**2. Materials and Methods**

#### *2.1. Experiment Design and Sample Collection*

Juvenile yellowfin tuna used in the experiment were cultured in offshore sea cages near Xincun Harbour, Xincun Town, Lingshui County, Hainan Province. It was temporarily raised for seven days in the Tropical Aquaculture Research and Development Center, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. A total of 60 fish were randomly collected before the experiment and transferred into two indoor cement tanks (5 m in diameter and 2.5 m deep) with a recirculating water system. The water temperature was controlled at 28.0 ± 0.5 ◦C, dissolved oxygen > 5.27 mg/L, ammonia nitrogen < 0.1 mg/L, pH 7.57 ± 0.12, salinity 32‰. Fish were fed daily from 08:30 to 09:00. Fresh miscellaneous fish (4 cm × 2 cm pieces; *Trachurus japonicus, Mene maculata*) were fed with 5–8% body weight daily by satiety.

Upon the experiment conducted, the mean body length and wet weight were 28.03 ± 1.78 cm and 503.23 ± 36.78 g, respectively. At the beginning of the experiment, 60 fish were randomly collected and divided into two groups. The temperature of the control group and the high-temperature group were set at 28 ◦C (control group) and 34 ◦C (HT group) in 3000 L tanks with three replicates each. The rising speed of water temperature is 2 ◦C/h, and the timing starts when the temperature reaches 34 ◦C. The water temperature of the HT group was raised and maintained by heating rods (Sensen Group Co., Ltd., Zhoushan, China)

The sampling time was at 0 h, 6 h, 24 h, and 48 h after stress. When the stress time was up, the samples were taken immediately. Three fish from each rearing tank were randomly collected and anesthetized with 0.03% MS-222, and body length and weight were measured. Liver, gill, red muscle, white muscle, and blood were taken. Blood was taken from the caudal vein, the white muscle from 2 cm below the dorsal fin, and the red muscle from near the spine perpendicular to the dorsal fin. Each tissue was stored in 2 mL RNA-free tubes at −80 ◦C until enzyme activity measurement. Store the extracted blood in a 2 mL centrifuge tube and use a desktop high-speed freezing centrifuge (EXPERT 18K-R) for centrifugation (temperature 4 ◦C, rotating speed 3000 R·min−1, lasting for 10 min) after standing for 1 h, and store it at −80 ◦C until analysis.

#### *2.2. Enzyme Activity Measurement*

All the tissue samples were partially thawed and homogenized mechanically using a tissue homogenizer on ice. The suspensions were centrifuged according to the requirements of the kits and the protein content in the supernatant was determined by the BCA Protein Assay kit (A045-4-2). The activities of SOD (A001-3-2) and MDA (A003-1-2) contents in the gill, liver, red muscle, and white muscle were measured. The concentration of COR (H094), triglyceride, cholesterol, ALP, osmotic blood glucose, lactic acid, K+, Na+, Cl−, C3, and C4 in the serum were measured, and the concentration of ALP in the liver was measured. All of the above assays were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in triplicates. Ion concentration, osmotic pressure, glucose, and lactic acid in serum were measured by a blood gas analyzer (PL2000 Plus, Nanjing Perlang Medical Equipment Co., Ltd., Nanjing, China), triglyceride, cholesterol, ALP, C3, C4, LDH, and liver antioxidant index ALP, LDH was measured by the biochemical analyzer (PVZS-300X, Beijing Prolong New Technology Co., Ltd., Beijing, China). The above measurements using the blood gas analyzer and biochemical analyzer were carried out in accordance with the instructions in triplicates.

#### *2.3. Calculations and Statistical Analysis*

Excel 2021 software was used for data sorting, Origin 2021 was used for drawing, and SPSS 26.0 software was used for significant difference analysis. Comparisons between different groups at the same time were conducted by independent *t*-test, comparisons between different times in the HT group were conducted by independent one-way ANOVA and least significant difference (LSD) test, and significant difference was set at *p* < 0.05.

#### **3. Results**

*3.1. Changes in Serum Indexes of Juvenile Yellowfin Tuna under an Acute Temperature Rise* 3.1.1. Changes in Ion Concentration, Osmotic Pressure, Blood Glucose, and Lactic Acid in the Serum of Juvenile Yellowfin Tuna under an Acute Temperature Rise

The osmotic pressure of the HT group (Figure 1a) showed a trend of increasing first and then decreasing with time, reaching the highest value at 24 h. There was a significant difference between 6 h, 24 h, and 48 h. At 6 h and 48 h, the osmotic pressure of the HT group was lower than the concentration in the control group and higher than the control group at 0 h and 24 h (Figure 1b). At 0 h, the osmotic pressure between the HT group and 28 ◦C had no significant difference, but there was a significant difference at 6 h, 24 h, and 48 h. The blood glucose in the HT group did not change with time (Figure 1c), there was a gradual increase, but it was not significant. At 24 h, the blood glucose in the HT group was higher than the concentration in the control group and lower than the concentration in the control group at other time points (Figure 1d). At 0 h and 24 h, there was no significant difference in blood glucose levels between the HT group and control group, but there was a significant difference at other time points. There was no significant difference between the lactic acid groups in the HT group (Figure 1e), but it fluctuated up and down, but not significantly (Figure 1f).

