Crowding stress-related protein markers: New candidates for assessing welfare of largemouth bass reared in an in-pond raceway system

Compared to traditional pond culture, in-pond raceway system (IPRS) is an advanced model, which can reduce waste of water resources and have the characteristics of high efficiency, high yield and environmental protection. However, how to determine the appropriate stocking density has not been studied using protein markers, especially from a welfare perspective. Therefore, this study mainly evaluated the effect of stocking density on the growth, meat quality and liver proteome of largemouth bass (Micropterus salmoides). Largemouth bass (average weight, 14.29 +/- 0.26 g) were randomly distributed into three in-pond raceways at three stocking densities: low stocking-density (LSD, 7000 individual/raceway), medium stocking-density (MSD, 20,000 individual/raceway) and high stocking-density (HSD, 30,000 individual/raceway). The growth performance, muscle quality and liver protein expression were analyzed after a 58-day feeding trial. The results showed that the LSD group obtained significantly higher specific growth rate compared to the HSD group. As the density increased, the survival rate of fish decreased from 92.56% to 86.33%, and the feed conversion ratio increased from 1.21 to 1.45. Different stocking densities can significantly affect the contents of essential fatty acids in muscle, but not have any obvious changes on the amino acid pattern in muscle. A total of 109, 35 and 209 differentially regulated liver proteins were detected in LSD vs. MSD, MSD vs. HSD, and HSD vs. LSD groups, respectively. Among them, Hsp70, Grp78, Erp57, Gfpt1, Pyg and Acc1 are involved in stress, glucose metabolism or lipid metabolism, which were confirmed by western blotting. Thus, these proteins could be selected as biomarkers to determine stocking density. The findings will benefit IPRS to balance the yield and welfare of fish according to the market and law changes, beyond profit-seeking in the future.