Effect Of Two Probiotics On The Growth Performance,immune Enzymes, Digestive Enzymes, Intestinal Microflora And TLR9 Gene Expression In Tissues Of Juvenile Cobia(rachycentron Canadum)

In order to exploit the effect of two probiotics, Bacillus subtilis and lactobacillus acidophilus as separate or mixed diets on growth performance, immune enzymes, digestive enzymes, intestinal microflora and TLR9 gene expression in tissues of juvenile cobia after continuous and non-continuous feeding, to provide guidance for the application of probiotics in aquaculture.Eight hundred and forty healthy juvenile cobia with an initial weight of 22.0±0.15 g were randomly divided into 7 groups(group number are T0, T1, T2, T3, T4, T5, T6,respectively) with 3 replicates per group and 40 fish per replicate. The fish in the control group was fed a basal diet and the fish in the experimental groups T1, T2, T3 were fed diets containing Bacillus subtilis 1×107cfu/g,Lactobacillus acidophilus 1×107cfu/g,or B.subtilis + L.acidophilus 2×107cfu/g for 30 days and then 20 days switched to basal diets,respectively. While the fish in the experimental groups T4, T5, T6 were fed probotic enriched diets as same as T1, T2, T3 for 50 days, respectively. Serum LZM, SOD, AKP and ACP activities in each group at 0d, 10 d, 20 d, 30 d, 40 d, 50 d, respectively were detected.At 0d, 30 d, 50 d the selected intestines were evaluated by high-throughput sequencing and polymerase chain reaction-denaturing gradient gel electrophoresis analysis of their structure and diversity of the intestinal flora, and the dominant microflora species annotation. At 0d and 50 d, the fish in each group were weighed to calculate the growth performance indices and test the amylase and protease activity in liver and intestine, TLR9 gene expression in liver, kidney, spleen and intestine of cobia were determined.The results showed that: WGR of T0-T2 groups have no significant difference(P<0.05) but lower than that of the T3-T6 groups, meanwhile, the T6 group has the maximum value which was significantly higher than that of other groups. The trend ofSGR is similar to the WGR. The FCR of T2-T6 groups were significantly better than that of the control group(P<0.05) and there is a best value in the T4, T6 groups which was significantly lower than that of other groups(P<0.05).Serum LZM of seven diets were first increased and then decreased during the whole experimental period, there was the maximum value at 10 d, significantly higher than that of other times(P<0.05). The activities of serum LZM in the probiotic groups were significantly higher than that in the control group(P<0.05). There were no significant difference about LZM at 10 d and 30 d among the probiotic groups(P>0.05). At 20 d, LZM of T3 and T6 group were significantly higher than that in the other groups(P<0.05) as well as the group of T2 and T3 at 40 d. There has significant difference among T1-T6 groups at50d(P<0.05) and the highest value was found in T6 group.The serum T-SOD was increased first and then decreased and reached the maximum value at 20 d. T-SOD in the group of T1-T6 was significantly higher than that of the control group(P<0.05), but no obvious difference was found among probiotics groups at 10d(P>0.05), and trend of 20 d was the same as 10 d, but T-SOD of T6 group significantly higher than that of T1 and T5 groups(P<0.05). At 30 d, T-SOD in the group of T2 and T5 was significantly lower than that in the control group(P<0.05), while other treatments showed no significant difference with control group(P>0.05). T-SOD in the T2 and T3 groups was significantly lower than that in control group at 40d(P<0.05), but there was no significance among other groups and control group(P>0.05). At 50 d, T-SOD in the T4-T6 groups was significantly higher than that of the T0-T3 groups, and the best value obtained in T6 group.Serum AKP appeared increased and then decreased from 0d to 50 d, and all groups have reached the maximum value at 40 d, but slightly lower in T1 and T3 group. At 10 d,serum AKP in the T1-T6 groups showed significantly lower than that in the control group(P<0.05), but no significant differences among treatments. There was no significant differences among treatments at 20d(P>0.05), while AKP in the T1-T6 groups was significantly lower than that in the control group at 30 d and 50d(P<0.05). Serum AKP of the T2 and T4 groups had no significant differences with the control group, but other groups were significantly lower than that in the control group at 40d(P<0.05).The trends of serum ACP increased at first and then decreased, and reached maximum values at 20 d. In addition to T3 and T6, serum ACP of the test groups was significantly higher than that of the control group(P<0.05) and ACP of T1 and T4 groups was significantly higher than that of other groups at 10 d. The group of T1 