Mechanisms Of Salinity Effects On Growth Performance And Isosmotic Point Calculation In Anadromous Fish, Chinese Sturgeon(Acipenser Sinensis)

Populations of A.sinensis, an anadromous fish species with Class-I state protection in China, grow in coastal waters (seawater) and enter into the Yangtze River (freshwater) to spawn after reaching sexual maturity, IUCN assessed as CR (Critically Endangered) status. A.sinensis subjecting to ex-situ conservation no longer live in brackish water, but spend their entire life cycle in freshwater (FW). The small size of the adults matured in freshwater was associated with decreased fecundity, resulting in more challenge to the conservation work. To restore the body size, this paper aimed to argue whether salinity affect sturgeon body size growth.5-month-old,9-month-old and12-15-year-old sturgeon were tested; Therefore, we constructed "specific collector combined count method in recirculating aquaculture system for feed conversion ratio determination" and the ’establishment of high grade continuous gradient salinity experiment apparatus and method for salinity preference in aquatic animal"; Explored the juvenile Chinese sturgeon salinity adaptation mechanisms; analyzed isosmotic points of5-month-old,9-month-old and12-15-year-old sturgeon; constructed relationships between fork length, body weight and age and serum osmolality, relationships between fork length, body weight and age and isosmotic points; Compared sturgeon behaviors, hormones, enzymes activities and tissue compositions between isosmotic environment and freshwater environment; Quantified fluorescence RT-PCR analysis of GHmRNA and IGF-1mRNA in tissues between isosmotic water (IW) and FW groups; Alalyzed the salinity and migration loss effects on the compositions of freshwater eggs and seawater eggs of Chinese sturgeon; The following conclusions were obtained:1. Establish two methods:’specific collector combined count method in recirculating aquaculture systems for feed conversion ratio determination’ and ’establishment of high grade continuous gradient dilution salinity experiment apparatus and method for salinity preference’; Count method could reduce physical, biochemical and mechanical error of quantifying uneaten feed collected from holding water. Simultaneously, it saved the disposals uneaten pellets after the uneaten pellets are collected, replaced siphoning by special designed collector and saved time and labors. In addition, the effects of the feces can be ignored. The former one was for calculation of feed conversion and provides a convenient feeding rhythm, meanwhile improved accuracy of FCR; the latter provide the equipment system and methodology for salinity preference experiments. The advantages lies in, on the one hand, the appetas promises long-time salinity gradients than former appatases. On the other hand, tested animals can swim free and rechoose their location.2. Five-month-old A.sinensis reared respectively in FW (0.41psu) and brackish water (BW,12.5psu) for26days. The first10days was adaptation period, the later16days were post acclimated period. In the26th day, one of filaments both in left and right second branchial arch was sampled for scanning electron microscopy and transmission electron microscopy examination, respectively. The gill filaments appeared in the mucous cells, pavement cell and MRC on the surface of filament. In terms of MRCs, there were two subtypes of MRCs in FW, one was the ball-shaped MRC, which was higher above the neighboring pavement cells, and the other one was triangular MRC, which was triangle and flat, but it depressed between pavement cells. Only ball-shaped MRCs emerged in filament of BW-acclimated fish. Furthermore, the density and distribution of ball-shaped MRCs did not differ between freshwater and brackish group. It was also found that transitional cells, from triangle to ball-shaped, existed in FW-acclimated filament. Serum PRL, GH and IGF-1levels did not differ between FW and BW groups, which also reflected fish developeded BW adaptation mechanism in FW. Five-month-old A.sinensis had formd adapted MRCs (mitochondria-rich cell) to BW, whose isosmotic point was9.52psu. Taken "establishment of high grade continuous gradient dilution salinity experiment apparatus and method for salinity preference" as a new experimental system and revealed that five-month-old A.sinensis prefer7.5-8.5psu. This agreed to that isosmotic point of5-month-old was9.52psu. As salinity increased, food intake decreased significantly at30psu and fish ceased feeding at38psu