Physiological Responses Of Silver Carp To Carbon Dioxide Anaesthesia And Its Mechanism

Silver carp?Hypophthalmichthys molitrix?is one of the four major freshwater farming fish and its annual yield ranks the second in freshwater cultured fish,mainly for fresh sale.in the process of alive fish transportation,silver carp are vulnerable to stress caused by hypoxia,crowding,shaking and other unfavorable environments,which leads to the change of the physiological state of fish body and even death,resulting in economic loss.Anesthetics can calm the fish body,reduce the metabolic rate of fish in the process of alive fish transportation,reduce the degree of stress,and thus improve the survival rate of transport.Contrast to other anesthetics,carbon dioxide?CO₂?is a metabolic gas,which has no toxicity and no drug residue on humans and fish.Therefore,it has become the only anesthetic agent permitted in edible fish in China.However,the concentration of CO₂ anesthesia is relatively narrow,with specific and size-dependent,and application research of alive fish transportation is insufficient.In particular,there are few reports about large-scale freshwater economic fish.Therefore,in this study,to explore the application of CO₂ anesthesia in the process of alive silver carp transportation,we measured the parameter change of main physiological and biochemical indicators,stress indicators,and neuromodulation related genes expression brain tissue after CO₂ anesthesia treatment for 15 hours and after resuscitation 24 hours,and compared with oxygenation and preservation treatment to understand the effects of CO₂ anesthesia on the physiological state of silver carp and neuromodulation related gene expression and understand preliminarily the mechanism of CO₂ anesthesia alive fish transpotation technology.The main results are as follows:?1?Establishment of CO₂ anesthesia method.First,the effects of different temperatures,CO₂ concentrations,and time?exposing time?on the survival rate of silver carp were investigated.Then on the basis of that,an orthogonal optimization experiment with three factors and two levels was designed to obtain the optimal anesthetic conditions for silver carp:when the carbon dioxide concentration was 155±15 mg/L and the temperature was 20?,the survival time of silver carp was as long as15 hours and the survival rate was 100%.?2?Physiological response to CO₂ anesthesia treatment.?1?After the experiment began 30 minutes,carbon dioxide anesthesia significantly reduced the breath rate,and decreased the response of silver carp.After 15 hours of anesthesia,the dissolved oxygen in the water decreased from 8.46±0.25 mg/L to 1.07±0.15 mg/L.?2?The concentration of serum potassium increased remarkably after anesthesia and recovered to normal level after 24 hours of recovery.there was no significant change in serum calcium concentration,but it decreased significantly at the time of recovery.the hemoglobin content and lactate dehydrogenase activity increased continuously.?3?Serum lactate concentration increased after anesthesia and decreased after recovery;urea nitrogen concentration continued to increase;hepatic glycogen content decreased significantly after anesthesia and rose after recovery but remained below the normal levels;muscle glycogen content did not change significantly.?4?The levels of epinephrine,aldosterone and cortisol in serum did not change much during the experiment.?3?Physiological response to oxygenation treatment.packing oxygenation treatment is as a control to co₂ anesthesia treatment.?1?After the experiment began 30minutes,there was no significant change in sputum respiratory frequency;after 15 hours of keeping alive,dissolved oxygen in water decreased from 9.74±0.64 mg/L to0.43±0.12 mg/L.?2?Serum potassium levels increased significantly after resuscitation,but decreased after reoxygenation but remained higher than normal levels;serum calcium did not change significantly after treatment and significantly decreased after reoxygenation;hemoglobin content increased after preservation and decreased after reoxygenation;dehydrogenase activity continued to increase.?3?The content of lactic acid in the serum increased significantly after treatment,and was still higher than the normal levels after reoxygenation;urea nitrogen content increased continuously;hepatic glycogen content decreased significantly after keeping alive and returned to the normal levels after reoxygenation;muscle glycogen content had little changed.?4?Adrenaline did not change much after keeping alive,and it significantly increased after reoxygenation;cortisol and aldosterone increased significantly after keeping alive and returned to the normal levels after reoxygenation.?4?The effect of carbon dioxide anesthesia on the content of neurotransmitters and the expression of related genes.After anesthesia treatment,the content of acetylcholine changed a little in brain,the content of dopamine and serotonin decreased,and the content of neurotransmitter increased after resuscitation.qPCR results showed that the expression of kcnk3a,kcnk9,HIF-1?,and EPO genes increased after anesthesia and the expression levels decreased after recovery.The above results suggest that CO₂ anesthesia treatment increased the concentration of potassium ions in the extracellular fluid or the excessive outflow of potassium ions in the nerve cells,resulted in hyperpolarization of the nerve cell membrane,inhibited the transmission of the membrane potential,hindered the conduction of nerve excitation,which reduced the fish body stress reaction,metabolic activity,performanced to reduce the frequency of breathing,decreased oxygen consumption,and made stress index change no significant.However,carbon dioxide anesthesia could extend the period of time which fishes returned to normal physiology state.It could also lead to hypoxia of fishes,resulting in increased anaerobic metabolism and cagravating the burden on the liver and kidneys.The results showed that CO₂ treatment was an effective and safe anesthetic method for silver carp.Compared with the sealed O₂ treatment,the metabolic rate and stress response could be reduced effectively.The 100%survival rate could be reached within a certain time and temperature range.The results of this study could provide basic information for the study of the mechanism of CO₂ anesthesia,provide guidance for the alive fish transport of silver carp,and also have reference value for the other alive fish transportation.