Effect Of Air Exposure And Transportation Stress On Plasma Biochemical Profiles And Hsp70 Mrna Expression Of American Shad(Alosa Sapidissima)Broodstock

Handling stress is an unavoidable fish stressor in aquaculture,including water changing,pollution absorbing,fish hauling and capturing,and transportation etc.These operations lead to a series of stress responses on fishes involving fish metabolism,endocrine,immune system and so on,which have a huge negative impact on the farmed fish.We chose the mature American shad(Alosa Sapidissima)Broodstock(body weight 683.53±22.10 g)cultured in the greenhouse cement pool as the the research object.Air exposure and transport stress experiments were performed respectively.To investigate the effect of air exposure and transport stress on biochemical profiles in American shad,plasma biochemical profiles,enzyme activities in tissues,hormones levels and hsp70 mRNA expression of American shad were measured.1.Air exposure had a significant influence on the physiological and biochemical profiles of American Shad.The experiment was divided into four groups,including three treatment groups(air exposure time of 15s,30s and 60s,respectively)and control group(without air exposure).Compared with the control group,the level of RBC,PCV,HGB,CAT and T4 were significantly decreased(P<0.05)after air exposure of 15s,30s and 60s.However,the levels of GLU,TC,TG,TP,K+,Ca2+,SOD,MDA,LZM,GST,ALT,AST and cortisol were significantly increased(P<0.05)in all air exposure groups.Meanwhile,the level of MCH,Na+,Cl-and T3 in the air exposure groups showed no significant difference(P>0.05)compared with the control.SOD and CAT activities in gill were decreased significantly(P<0.05)after air exposure of 15s and 60s.The ALT and AST levels in the liver were increased significantly(P<0.05)after air exposure of 30s and 60s,and the content of glycogen in the liver was decreased significantly(P<0.05)in all air exposure groups.CK in heart only significantly increased(P<0.05)after air exposure of 60s and AChE activity was significantly decreased(P<0.05)after air exposure of 15s and 60s.2.Transportation stress also had a significant influence on the physiological and biochemical profiles of American Shad.The results showed that the levels of RBC,PCV,HGB,TG,SOD and T4 were decreased significantly during 2h or 4h after transport(P<0.05)compared with the precursor transport(0h).However,the levels of GLU,TC,K+,Cl-,MDA,LZM,GST,ALT,AST,cortisol and T3 were increased significantly during 2h or 4h after transport(P<0.05).Meanwhile,the levels of MCH,Na+,Ca2+ and CAT had no significant difference compared with the precursor transport(P>0.05).The activities of Na+-K+-ATP and CAT enzyme in the gill were decreased significantly(P<0.05)during 2h or 4h after transport.CAT activity and glycogen content in the liver were decreased significantly(P<0.05),but the levels of ALT,AST were increased significantly after transportation(P<0.05).The level of CK was significantly increased(P<0.05),but the activity of AChE was obviously decreased(P<0.05)after 4h transportation.Therefore,transportation stress led to the certain damage in the gill,liver and heart,influencing the physiological performance of American shad broodstocks.3.The full-length HSP70(Heat Shock Protein70)cDNA of American shad was cloned by RT-PCR(reverse transcription-polymerase chain reaction)and RACE(rapid amplification of cDNA ends)methods.The full-length of HSP70 cDNA was 2,545 base pairs(bp)long and contained a 1,914 bp open reading frame(ORF)encoding a peptide of 637 amino acids.The protein secondary structures contained three characteristics sequences of HSP70 family.The HSP70 amino acid sequences of American shad showed more than 84.0%identity with Astyanax mexicanu,73%and 38%identity with Drosophila auraria and Escherichia coli respectively by homology analysis,suggesting a highly evolutional conservation.4.The tissue distribution expressions of hsp70 mRNAs were detected in American shad by real-time PCR.Elevated levels of hsp70 mRNA expression were observed in the gill,muscle and head-kidney,while relatively high expression levels were present in the blood and heart,and lower levels were present in the liver,spleen,intestine and kidney.The hsp70 mRNA level was no significant change(P>0.05)in the gill and head-kidney after air exposure compared with the control group.The expression of hsp70 mRNA in the liver was significantly higher(P<0.05)after 15s,30s and 60s of air exposure.The raise of hsp70 mRNA level in the liver was not positively correlated with the intensity of handling stress.The expression of hsp70 mRNA presented a trend from increase to decrease in the gill and liver.While,it showed an increasing tendency in the head-kidney.In conclusion,air exposure and transportation stress had influences on physiological and biochemical profiles and tissue enzyme activities in the gill,liver and heart of American shad broodstocks.The function of the respiratory and circulatory system weakened when suffered stress.Oxidative stress led to the certain damage in the liver,gill and heart.The expression of hsp70 mRNA in the liver has a prompt response under air exposure and transportation stress.In view of the response complexity of hsp70 gene to different stressors,it still needs to further study of the response under various stressors,improving fish stress resistance for reasonable application.