3. Results
3.1. Elevated CO2 significantly inhibited growth.
CO2 concentration affected the weight gain rate (WGR), specific growth rate (SGR), feed coefficient (FCR), and condition factor (CF) (Fig. 1). At the initial stage of culture (at 7 d), there was no significant difference among the groups. Over time, differences in growth among the treatments became evident. WGR and SGR, which characterize juvenile growth, declined linearly with increasing CO2 exposure (Fig. 1A, B). At 14 d, growth stratification appeared between the groups, increasing with concentration and time (Fig. 1). At 60 d, the differences between the 32 mg/L treatment and control were almost three-fold for WGR (27.86% ± 3.32% and 118.39% ± 6.89%, respectively) and SGR (0.41% ± 0.04% and 1.30% ± 0.05%, respectively). In contrast, FCR was positively associated with CO2 concentration (Fig. 1C), being highest at 32 mg/L. CF, which varied slightly among the treatments and over time, was generally poor at high CO­2 levels.
Elevated CO­2 caused stress that reduced fish health. CSR declined with increasing CO2 concentration (Fig. 2A), with significant differences between the control and treatments. CSR was lowest at 32 mg/L, at only 68.42%, which was 29.83% lower (P < 0.05) than in the control group (Fig. 2A).
In the fish sampled on day 30, HSI and KSI decreased with increasing CO2, and were significantly lower at 32 mg/L than at 0 and 8 mg/L (Fig. 2B, C). In contrast, SSI was significantly higher at 16, 24, and 32 mg/L than at 0 and 8 mg/L (Fig. 2D).
3.2. CO2 concentration affected Hb oxygen-carrying capacity.
Plasma Hb (in g/L) decreased significantly with increasing CO2 (Fig. 3A), from 76.69 ± 3.81 in the control, to 74.23 ± 3.80 at 8 mg/L CO2, 71.42 ± 2.21 at 16 mg/L, 70.72 ± 2.90 at 24 mg/L, and 70.15 ± 3.78 at 32 mg/L, which had the most highly significant change (Fig. 3). Total methemoglobin (in g/L) increased significantly with increasing CO2concentration: it was 0.29 ± 0.05 at 32 mg/L CO2, the most significant difference relative to the control (0.15 ± 0.01).
CAT, SOD, and GPX are important antioxidant enzymes that protect cells from oxidative damage caused by ROS. On day 60, SOD, GPX, and CAT levels did not differ significantly between 0 and 8 mg/L CO2; GPX levels did not differ significantly between 0 (or 8) mg/L and 16 mg/L, but did differ significantly between 24 and 32 mg/L (Table 2). SOD activity was significantly lower at 16 mg/L than at 24 and 32 mg/L. CAT activity was significantly lower at 16 and 24 mg/L than at 32 mg/L. Overall, antioxidant enzyme activity increased with CO2concentration. Lysozyme levels increased significantly with increasing CO2: they were not significantly different between 0 and 8 mg/L CO2, but were significantly higher in the other groups.
Blood pH first declined with increasing CO2concentration, then increased significantly, with the most significant increase at 32 mg/L (Table 3).
3.3. Liver injury became more pronounced with age.
Relative to the control (Fig. 4A), elevated CO2 caused varying degrees of liver damage (Fig. 4B, C, D, E), predominantly involving hepatocyte vacuolization, nuclear atrophy and deformation, and vascular congestion in the sinus or portal vein. Relative to the control, plasma glutamate pyruvate transaminase (GPT) and glutamate oxalate transaminase (GOT) were significantly higher following CO2 treatment; GPT was highest at 32 mg/L (Fig. 5A, B), consistent with the liver injury results (Fig. 4).
3.4. CO2 stress reduced fish growth.
To assess whether elevated CO2 affects GH/IGF-1 levels, we measured the expression of four genes, GHR, IGF-1, IGF-1R, and thyroid hormone receptor (THR). GHR expression was significantly lower following CO2 treatment than in the control, and was lowest at 32 mg/L (Fig. 6C). IGF-1 and IGF-1R expression decreased similarly with increasing CO2 (Fig. 6A, B), and was lowest at 60 d at 32 mg/L CO2. THR expression declined substantially with CO2 concentration: at 32 mg/L, it was significantly lower than in the other groups (Fig. 6D). These results indicate that CO2 stress substantially inhibited fish growth.