Tilapias were acclimatized for 7 days at 30 C and were
randomly divided into injection and oral groups. For the injection
group, two hundred and forty tilapias were randomly divided into
12 subgroups, twenty each. Fishes in nine test subgroups were
intraperitoneally injected with 0.5, 1 and 2 mg L-proline, which was
dissolved in 40 mL of sterile PBS, once daily for 3 days, which was
determined by pre-test and three repeated subgroups each as
described previously [14]. The other three subgroups were used as
control fishes, which were injected with 40 mL of PBS only. Meanwhile,
the same approach was carried out except for the replacement
of 2 mg L-proline with 2 mg L-threonine or L-aspartate. For the
oral group, eighty tilapias were randomly divided into 4 subgroups,
twenty each. L-proline was diluted with 0.85% sterile saline to a
working solution at 50 mg/mL and then was administered 40 mL
directly into gastral cavity using a 100-mL syringe as the test group
once daily for 3 days with three replicas. The left twenty tilapias
were treated with the same volume of sterile saline and were used
as a control group. In the third days after the last injection, these
tilapias were challenged by intraperitoneal inoculation of
2.0 103 CFU/fish of S. agalactiae at 30 C. In addition, ninety tilapias
were randomly divided into 3 subgroups and were challenged
by intraperitoneal inoculation of 2.0 103 CFU/fish of
S. agalactiae separately cultured at 20, 25 and 30 C for investigation
of virulence of bacteria cultured in the different temperatures. The
tilapias were observed twice daily for 15 days.