10℃,20℃,33℃)on the CORT level,immune biochemical indexes and antibacterial effect of plasma in Chinese turtle,which have little difference in weight between 1winter age individuals,was studied with thermostatic control group of 28℃.The preliminary studies on the expression of CORT-related gene MC2R(melanocortin-2 receptor,also known as Adreno Cortico Hormones Receptor,ACTH-R)and NR4A1(Nuclear Receptor Subfamily 4 Group A Member 1,One of the transcription factors of corticosterone synthase)in Chinese sturgeon under different temperature and environmental conditions were also conducted.The experiment is intended to provide basic data for the research of temperature immunity of Pelidiscus sinensis and breeding production.The specific research content of the experiment is as follows:1.Study on the background level of Pelidiscus sinensisThe body weight of Chinese soft-shell turtle at different months ages showed a significant upward trend with the increase of the ages,and there was a significant difference in the body weight of Chinese turtle among different ages(P<0.01).With the increase of age,the content of CORT showed a decreasing trend,and the plasma CORT content of 12 months old individuals was significantly lower than that of 24 months(P<0.01).There was a extremely significant difference in body weight between male and female of different ages of Chinese turtle(P<0.01),but there was no significant difference in body weight between individuals of different genders within the same age(P>0.05).A linear analysis of the body weight and CORT content of Chinese turtle in each months and overall months(12,18,and 24 months of age)of age showed that there was a certain correlation between body weight and CORT content,and the content of CORT showed a downward trend with the increasing weight,but the correlation between them was not significant.2.Effect of temperature changes on plasma corticosterone content in Pelidiscus sinensisWhen the environmental temperature changed,the CORT content in plasma of Chinese soft-shell turtle at 10℃ and 20℃ changed first and then decreased with theincrease of treatment time,and there was a significant difference with the control group after 4 and 6 hours processed(P<0.05).The plasma CORT content in the 33℃ treated group fluctuates with the increase of treatment time,and there is no significant difference when compared with the control group(P>0.05).In addition,the content of CORT in each treatment group was higher than that in the control group except the group of 33-12(treated at 33℃ for 12 hours)which was slightly lower than that in the control group.3.Effects of temperature changes on plasma biochemical immune indices and bacteriostasis of Pelidiscus sinensisWith the increase of processing time of Chinese turtle under different temperature conditions,the plasma enzyme activity and protein content of turtle increased at first and then decreased,but there was no significant difference between treated groups and the control(P>0.05).Compared with the control group,the total content of lysozyme(LZM)increased,and the content of total protein(TP),albumin(ALB)and the activity of acid phosphatase(ACP)and alkaline phosphatase(ALP)have decreased overall.The antibacterial effect of plasma that come from the turtle of thermostatic control group(28℃)was the best in vitro.At the same time,the bacteriostatic effect gradually weakened with the increase of temperature,and the inhibitory effect of the 10℃ group was the worst.At the same temperature,with the increase of treatment time,the antibacterial effect gradually deteriorated and then recovered.What’s more,the antibacterial effect was worst after processed for 4 and 6 hours.We can see that the bacteriostatic effect of plasma is associated with changes in CORT levels,and with the increase of corticosterone levels the antibacterial effect of plasma will decrease.4.Effects of temperature changes for the expression of MC2 R and NR4A1 genes of Pelidiscus sinensis The expression of MC2 R gene in the adrenal gland of Chinese turtle is much higher than that in the heart,liver,spleen,kidney,muscle and brain,and that shows its specific expression pattern.The relative expression levels of MC2 R gene in the adrenal glands of each temperature-treated group were greater than that in the isothermal control group(28℃),and the gene expression of tissue increased with time.The expression of MC2 R gene and the CORT content of Chinese turtle in each temperature treatment groups showed a certain lagging tendency with the increase of temperature treatment time,butthe expression level of MC2 R gene was obviously increased compared with the control group.In addition,the relative expression levels of NR4A1 gene in the adrenal tissue of all temperature-treated groups showed a trend of decreasing first and then increasing.The expression levels of the 10℃ and 20℃ temperature groups decreased after treated for 4 hours,but then increased at 6 hours treating;What’s more,after treated for 12 hours the expression levels was close to the isothermal control group(28℃).The expression results of NR4A1 is basically the same with the changes in plasma corticosterone under different temperature conditions.The results of MC2 R and NR4A1 gene expression further confirmed from the molecular level that the temperature change did give a stress response to the Chinese soft-shelled turtle. |
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