compared to food intake at12.5psu.3. For9-month-old sturgeon, isoionic points were analyzed in the same way as the isosmotic point was determined: K+:9.65±1.01psu; Cl-:6.82±0.37psu; Na+:9.40±0.58psu; Ca2+:1.62±0.71psu. The trend of Na+concentration as salinity increased was not pronounced (P=0.250), but Cl-tended to pronouncedly higher levels as salinity increased (P=0.021). NaCl contributed to total osmolality and the real NaCl percentages had the same levels in all salinity treatments (OsmNaCl=224.27±3.17-233.41±2.63mOsm/kg;93.39±2.22%-96.79±4.17%) as it did at the isosmotic point (OsmNaCl=227.45±8.04mOsm kg-1;89.93±4.09%). Serum Mg2+level were far lower than the water Mg2+level at any salinity treatment. Nevertheless, serum P was much higher than water at any salinity treatment. Serum Mg2+and K+levels were significant higher in as salinity increased.4. Salinity changed the gill ventilation rate, tail beat frequency and daily feeding rhythm.For9-month-old A.sinensis, the lowest gill ventilation rate (74±2min-1) and the lowest tail beat frequency (51±3min-1) were observed in IW. The feed intake had no difference at8:00,15:00and20:00in0.25,6.32and25psu groups. FCRs (1.14-1.46) and SGRs (0.85-1.31) were comparable at all salinity groups, including the isosmotic point (PFCR>0.05and PSGR>0.05), but FCR was smaller at25psu and IW groups. In long term salinity tolerance, the condition factor was markedly higher after22d for all15sturgeon compared to the values before the test (P<0.05). However, the feeding rhythm at20:00of12psu-acclimated fish was significantly more than15:00. In19psu group, the feed intake at8:00was significantly more than at20:00. The difference resulted from individual difference. In general, there was no difference in feeding rhythm of each feeding at any salinity group. But the daily feed intake in25and0.25psu groups were significantly higher than6.32and12psu groups.5. For9-month-old A.sinensis, in the acclimation period, PRL increased slightly at12.7psu compared to5.98,6.32and9.69psu groups, but it declined again at19.1and25psu. In terms of GH, this hormone gradually increased as salinity increased and was the highest at25psu. The trend of IGF-1was contrary to that of GH, with the lowest IGF-1level observed at25psu. In long term salinity tolerance, the three hormone levels exhibited no significant differences among salinities. As time went on, no obvious changes were observed in any hormone. In IW group and FW group comparison test, the PRL level was significantly higher at the isosmotic point than in freshwater (P<0.01). However, GH and IGF-1exhibited no significant differences. For5-month-old A.sinensis, PRL, GH and IGF-1showed no trend in both acclimated period and post-acclimatedion. RT-PCR analysis of related expression of pituitary GH and IGF-1in different issues showed: pituitary GH mRNA and liver IGF-1mRNA expression in FW-acclimated group were significantly higher than IW-acclimated group; Muscle and kidney IGF-1mRNA levels had the same level between FW and IW groups.6. The digestive enzymes and Na+/K+-ATPase consumed more energy instead of energy for growth group in FW than in IW group. The in the gill (P<0.05), foregut (P<0.01) and spiral valve (P<0.01) in FW were approximately7%higher than in IW. The lipase activities in the foregut, spiral valve and stomach in FW were10%higher than in the IW group (P<0.05), and the activities were similar in the other examined issues. In addition, ALT (alamine transaminase) activity in the foregut was significantly higher in FW than in IW, showing a difference of approximately8%compared to IW group (P<0.05). Amylase activity in the liver was also higher in FW than in IW (P<0.05), while these values were equal in other tissues. Furthermore, the protease activity in the spiral valve was18.1%lower in IW than in FW, while in the mid-gut, it was13.2%higher in IW than in FW (P<0.05).7. Comparisons of feces protein and amino acids composition showed no difference, as well fatty acids. Muscle protein and amino acids had the similar levels between IW-acclimated fish and FW-acclimated fish. Both groups had the same level fat content (1.92±2.69) g and similar fatty acids compositions. However, in terms of minerals of muscle, Ca content in IW acclimated fish was significantly higher than in in FW-acclimated fish, P and Mg contents had the same levels. Different conditions about Ca, P, Mg contents were present in scutes and cartilage. In abdominal scute and cartilage, Ca levels were higher in FW-acclimated fish than IW-acclimated fish, but in lateral