The effects of chronic immune stimulation on muscle growth in rainbow trout

Successful production of aquaculture species depends on efficient growth with low susceptibility to disease. Therefore, selection programs have focused on rapid growth combined with disease resistance. However, chronic immune stimulation diminishes muscle growth (a syndrome referred to as cachexia), and decreases growth efficiency in production animals, including rainbow trout. In mammals, recent results show that increased levels of pro-inflammatory cytokines, such as those seen during an immune assault, specifically target myosin and MyoD and inhibit muscle growth. This suggests that increased disease resistance in fish, a desired trait for production, may actually decrease the growth of muscle, the main aquacultural commodity. To test this possibility, a rainbow trout model of cachexia was developed and characterized. A six-week study was conducted in which rainbow trout were chronically immune stimulated by repeated injections of LPS. Growth indices were monitored, and whole body and muscle proximate analyses, real-time PCR, and immunohistochemistry were conducted to examine the resulting cachectic phenotype. Muscle ratio was decreased in fish chronically immunostimulated, however expression levels of MyoD2 and myosin were not decreased compared to fish that were not immunostimulated, indicating that while muscle accretion was altered, the mechanism by which it occurred was somewhat different than that characterized in mammals. Microarray analysis was used to compare gene expression in fish that had been chronically immunostimulated versus those that had not to identify possible alternative mechanisms of cachexia in fish. Keywords: muscle, cachexia, rainbow trout, chronic immune stimulation Overall design: Fish of approximately 60g were separated into either an experimental group receiving an LPS injection or a control group receiving a sterile PBS injection. The LPS group was injected with 10 mg/kg bw (body weight) LPS in a volume of 0.1ml sterile PBS, and the PBS group was injected with an equal volume of sterile PBS. Liver, and muscle tissue were harvested from 10 fish from (saline and LPS injected) at day 43 of the experiments. During the experiment, fish were fed to satiation once daily with a commercial feed (Silver Cup, Murray, UT), and feed consumption was recorded daily. In the first week following injection, fish injected with LPS consumed 45-70% of the amount of feed consumed by the saline-injected fish. The following week, LPS-injected fish consumed 80-90% of the amount of feed consumed by the saline-injected fish.Total RNA from either muscle or liver was pooled by treatment (saline- or LPS-injected, 9 fish per treatment). cDNA was prepared and either Cy3- or Cy5-labeled using Superscript II Reverse Transcriptase (Invitrogen) and Genisphere’s Array 50 Kit.Scanning was performed using a Perkin Elmer ScanArray 5000 and images and data were processed using ScanArray Express software (Perkin Elmer, Wellesley, MA). The adaptive circle method was used for quantitation, and mean background of spots was subtracted and LOWESS normalization was performed. Data was filtered by eliminating sequences that did not have a minimum of 2-fold difference in expression on at least one of the dye-flip slides and that did not have a spot mean minus background pixel intensity of at least 1000. For the microarray work presented in the paper, only the samples from day 43 were analyzed, because day 43 is the relevant time point for testing the hypothesis that chronic immune stimulation suppresses muscle growth.