| One of the major problems facing the culture of Nile tilapia,Oreochromis niloticus is its susceptibility to pathogen infection especially Streptococcus agalactiae and environmental stressors,for instance,hypoxia,cold,and heat stress.Currently,the fore of research indicates that the use of natural immunostimulants has the potential in boosting the immune function,and tolerance to environmental perturbations in many organisms such as cultured fish.Therefore,in the present research,the efficacy of traditional Chinese medicine [TCM](i.e.,a mixture of Astragalus membranaceus,Angelica sinensis,and Crataegus hupehensis of ratio 1:1:1 w/w),and or Bacillus species [BS](i.e.,a mixture of Bacillus subtilis and Bacillus licheniformis of ratio 1:1 w/w)were examined to ascertain their contribution in improving the growth and health of Nile tilapia.It is hopeful that the results of this study will contribute to understanding better the mechanism of action and contribution of multispecies TCM and or BS combination in improving the growth and health of tilapia.Also,the data in this study could help suggest the optimal dose of TCM and or BS supplements that can be considered as a potential immunopotentiating agent in tilapia culture.The study has been carried out by firstly evaluating the effects of BS only,TCM only,and TCMBS mixtures against a control(CT)treatment to determine their effects on growth,immune response,and disease resistance of Nile tilapia in a 4 weeks of feeding trial.Secondly,the study has been conducted to examine Nile tilapia's tolerance after exposure to some physical stressors in an 8 weeks trial.In the individual(TCM only or BS only)test,fish were fed diets containing TCM supplements at 10 g kg-1 or BS supplements at 3(BS3),5(BS5),7(BS7),and 10(BS10)g kg-1 and tested against fish fed a CT diet.In the mixture test(i.e.TCMBS combinations),fish were fed diets containing TCMBS1 [TCM at 3 g kg-1 and BS at 7 g kg-1],TCMBS2 [TCM at 5 g kg-1 and BS at 5 g kg-1],TCMBS3 [TCM at 7 g kg-1 and BS at 3 g kg-1] and tested against those fed the CT diet.The main results are as follows: Effects of TCM,BS,or TCMBS on Growth and the Immune Response of TilapiaAt the termination of experiments after 4 weeks,analysis of the growth indices [i.e.,weight gain(WG),specific growth rate(SGR),and feed conversion ratio(FCR)] showed that the application of TCM only at 10 g kg-1 and BS(BS10)at 10 g kg-1 when used individually significantly(P < 0.05)increased the WG and SGR,and lowered FCR compared to the CT group.Concerning the use of various TCMBS combinations,the TCMBS3 group was found to have significantly(P < 0.05)higher effect on these growth indices compared to the CT.Among the all the treated groups(i.e.TCM,BS,and TCMBS combinations),the TCMBS3 showed the highest(P > 0.05)values in these growth indices compared to the CT group.The effects of the application of individually mixed immunostimulants(TCM or BS)on humoral immune response upon termination of the feeding trials after 4 weeks revealed significant(P < 0.05)enhancement in serum lysozyme(LYZ),superoxide dismutase(SOD),catalase(CAT),protease,and anti-protease activities and immunoglobulin(Ig)M levels in the TCM group and all the BS groups compared to the CT group.A similar trend was observed in all mixed preparations(TCMBS)in which all treated groups significantly(P < 0.05)enhanced LYZ,SOD,CAT,protease,and anti-protease activities,but with highest values in CAT and anti-protease activities in TCMBS3 compared to control.Furthermore,research results from the skin mucus of Nile tilapia,after 4 weeks of feeding with different TCMBS combinations showed significant(P < 0.05)enhancement in total protein concentration,and LYZ,proteases,anti-protease,and peroxidases activities,but no significant(P > 0.05)change in p H and alkaline phosphatase(ALP)activity was observed in all mixture treated groups compared to the control group.Interestingly,among the treatment groups,the TCMBS3 group demonstrated significantly(P < 0.05)the highest effects in all the indices measured above in the skin mucus compared to the CT group.Also,in the skin mucus especially that of the TCMBS3 group showed strong(P < 0.05)bactericidal activity against Streptococcus agalactiae and Aeromonas hydrophila growth.Concerning the expression of immune-related genes ?-defensin,heat shock protein(HSP)70,and C-lysozyme(C-LYZ)were significantly(P < 0.05)up-regulated following individual TCM or BS application and mixtures of TCMBS application compared to the CT in the mid-intestine(MINT)and head-kidney(HK).However,among all the treatment groups,expression values of the immune-related genes in this study seemed higher(P < 0.05)in TCMBS3 group.Also,in the skin tissue,the TCMBS3 group showed significant(P < 0.05)up-regulation of the expression of interleukin 1 beta(IL-1?),interleukin 10(IL-10),major histocompatibility complex 1(MHC1),interferon gamma(INF-?),Caspase-3,and immunoglobulin(Ig)D compared to the other treatment groups and the control group.In the present study,after challenge with S.agalactiae,survival among the best-performing individual and combination groups were as follows TCMBS3(97 %),BS10(80 %),TCM(70 %),and CT(35 %).Given the results of the above experiments using TCM only or BS only or a combination of TCMBS mixtures suggested that the mixed formulation(i.e.,TCMBS3)have a higher immune potentiating effect on Nile tilapia.Therefore,to deepen knowledge about the immune influences of TCMBS3 in Nile tilapia,a 2 week follow-up experiment was conducted to examine its application on the expression of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase genes,antioxidant genes,and production of reactive oxygen species(ROS)in the neutrophils of Nile tilapia.The results of TCMBS3 application showed significant up-regulation in expression of NADPH oxidase genes(i.e.,gp91 phox,p22phox,p40 phox,p47phox and p67 phox,antioxidant genes(i.e.,SOD,CAT,and GST)and an increase in the production ROS compared to the control in splenic neutrophils of tilapia.Taken all of the above into consideration,it can be concluded that there is justification for using TCM,BS,or in combination TCMBS as immune potentiating immunostimulants in Nile tilapia farming.Overall,TCMBS3 is suggested as the best to improve growth and the immune status of Nile tilapia.Effects of TCM and or BS on the Stress Tolerance of Nile TilapiaConcerning the influence of TCM and or BS on the physiological response of Nile tilapia,an 8 weeks trial with all 6 diets including a CT diet enriched with TCM,BS,TCMBS1,TCMBS2,and TCMBS3 were used to evaluate some hemato-immunological,stress,and antioxidant indices,as well as stress-related genes evaluated before and after exposure to hypoxia,cold,and heat stressors.The results after the experimental trial for 8 weeks and afterwards exposure to physical stressors including hypoxia,cold,and heat stress,showed increased(P < 0.05)hematocrit,red blood cells,hemoglobin,and white blood cells in fish blood in most of the herbal-probiotic groups compared to the control group.Lowered(P < 0.05)cortisol,glucose,as well as pyruvate kinase,alanine aminotransferase,aspartate aminotransferase,and lactate dehydrogenase,and increased(P < 0.05)myeloperoxidase,total antioxidant capacity,superoxide dismutase,and catalase activities were observed in the serum of all herbal-probiotic groups over the control group.Besides,all herbal-probiotic groups showed increased(P < 0.05)expression of HSP 70 and hypoxia-inducible factor 1 alpha(HIF-1?)m RNAs in the livers of fish.However,among the herbal-probiotic groups,Nile tilapia fed with TCMBS2 diet showed enhanced(P < 0.05)values in most of the indices measured under the different stress conditions.Collectively,tolerance to physical stressors,after 8 weeks of feed trial favours the application of TCMBS2 in the diet of Nile tilapia. |
PDF Full Text Request