Effects of hypoxia and reoxygenation on antioxidation, respiratory 

metabolism and tissue structure of Marsupenaeus japonicus

ZHAO Sizhe1,6， HUA Songsong1， LI Yongchuang1， JIANG Haiyi1， 

ZHANG Qingqi5， WANG Panpan1,2,3， GAO Huan1,2,3,4， YAN Binlun1,2,3,4

(1. Jiangsu Key Laboratory of Marine Biotechnology, College of Marine Science and Fisheries, Jiangsu Ocean 

University, Lianyungang Jiangsu222005, China; 2. CoInnovation Center of Jiangsu Marine Bioindustry Technology, 

Huaihai Institute of Technology, Lianyungang Jiangsu222005, China; 3. Jiangsu Provincial Infrastructure for 

Conservation and Utilization of Agricultural Germplasm, Nanjing210014, China; 4. Jiangsu Institute of 

Marine Resources Development, Lianyungang Jiangsu222005, China; 5. Lianyungang Qiming Aquatic Products Co., 

Ltd., Lianyungang Jiangsu222005, China; 6. Jiangsu Haorun Biological Industry 

Group Co., Ltd., Taizhou Jiangsu225300, China)

Abstract: As one of the necessary factors for the survival of aquatic animals, dissolved oxygen (DO) plays an important role in growth, development and reproduction. Human activities, global warming, water eutrophication and other phenomena have led to the formation of “low oxygen zones” in the ocean, which have seriously damaged the marine ecological environment and endangered the health of aquatic animals. At the same time, in today’s highdensity breeding mode, excessive breeding density and excessive feed feeding are easy to cause transient or persistent hypoxia in the water body. However, relying on the traditional aeration and aeration methods alone can only solve the problem fundamentally and increase the cost of breeding. And with the development of society and the continuous improvement of human living standards, people’s demand for fresh aquatic products is increasing day by day. In the process of transportation, hypoxia is very likely to occur, which will make aquatic animals have a stress response and eventually lead to death, resulting in serious economic losses. Therefore, studying the effects of hypoxic stress on aquaculture species is very necessary to guide the healthy farming and transportation of aquatic animals. Generally speaking, we refer to the water body with dissolved oxygen content below 2 mg· L-1 as hypoxic water body, and in normal water body, the dissolved oxygen content of lower layer water is often the lowest. Marsupenaeus japonicus is an important economically cultured shrimp in China. Compared with Litopenaeus vannamei, the yield per unit area of M. japonicus has always been at a low level. The reason is that its habits of burrowing sand and other benthic lives restrict its highdensity cultivation. The lower dissolved oxygen content in the bottom water will not only affect the survival rate of its breeding, but also affect the growth rate and breeding density to a certain extent. Therefore, in order to explain the effects of hypoxia reoxygenation process on the physiology of M. japonicus, this study took M. japonicus as the research object, carried out an acute hypoxia reoxygenation stress experiment, measured the changes of antioxidant enzymes(CAT,GSHPX,SOD) and respiratory metabolic enzymes(PK,PFK,SDH,HK) in the hepatopancreas, gills and muscle tissues of M. japonicus at different time points, and observed the changes of body tissue structure. In this experiment, healthy and vigorous M. japonicus with an average body weight of (0.80±0.06) g and an average body length of (4.7±0.2) cm was selected for temporary culture for 3 days at (25.0±0.3) ℃, with a salinity of 28.0±0.5, pH of 8.0 ± 0.2. During the temporary culture, oxygen was continuously charged to maintain the DO content at (6.7 ± 0.2) mg· L-1. After the temporary culture, 100 shrimps with similar size and good vitality were selected and distributed evenly into 5 cultivation tanks (60 L, 50 cm × 40 cm × 30 cm) for experiments. The experiment was conducted in the control group (0 h) and treatment groups. The DO content in the water of the control group was (6.7 ± 0.2) mg· L-1. The treatment group included two stages: hypoxia (024 h) and reoxygenation (2436 h). In the hypoxia stage, DO content was reduced by adding Na2SO3, DO content was reduced to (1.6 ± 0.2) mg· L-1 within 30 min, and was restored to (6.7 ± 0.2) mg· L-1 by continuous aeration in the reoxygenation stage. The safety and stability of the hypoxic treatment with Na2SO3 was proved by preexperiment before the experiment. Na2SO3 was added to the water before the experiment officially started, and the experiment started immediately when the DO content dropped to (1.6 ± 0.2) mg· L-1, and DO content was detected every 30 min, and DO level of water body was maintained by aeration and adding Na2SO3. After the start of the experiment, 9 shrimps were randomly selected at each time point of 0, 3, 6, 9, 12, 24, 36 h, and the hepatopancreas, muscle and gill tissues were collected for antioxidant and respiratory metabolism. The hepatopancreas and gill tissues of 3 shrimps were taken at 0, 24, 36 h for section observation. The results showed that the antioxidant capacity of M. japonicus had significant changes at different time points (P<0.05). CAT, GSHPX, SOD activity and MDA content in hepatopancreas tissues in hypoxia stage showed a trend of first rising and then declining, and their activities decreased to the lowest level in 24 h, while the activities of antioxidant enzymes and MDA content in reoxygenation stage showed an upward trend. In muscle and gill tissue, GSHPX, SOD activity and MDA content increased first and then decreased with continuous hypoxia, while CAT activity continued to decrease. During the reoxygenation stage, the activities of various enzymes and the content of MDA increased significantly (P<0.05). The effect of hypoxiareoxygenation on the respiration and metabolism of M. japonicus was also significant (P<0.05). The activities of PFK and HK in the hepatopancreas and gill tissues first increased and then decreased with the continuous hypoxia, while the activities of SDH decreased significantly. In muscle tissue, the activities of PFK, HK, SDH and PK all showed a downward trend with the continuous hypoxia, and reached the lowest value at 24 h. During the reoxygenation stage, the activities of PFK, HK, SDH and PK in hepatopancreas and gill tissues all showed an upward trend, while PFK in muscle tissue decreased significantly (P<0.05). Under hypoxic stress, LD content increased significantly in all tissues (P<0.05), and decreased significantly in the reoxygenation stage (P<0.05). It can be seen from the results of tissue sections that with the continuous hypoxia, the hepatopancreas basement membrane is damaged, the volume of transport vacuoles significantly increases, the lumen is deformed, the shape is distorted and irregular, and there are significant granular substances in the lumen, the number of storage cells is significantly reduced, and the hepatopancreas structure is further damaged after reoxygenation. At the same time, the gill tissue structure is damaged under hypoxia: the gill filament is bent and swollen, the cuticle is broken, the secondary lamella is swollen, the epithelial cells are partially shed and swollen, and the lymphocytes are arranged loosely and disorderly. The damage of the gill tissue structure is improved after reoxygenation. By exploring the adaptation and regulation mechanism of M. japonicus to hypoxia stress, this study will help in providing some guidance and suggestions for the culture and management strategy of M. japonicus, improving the survival rate and culture density of M. japonicus, and pointing out the direction for breeding new varieties resistant to hypoxia in the future.

Keywords: Marsupenaeus japonicus；hypoxia stress；reoxygenation；enzyme activity；tissue structure