scute and back scute, Ca, Mg and P levels in IW group were higher than in FW group. In cartilage, Mg was not detected both in IW and IW groups. Overall, Ca and P levels in the total scutes and head cartilage in FW-acclimated fish were higher than in IW-acclimated fish, but Mg level in IW (2019.7±183.4mg/100g) was higher than in FW (1827.8±107.8mg/100g). The liver lipid contents (60.96±5.30mg/100g,45.29±18.34mg/100g), VA contents (31.22±9.41mg/100g,24.43±5.15mg/100g) had similar level in the two groups (FW was former, IW was latter). VE values were not detected in liver.8. Eggs biochemical contents from wild origin and remature A.sinensis in freshwater were compared, which were related to induce spawning success. The results included dry weight, lipids, fatty acids, protein, amino acids, vitellin (Vn), phosphorus (P), vitamin A (VA) and vitamin E (VE) as well as the concentrations of relevant trace elements. The dry weights of the eggs did not differ between the sturgeons. The wild sturgeon eggs had significantly higher protein content but lower lipid content than the remature sturgeon eggs. There were multiple and significant differences in the fatty acids. Vn showed no difference. The P, magnesium (Mg) and VE contents in seawater eggs were significantly higher than in freshwater eggs. The calcium (Ca) and zinc (Zn) contents showed no difference between both batches. Cadmium (Cd) was only detected in one sample of freshwater eggs. These results helped to discriminate between remature (farmed) and wild origin Chinese sturgeon eggs. It is important to take measures to decrease lipid percentages and to increase protein contents of eggs for remature Chinese sturgeon or even sturgeon matured in freshwater.9. A.sinensis were acclimated in0.27,6,12,19and25psu, relationship between water osmolality and salinity was determined by linear fitting. Second-order polynomial fitting were used to assess relationship between serum osmolality and salinity. Isosmotic point was got (9.57±0.06psu) through solution of the two equations. At isosmotic salinity environment, serum osmolality equaled to environment osmolality, sturgeon faced no osmotic pressure. The results of analyzing serum osmolality of5-month and9-month-old A.sinensis showed: there were no significant differences of serum osmolality under FW or BW, including IW. Accordingly, the results provide method to assess isosmotic point of larger ex-situ conservation subadult sturgeon in FW. Regard as the value of serum osmolality in FW as serum osmolality in BW. Serum osmolality in FW-acclimated fish had the same level as serum osmolality in IW-acclimated fish. Simultaneously, we could also take serum osmolality in FW-acclimated fish as serum osmolality in IW-acclimated fish. Take the value of serum osmolality in FW group into the equation of relationship between salinity and water osmolality, and then get the salinity i.e. isosmotic point. The order-two polynomial fitted best in relationship between age and serum osmolality, the correlation coefficients was highest. Therefore age was considered as the most relevant factor to serum osmolality:Y=278.061-11.3127x+0.6587X^2. Derived the equation and the X corresponding to value of derivative equled to zero was8.6years, i.e. from birth to age9the serum osmolality decreased and serum gradually began to increase with age since9-year-old. Similarly, isosmotic osmolality and age were most relevant:Y=9.79431-0.35711X+0.0213X2. Derived the equation and the X corresponding to value of derivative (0) was8.6years. This was similar to serum osmolality equation, i.e. from birth to age9-years, the Chinese sturgeon isosmotic point decreases, from9years on, the isosmotic point began to gradually increase with age.In conclusion, the Chinese sturgeon developed seawater-entry readiness in freshwater and the fish preferred isosmotic salinity compared to other salinities. Considering the obviously lower gill ventilation rate, tail beat frequency, Na+/K+-ATPase activity in the gills as well as the lower lipase, ALT and amylase activities observed, lower daily feeding rhythm but same SGR levels, significantly higher Ca content in muscle and increased Mg content in scutes and cartilage in IW compared to FW a salinity corresponding to the IW is ideal for the growth of naive A. sinensis. A. sinensis is suggested to rear in8.3-11.5psu. Besides, Ca2+and Mg2+should be compensated in FW culture. Meanwhile, the plasma osmolality and isosmotic point may vary with the size and age of fish, and salinity may need to be adjusted as naive A. sinensis grow.