| Litopenaeus vannamei is one of the main cultured shrimp species in China.Under the condition of high density culture,the high concentration of ammonia nitrogen and nitroso nitrogen often affects the growth and survival of Litopenaeus vannamei.This study investigated the response of Litopenaeus vannamei to ammonia and nitrite stress in terms of growth performance,activities of ammonia metabolic enzymes,antioxidant enzymes and hepatopancreas cell apoptosis.1.Effects of ammonia subacute stress on growth,ammonia metabolism,antioxidant and apoptosis related indexes of Litopenaeus vannameiSet ammonia nitrogen concentration 0.02mg/L as control group(C_A).According to the set concentration of 0.9mg/L(S1),5.2mg/L(S2)and 12mg/L(S3),10g/L mother liquor of 9ml,52ml and 120ml were added into 100L test water,respectively,and three ammonia nitrogen stress treatment groups were set up.Litopenaeus vannamei were placed in each ammonia nitrogen concentration groups under stress for 40d,and sampled at 20d and 40d.After 20 days of stress treatment,the body weight growth rate and survival rate decreased,the contents of ammonia nitrogen and urea nitrogen in hemolymph increased,and the activities of glutamate dehydrogenase(GDH),glutamine synthase(GS)and catalase(CAT)and the relative expressions of cyt c and caspase 3gene in hepatopancreas increased with the increase of ammonia nitrogen concentration.After 40 days of stress treatment,the activities of GS,T-SOD and CAT in hepatopancreas in S2 and S3 groups were significantly lower than C_A group(P<0.05),the relative expression levels of cyt c and caspase 3 genes in hepatopancreas were significantly higher than C_A group(P<0.05).TUNEL showed that the number of hepatopancreas apoptosis cells increased with the increase of ammonia stress time and concentration.The results showed that ammonia subacute stress adversely affected ammonia metabolism and antioxidant system of hepatopancreas of Litopenaeus vannamei,induced hepatopancreas cell apoptosis,and caused certain damage to hepatopancreas.2.Effects of time after ammonia subacute stress on ammonia metabolism,antioxidant and apoptosis related indexes of Litopenaeus vannameiSet ammonia nitrogen concentration 0.02mg/L as control group(C_A’).According to the set concentration of 0.9mg/L(S1’),5.2mg/L(S2’)and 12mg/L(S3’),10g/L mother liquor of 9ml,52ml and 120ml were added into 100L test water,respectively,and three ammonia nitrogen stress treatment groups were set up.Litopenaeus vannamei were placed in each ammonia nitrogen concentration group for 10 days.Samples were taken at 0h,1h,6h and 12h,respectively.After stress treatment,the contents of ammonia nitrogen and urea nitrogen in hemolymph increased with the increase of treatment concentration,and the activities of hepatopancreas GDH,GS,T-SOD,CAT and total antioxidant capacity(T-AOC)in S1’group were significantly higher than C_A’group(P<0.05),the relative expression levels of hepatopancreas cyt c and caspase 3 gene in the stress treatment group were significantly higher than C_A’group(P<0.05).After stress,the contents of ammonia nitrogen and urea nitrogen in hemolymph and the relative expression level of hepatopancreas cyt c and capase 3 gene in S1’group were not significantly different from those in the control group at 6h(P>0.05),there was no significant difference in hepatopancreas GDH and GS activities between S1’group and C_A’group at 12h(P>0.05);There was no significant difference in haemolymph ammonia nitrogen content and hepatopancreas GDH and GS activities between the S3’group and the control group at 12h(P>0.05),but the hemolymph urea nitrogen content,hepatopancreas T-SOD,CAT enzyme activities and hepatopancreas cyt c,caspase 3gene relative expression level at 12h were significantly higher than C_A’group(P<0.05).In conclusion,S3’ammonia subacute stress has certain effect on hepatopancreas ammonia metabolism,antioxidant system and cell apoptosis of Litopenaeus vannamei,and 12h stress time cannot restore the normal level.3.Effects of time after nitrite subacute stress on immune,antioxidant and apoptosis related indexes of Litopenaeus vannameiSet nitroso nitrogen concentration 0.02mg/L as control(C_N),three nitrogen-nitrogen stress treatment groups were set up by adding 5ml,14ml and 40ml of10g/L mother liquor to 100L test water at the concentration of 0.5mg/L(S_N1),1.4mg/L(S_N2)and 4mg/L(S_N3),respectively.The experimental group was divided into control group and stress treatment groups.The concentration of nitroso nitrogen in stress treatment group was 0.5,1.4 and 4mg/L.Litopenaeus vannamei were placed in each nitroso nitrogen concentration group for 10 days,and sampled at 0h,1h,6h and 12h,respectively.After stress treatment,the content of nitroso nitrogen in hemolymph and the relative expression of hepatopancreas cyt c and caspase 3 gene increased with the increase of treatment concentration,while the activities of hepatopancrea acid phosphatase(ACP)and alkaline phosphatase(AKP)decreased with the increase of treatment concentration.Hepatopancreas T-SOD,CAT and T-AOC enzyme activities in S_N1 and S2 groups were significantly higher than C_N group(P>0.05).After stress treatment,there was no significant difference in the content of nitrosonitrogen in hemolymph between the stress groups and C_N group at 6h(P>0.05),the relative expression levels of hepatopancreas ACP,AKP and hepatopancreas cyt c gene in S_N1group were not significantly different from C_Ngroup at 6h(P>0.05),the activities of hepatopancreas T-SOD,CAT and T-AOC in S_N1 group were not significantly different from C_N group at 12h(P>0.05),the relative expression levels of hepatopancreas ACP,AKP,CAT and hepatopancreas caspase 3 genes in S_N2 group were not significantly different from the control group at 12h(P>0.05).In conclusion,when the concentration of nitroso nitrogen was higher than S_N2,the subacute nitrite stress had certain effects on hepatopancreas immune system,antioxidant system and cell apoptosis of Litopenaeus vannamei,and 12h could not restore the normal level. |
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