Physiological Response Under Hypoxia Stress And The Regulation Effect Of Feeding Earthworm Liquid In Genetically Improved Farmed Tilapia(Oreochromis Niloticus)

Dissolved oxygen is an essential environmental factor for aquatic organisms.Hypoxia stress can affect fish homeostasis,leading to tissue and cell damage,thus endanger fish health and seriously restricts the development of aquaculture.Therefore,it is of great significance to explore the adaptive mechanism and mitigation methods of fish under hypoxia stress.In this study,Genetically improved farmed tilapia(GIFT,Oreochromis niloticus)was selected as the research object.We explored the effects of hypoxia stress on serum biochemical and liver antioxidant indexes,tissue structure and cell apoptosis of GIFT,and whether different levels of earthworm liquid supplemented in diets can alleviate hypoxia stress of GIFT to some extent.The main results of this study are as follows:1.Effects of hypoxia stress on antioxidant and immune response in liver of GIFTThis study selected 240 GIFT with an average body weight of 30 ± 0.22 g for acute hypoxia stress,setting the hypoxia group(DO = 0.55 mg/L)and the control group(DO =5.0 mg/L),each group of three repeats.Samples were taken at 0,2,8,24,48 and 96 h to determine the effects of antioxidant indexes,tissue structure and cell apoptosis in liver of GIFT under hypoxia stress by ELISA method,HE and TUNEL staining method.The results showed that with the prolongation of hypoxia stress time,the activities of SOD,CAT and GSH-Px in liver of GIFT were gradually increased;disordered liver cells,significantly reduced cytoplasm,and significant vacuolization in liver cells;the number of apoptotic cells in liver of the hypoxia group was significantly higher than that of the control group.These results indicated that hypoxia stress could cause liver injury and promote cell apoptosis of GIFT.2.Cloning,distribution and expression analysis of CaSR gene of GIFT under hypoxia stressIn this study,the c DNA full-length sequence of CaSR gene of GIFT was cloned by RT-PCR,and the distribution in tissues of CaSR gene was detected by quantitative real-time PCR(q RT-PCR).The results showed that the c DNA full-length sequence of CaSR gene of GIFT was 3265 bp in length,including 21 bp at the 5′-UTR,421 bp at the3′-UTR,and a 2823 bp open reading frame(ORF)encoding 940 amino acids.It has high similarity to Nile tilapia and Oreochromis mossambicus,and the homology of nucleotide and amino acid reached the 99 %.The overall structure of the predicted protein was consistent with that of known species CASR,indicating that the expression of CaSR gene may be highly conserved among different species.The q RT-PCR result showed that CaSR gene mRNA was expressed in brain,gill,heart,liver,kidney,spleen,stomach,intestine and muscle of GIFT,with the highest expression in muscle,followed by kidney.In order to better explore the adaptation mechanism of GIFT under hypoxia stress,q RT-PCR was used to detect the expression changes of CaSR and apoptosis-related genes(Bcl-2,Caspase-3and P53)mRNA in liver of GIFT under hypoxia stress(DO = 0.55 mg/L).The results showed that with the prolongation of hypoxia stress time,the mRNA expression levels of CaSR,Bcl-2,Caspase-3 and P53 genes of the hypoxia group were firstly increased and then decreased.These results suggested that CaSR might induce changes of apoptosis-related regulatory factors by regulating calcium homeostasis,and mediate apoptosis,thus participating in the molecular mechanism of hypoxia response of GIFT.3.Effects of adding different levels of earthworm liquid supplemented into diets on hypoxia stress of GIFT240 healthy GIFT juveniles with an average body weight of about 3.0 ± 0.16 g were selected and randomly divided into four groups,three of the group for earthworm liquid adding level(the proportion of adding earthworm liquid in diets was 0 %,0.05 % and 0.4 %,respectively),the other as the negative control group,each group of three repeats.After feeding for 60 days,acute hypoxia stress(DO = 0.55 mg/L)was applied to three earthworm liquid supplemental groups,and 5.0 mg/L was applied to the control group.Samples were taken at 0,24 and 96 h to determine the effects of different levels of earthworm liquid on serum biochemical and liver antioxidant indexes of GIFT under hypoxia stress by biochemical method and ELISA method.These results indicated that hypoxia stress could damage liver tissue of GIFT,make its metabolism disorder,affect blood glucose,lipid metabolism and antioxidant capacity,and reduce the body immunity.However,adding earthworm liquid in diets could slow down the decrease of serum total protein(TP),alkaline phosphatase(ALP)and lysozyme(LZM)levels,but increase the activity of alanine aminotransferase(ALT)in serum at 0 h.The content of MDA in 0.4 % earthworm liquid supplemental group decreased to the level of control group at 96 h,and the activity of SOD increased.These results indicated that adding earthworm liquid in diets could improve antioxidant and immune function of GIFT to some extent,and when the supplemental level of earthworm liquid was too high,it might cause damage to liver of GIFT.At the same time,in order to further explore the role of earthworm liquid in liver damage induced by hypoxia stress,we detected the effects of different levels of earthworm liquid supplemented in diets on the expression of CaSR gene in liver of GIFT under hypoxia stress.The results showed that the mRNA expression level of CaSR gene of 0.05 % and 0.4 % earthworm liquid supplemental groups was significantly lower(P < 0.05)than that of the other two groups at0 h;the mRNA expression level of CaSR gene of 0.4 % earthworm liquid supplemental group was lower than that of 0 % and 0.05 % at 24 and 96 h,and had no significant difference compared with the control group(P > 0.05).These results indicated that the addition of earthworm liquid in diets could inhibit excessive cell apoptosis caused by tissue damage.