The K+ concentration in the HT group (Figure 1g) showed a trend of first decreasing and then increasing with the prolongation of time. There was no significant difference between 0 h and 6 h, 24 h, and 48 h. At 48 h, K+ in the HT group was lower than in the control group and higher than in the control group at other time points (Figure 1h). The concentration of Na<sup>+</sup> and Cl<sup>−</sup> increased first and then decreased with the extension of stress time, with the same trend, and reached the peak at 24 h. Na<sup>+</sup> concentration in the HT group (Figure 1i) had significant differences at 6 h, 24 h, and 48 h. At 24 h, the Na<sup>+</sup> concentration in the HT group was higher than that in the control group and lower than that in the control group at other times (Figure 1j). The concentration of Cl− in the HT group (Figure 1k) was significantly different between adjacent groups. At 24 h, the Cl− concentration in the HT group was higher than that in the control group and lower than that in the control group at other times (Figure 1l). The concentration order of three ions in each group was Na+ > Cl<sup>−</sup> > K+.

**Figure 1.** *Cont*.

**Figure 1.** *Cont*.

**Figure 1.** Changes in osmotic pressure (**a**), osmotic pressure value (**b**), blood glucose (**c**), blood glucose value (**d**), lactic acid (**e**), lactic acid value (**f**), K+ concentration (**g**), K+ value (**h**), Na+ concentration (**i**), Na+ value (**j**), Cl<sup>−</sup> concentration (**k**) and Cl<sup>−</sup> value (**l**) in the serum of young yellowfin tuna under acute high–temperature stress. The value is the gap of the experimental group minus the control group. Red means down and green means up. The significantly different between the control group and the HT group was represented by a -. Different and the same letters indicate a significant difference (*p* < 0.05) and insignificant difference (*p* > 0.05).

3.1.2. Changes in Serum Cortisol in Juvenile Yellowfin Tuna under an Acute Temperature Rise

After the acute temperature increase, the cortisol in the HT group first decreased and then increased with the prolongation of stress (Figure 2a), and there was no significant difference between adjacent groups. Among them, the difference between the HT group and the control group (Figure 2b), during 48 h, the HT group was higher than the control group. There was no significant difference in serum cortisol concentration between the control group and the HT group at 0 h, 6 h, and 24 h, but there was a significant difference between the control group and the HT group at 48 h.

**Figure 2.** Changes of serum cortisol (**a**) and cortisol value (**b**) in young yellowfin tuna under acute high-temperature stress. The value is the gap of the experimental group minus the control group. Red means down and green means up. The significantly different between the control group and the HT group was represented by a -. Different and the same letters indicate a significant difference (*p* < 0.05) and insignificant difference (*p* > 0.05).

3.1.3. Changes of Metabolic Indexes in Serum of Juvenile Yellowfin Tuna under an Acute Temperature Rise

The serum triglyceride and cholesterol concentration in the HT group (Figure 3a,c) showed a trend of first decreasing and then increasing with time, there were significant differences between the groups. The concentration of serum triglycerides in the HT group was lower than the corresponding concentration in the control group at four times point (Figure 3b). There was no significant difference between the HT group and the control group at 0 h, but there were significant differences at the other points. The cholesterol concentration in the HT group was lower than the control group at 6 h and higher than at the other points (Figure 3d). There was no significant difference in serum cholesterol concentration between the HT group and 28 ◦C at 0 h, but there was a significant difference between 6 h, 24 h, and 48 h. The activity of alkaline phosphatase in the serum of the HT group (Figure 3e) showed a trend of first decreasing and then increasing. The lowest value was reached at 6 h, and it reached to gradually stable after 6 h. The activity of alkaline phosphatase of the HT group was higher than the control group all the time. There was no significant difference in serum alkaline phosphatase concentration between the HT group and the control group at 0 h, but there was a significant difference at the other times point.

**Figure 3.** Changes in triglyceride (**a**), triglyceride value (**b**), cholesterol (**c**), cholesterol (**d**), alkaline phosphatase (**e**), and alkaline phosphatase value (**f**) in serum of young yellowfin tuna under acute high–temperature stress. The value is the gap of the experimental group minus the control group. Red means down and green means up. The significantly different between the control group and the HT group was represented by a -. Different and the same letters indicate a significant difference (*p* < 0.05) and insignificant difference (*p* > 0.05).

3.1.4. Changes of Immune Indexes in Serum of Juvenile Yellowfin Tuna under an Acute Temperature Rise

After acute temperature increase, the concentration of C3 (Figure 4a) in serum in the HT group showed a downward trend, and it remained stable with time, there was no significant difference between 6 h, 24 h, and 48 h. At 0 h, there was no significant difference between the HT group and 28 ◦C, and there was a significant difference at other time points. The C3 concentration in the serum of the HT group was higher than the corresponding concentration in the control group at 0 h and 6 h, and lower than the corresponding concentration in the control group at 24 h and 48 h. The concentration of complement C4 (Figure 4c) in the serum of the HT group showed a trend of first decreasing and then increasing with the prolongation of time, and there was a significant difference between each time point. At 0 h, there was no significant difference between the HT group and 28 ◦C, and there was a significant difference between 6 h, 24 h, and 48 h.

**Figure 4.** Changes in complement C3 concentration (**a**), C3 value (**b**), complement C4 concentration (**c**) and C3 value (**d**) in serum of young yellowfin tuna under acute high–temperature stress. The value is the gap of the experimental group minus the control group. Red means down and green means up. The significantly different between the control group and the HT group was represented by a -. Different and the same letters indicate a significant difference (*p* < 0.05) and insignificant difference (*p* > 0.05).
