The inclusion of crystallized methionine (Met) in plant-derived diets has been commonly practised for optimal growth and health of animals. Nutrient absorption begins in the intestine, but little has been known about the Met absorptive mechanism in fish. How much and how fast Met absorbed largely relies on transporters located in the apical and basolateral side of the intestinal membrane. Understanding the mechanism behind Met transport is important to develop a cost-effective feed formulation. The objective of this study was to characterize Met transport pathways and transporters involved in Met uptakes in rainbow trout gut.
Rainbow trout were housed in a recirculating system with optimal growth conditions (dissolved oxygen above 6 mg/L and temperature around 120 C). Healthy fish were sacrificed. Freshly-collected gastrointestinal tracts including pyloric caeca (PC), midgut (MG) and hindgut (HG) were mounted in experiment Ussing chambers. Radioactively-labelled substrate DL-[14C]Met was used as a tracer to measure unidirectional flux from mucosal (apical) to serosal (basolateral) side of the intestine. The experiment was carried out with physiological buffer in the presence or absence of sodium to characterize Na+-dependent and Na+-independent transport mechanisms. Data were fitted to the classic Michaelis-Menten equation to determine the maximal flux rate (Jmax) and affinity (Km). Gene expression using RT-qPCR was performed to identify candidate genes associated with Met flux kinetics. The experiments were performed in both triploid (3N) and diploid (2N) trout as tools to determine the Met transport mechanism in trout gut.
The results demonstrated that DL-Met transport was Na+-dependent and concentration-dependent at the concentration of 0.2-20 mM. Calculated Km values around 0.6-1 mM and no significant differences found among segments, suggesting the presence of a similar Na+-dependent low-affinity transporter along the intestinal tract. Jmax in PC & MG of 2N trout were 0.0014 ± 0.0002 and 0.002 ± 0.0002 µmol/cm2.hr, respectively. These were significantly higher than Jmax in PC & MG of 2N trout with 0.0009 ± 0.0001 and 0.0013 ± 0.0001 µmol/cm2.hr in PC & MG respectively (student's t-test, p < 0.05). These differences could be due to lower mRNA expression of low-affinity Na+-dependent transporter B0AT1-like (SLC6A19-like) in 3N trout compared to 2N trout.
Observations in this study confirmed that fish growth could be improved via ploidy and genetic manipulation. Additionally, knowledge of Met transport may contribute to understanding amino acid interaction, dietary design, and study fish diseases associated with transporter disorders