Growth And Physiological Adaptation Of Grass Carp At High NaHCO₃ Alkalinity Stress

There are about 6.9×10⁸ hm²of low-lying saline waters in China,and the main characteristics of these water resources are high salinity and poor buffering capacity,which often adversely affect the growth,development and reproduction of fish.This has led to a general lack of aquatic biological resources in saline-alkali waters,and fishery production has been at a low level.In addition,with the continuous compression of freshwater aquaculture space in China,the development and utilization of new aquaculture space,especially the rational utilization of saline-alkali water resources,has become a new and important research direction.During the period of fish growth and development,if the alkalinity in the water is high,it will produce irreversible stagnation in the development of embryonic neural tissue,resulting in increased fertilization rate and malformation rate,decreased hatchability rate and embryonic death,juvenile survival rate and growth arrest.Therefore,it is important to master the tolerance and regulatory mechanisms of salinity of different fishes for the development and utilization of these waters.In this study,a pre-experiment was done based on the measured semi-lethal concentration of 7.8g/L of sodium bicarbonate in grass carp.Based on the pre-test grass carp minimum lethal concentration of 37mmol/L,alkalinity levels of 18mmol/L and 32mmol/L were finally selected for a 7d short-term stress and a 60d long-term growth experiment on grass carp.We evaluated the effects of NaHCO₃stress on antioxidant enzyme activities,tissue ultrastructure and genes related to tissue apoptosis in gill,liver and kidney of grass carp,as well as on intestinal structure and intestinal microorganisms of grass carp.In this study,we found that NaHCO₃ alkaline stress significantly（P<0.05）reduced the growth performance of juvenile fish in terms of body weight,weight gain rate and specific growth rate with increasing alkalinity,and the bait coefficient increased with increasing alkalinity by comparing the 60d NaHCO₃ stress growth experiment.Intestinal histological and histochemical studies revealed that high alkalinity（32mmol/L）NaHCO₃ alkaline stress significantly reduced（P<0.05）the trypsin,amylase and lipase activities of grass carp,while low alkalinity（18mmol/L）significantly reduced the trypsin and lipase activities of grass carp.This suggests that NaHCO₃ base stress has a more severe effect on the ability of grass carp to digest and absorb protein and fat.The crease width was significantly smaller in the alkalinity group than in the control group,and the height of the intestinal crease and the thickness of the muscle layer were significantly（P<0.05）smaller in the high alkalinity group than in the low alkalinity and control groups.And the cuprocyte density 651±17.21 cells-mm^-2 and mucus cell density 1597.57±43.26 cells-mm^-2 were significantly（P<0.05）larger in the control group than in the alkalinity group.The density of type II,type III and type IV mucus cells in the alkalinity group were significantly smaller than that in the control group,and these may be important factors affecting the bait utilization and growth performance of grass carp.A high-throughput sequencing pair study of the intestinal flora of grass carp under NaHCO₃ alkaline stress for 60d revealed that at the phylum level,the dominant populations in all groups were Clostridiales,Aspergillus and Thickwalls,but the abundance of each dominant phylum did not differ,with the LAW,HAW groups having significantly lower Aspergillus phylum than the FW group（P<0.05）.The abundance of thick-walled clades was significantly higher in both the HAW and LAW groups than in the FW group（P<0.05）.The HAW group was significantly lower than the LAW and FW groups（P<0.05）in both the anaphylaxes phylum.At the genus level,Cetobacterium spp.and Synechococcus spp.were significantly lower in the HAW group than in the LAW and FW groups（P<0.05）.The functional prediction of intestinal flora genes based on the Greengene database yielded that the abundance of metabolic functional flora such as proteins,amino acids and carbohydrates,functional flora involved in cellular transport and catabolism,and functional flora involved in environmental processing were lower in the LAW and HAW groups than in the FW group.The abundance of lipid metabolism,energy metabolism,organic systems and disease-related functional flora was higher in the LAW and HAW groups than in the FW group.In conclusion,NaHCO₃ alkaline stress altered the structural and functional status of the grass carp intestinal microflora,which may be an important factor in reducing the growth performance of grass carp.NaHCO₃ alkali stress for 7d.The results observed by scanning electron microscopy revealed significant lesion damage in gill and kidney histological structures at 24-96 h of high alkalinity stress.At 24h of stress,gill filaments were folded and gill spacing increased;at 48h of stress,gill filaments were folded severely.At 72h and 96h of stress,the gill filament spacing increased and folding was serious.At 7d of stress,the gill filaments recovered,slightly folded,but the gill filaments were evenly spaced.In addition,the renal tubules of juvenile fish under high alkalinity conditions at 24h of stress were infiltrated,with blurred structural contours and a vesicular,water droplet-like degenerative necrosis on the tissue surface,and then the lesion damage moderated,and by 96h most of the outline of the renal tubules could be clearly seen.However,studies on the relative expression of caspase-7and Bcl-2,genes related to apoptosis genes in the gills and kidneys of juvenile fish in the high alkalinity group,revealed that the relative expression of Caspase-7 and Bcl-2genes increased abruptly and significantly（P<0.05）higher than that of the control group at the initial 24-72h of stress,and then began to show a decreasing trend.This is consistent with the scanning electron microscopy observations,suggesting that high alkalinity stress may have caused apoptosis in gill and kidney tissue cells.In contrast,NaHCO₃ alkaline stress at 60d showed that with increasing alkalinity,the gill filaments of juvenile fish shed interlamellar cell clusters,increased the spacing of gill lamellae,thinned the thickness,and severely shed epithelial cells,resulting in elongation of gill lamellae in the low alkalinity group and contraction and thinning in the high alkalinity condition.The Na⁺/k⁺-ATPase,alkaline phosphatase（AKP）,acid phosphatase（ACP）and total antioxidant enzyme（T-AOC）activities of grass carp gill,liver and kidney tissues peaked around 24-48h during the initial period of NaHCO₃ alkaline stress for 7d,and were significantly higher than those of the control group（P<0.05）,and then began to show a decreasing trend.In summary,NaHCO₃ alkaline stress caused oxidative stress and apoptosis in grass carp for a short period of time,but the gill,liver and kidney related indexes of grass carp returned to normal in the late stage of stress under low alkalinity conditions,indicating that grass carp have certain resistance and self-healing ability in the face of low NaHCO₃ alkalinity stress.However,under high alkalinity conditions,it was difficult to restore all relevant indicators to normal levels,indicating that high alkalinity stress causes irreversible damage to juvenile fish and therefore affects the growth of juvenile fish.