and T4 showed nosignificant differences with the control group at 20 d, while the rest groups were significantly higher than that of the control group(P<0.05). T1 and T4 groups were no significant differences with the control group, but the other groups were significantly lower than that of the control group at 30d(P<0.05). At 40 d, serum ACP of T1-T6 groups were significantly lower than the control group, and the ACP of the T6 group was significantly higher than that of T3 and T4 groups(P<0.05). At 50 d, serum ACP of T1 and T2 groups were significantly higher than that of T0 group(P<0.05), while ACP of T6 group was no significant difference with T0 group(P>0.05), and the rest of groups showed significantly lower than that of T0 group.The amylase activity in liver of T1-T6 groups showed significantly higher than that of the control group(P<0.05) and T6 is the highest. On the contrary, the amylase activity in midgut of T1-T6 groups were significantly lower than that of the control group(P<0.05)and T5 is the lowest. The amylase activity in foregut of T5 and T6 groups were significantly higher than that of the control group and other groups(P<0.05). There was no significant difference between T4 group and T3, T6 groups in the activity of hindgut amylase, but significantly higher than that of the rest groups. The protease activity in liver of T1-T6 groups showed significantly higher than that of the control group(P<0.05), and T5-T6 groups are the best. The foregut, midgut, hindgut protease activity of T4-T6 groups showed significantly higher than that of other groups(P<0.05), and maximum value was found in T6 group.Foregut sample shannon index of T6 group in 30 d and 50 d was significantly higher than that of the control group and other groups(P<0.05), suggesting that both two samples of flora diversity were higher than other groups. There was significant difference in the beta diversity between 50 d T0 group and 0dY(0d foregut sample) or 30 d T0 or 50 d T6group as well as between 50 d T1 group and 30 d T1 or 30 d T6 group. The superiority bacteria of foregut in 0dY and 50 d T0-T4 groups is photobacterium, and the proportion in the intestinal microflora is 91.24%, 76.77%, 75.89%, 94.92% and 99.56% respectively.The superiority bacteria of 30 d T0 and 30 d T5 is Acinetobacter, and the proportion is73.73%, 95.6%, respectively. The superiority bacteria of T5-T6 in 50 d is Serratia, and the proportion is 69.85%, 62.73%, respectively. The superiority bacteria of T4 and T6 in 30 d is Vibrio, Staphylococcus, and the proportion is 89.41% and 53.55% respectively.The bands of hindgut samples in each group had no significant difference at 0d and30 d, which has 6.38 strips. There are two common bands 18 and 19 among T0, T4-T6, and the band of 19 is superiority flora which its proportion is 74.13%. The similarity of intestinal microflora in T0 and T4, T5, T6 groups is 64%, 67% and 40% respectively. At50 d, T1-T6 groups of hindgut diversity of average bands was 15 and all of them were no significant difference, except T3 group that had 10 bands. T0-T3 had 10 common strips and band 6, 28, 35, 40 are the major superiority flora in the hindgut, which the proportion is 36.1%, 15.75%, 15.27%, 11.74%, respectively. The similarity of intestinal microflora in T0 group and T1-T3 group is 67%, 66%, 52%. T4 and T6 groups had 10 common bands and band 5, 32, 37, 40 are the major superiority bacteria that the proportion is 42.16%,14.97%, 10.36% and 10.68% respectively. The similarity intestinal microflora of T4 group andT5, T6 group intercomparison is 58%, 46% and 64%.The expression of TLR9 gene in liver of T2 group was no significant difference with the control group(P>0.05), but TLR9 gene expression in liver was significantly improved of other groups, especially T4-T6 groups were significantly higher than that of T0-T3groups(P<0.05), but no significant difference among T4-T6 groups(P>0.05). The expression of TLR9 gene in kidney of T3-T6 groups showed significantly higher than that of the control group(P<0.05), and T6 group was significantly higher than that of T3-T5groups(P<0.05), while T1 and T2 groups were significantly lower than that of the control group(P<0.05). The expression of TLR9 gene in spleen is similar to liver(P<0.05). The expression of TLR9 in intestine of T1-T6 groups were significantly higher than the control group(P<0.05), while T1-T3 groups were no significant difference each other as well asT4-T6 groups(P>0.05).Therefore, dietary administration of Bacillus subtilis, Lactobacillus acidophilus as separate or compounds(1:1), could promoted the growth performance, immune enzymes,digestive enzymes, improved the intestinal microflora and increase the expression quantity of TLR9 of juvenile cobia. Continuous feeding is better than non-continuous feeding and the mixed diet had the best effect.