Effect Of Dietary Threonine On The Growth, Digestion Capacity And Immunity Of Juvenile Blunt Snout Bream (Megalobrama Amblycephala)

The contribution of aquaculture to world food production has increased significantly over the last few decades and this sector now supplies nearly half of the total fish and shellfish used for human consumption. Advances in culture techniques and the introduction of new species have contributed to the rapid growth of the aquaculture industry.Considering its importance in the world food sector, it is widely recognized that the industry should become sustainable from every angle.Blunt snout bream (Megalobrama amblycephala) is one of the major aquaculture species in China with extremely high economic value. In the last decade, its production in China has been rapidly increased and reached approximately 0.7 million tons in 2012. It is also introduced to North America (northern Canada to southern Mexico),Africa and Eurasia.This fish has a bright future in aquaculture worldwide because of its adaptability to local environment, compatibility with native species, good candidate freshwater intensive culture,fast growth, high larval survival rate, tender flesh, and disease resistance.In intensive aquaculture, specific understanding of the ten dietary indispensable amino acids (IAAs) requirements is essential to formulate amino-acid balanced, cost-effective and eco-friendly practical diet of a candidate fish species. Indeed, many studies have been hammered at searching the appropriate rations of IAAs in aqua-feeds and investigated their relationship in fish nutrition research. Among IAAs, threonine has been proved to be an indispensible amino acid for optimal growth of fish; and it is extracted in greater proportion by the small intestine in animals, suggesting that threonine is involved in intestinal functionality and maintenance. In higher animals, threonine is used to the greatest extent by the portal-drained viscera, and 60-80% of dietary threonine is extracted by the portal drained viscera on the first pass, which could utilize dietary threonine for protein synthesis.The present study conducted a 9-week feeding trial to investigate the effects of threonine on the growth, digestive, absorptive, antioxidative and immune functions and protein synthesis-antioxidant-immune-cytokine-related gene expressions of juvenile blunt snout bream. For this purpose, three tanks (300 litres/ tank) were randomly arranged and assigned to each experimental diet. Juvenile fish were fed with diets containing graded threonine levels (0.58, 1.08, 1.58, 2.08 or 2.58% of the diet) to apparent satiation four times daily. This study includes five key aspects:1. Dietary threonine requirement of juvenile blunt snout bream (Megalobrama amblycephala)This study was conducted to determine the dietary threonine requirement of juvenile blunt snout bream and to assess the effects of graded dietary threonine levels (0.58 - 2.58%)on the growth performance, feed utilization, whole body composition, plasma composition and target of rapamycin (TOR) pathway gene expression. The results indicated that final body weight, weight gain rate (WGR), specific growth rate (SGR), feed efficiency ratio(FER), protein efficiency ratio (PER), protein retention value (PRV), fulton condition factor(K factor), and whole body protein content significantly (P<0.05) increased with increasing dietary threonine levels up to 1.58% and thereafter decreased. Plasma free threonine concentration and total protein content rose as dietary threonine levels increased (P<0.05).High dietary threonine level (2.58%) triggered plasma urea content (P<0.05). Dietary threonine regulated the target of rapamycin (TOR) and eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) gene expressions in the proximal-intestine (PI),mid-intestine (MI) and distal-intestine (DI) of juvenile blunt snout bream, which may explain further that dietary threonine enhanced growth performance and protein retention.Based on the second-degree polynomial regression analysis of SGR against dietary threonine levels, the optimum dietary threonine level for juvenile blunt snout bream was estimated to be 1.57% of the diet, corresponding to 4.62% of dietary protein.2. A deficiency or an excess dietary threonine level affects weight gain, enzyme activity, immune response and immune-related gene expression in juvenile blunt snout bream (Megalobrama amblycephala)This study was carried-out to investigate the impacts of deficient and excess dietary threonine levels on weight gain, plasma enzymes activities, immune responses and expressions of immune-related genes in the intestine of juvenile blunt snout bream.Triplicate groups of fish were fed with deficient (0.58%), optimum (1.58%) and excess(2.58%) threonine level diets to near satiation four times a day for 9 weeks. The results showed that both deficiency and excess threonine level diets significantly (P<0.05) reduced the weight gain of blunt snout bream. Excess dietary threonine level triggered plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities (P<0.05);whereas superoxide dismutase (SOD) activity was not significantly influenced by imbalanced-dietary threonine level (P>0.05). Plasma complement component 3 (C3) and component 4 (C4) concentrations were significantly depressed by the deficiency of dietary threonine (P<0.05). Dietary threonine regulated TOR, 4E-BP2, tumour necrosis factor alpha (TNF-a) and copper-zinc superoxide dismutase (Cu/Zn-SOD) gene expressions in the intestine of blunt snout bream,which may go further to explain the adverse effects of a deficient and/or an excess dietary threonine level on growth, immunity and health of fish.Furthermore, the present study also suggests that an optimum dietary threonine could play an important role in improving growth, enhancing immune function and maintaining health of fish.3. Threonine affects digestion capacity and hepatopancreatic gene expression of juvenile blunt snout bream (Megalobrama amblycephala)This study was conducted to investigate the effects of graded dietary threonine levels(0.58 - 2.58%) on the digestion capacity and hepatopancreas gene expression of juvenile blunt snout bream. At the end of the feeding trial the results indicated that hepatopancreas weight (HW), hepatosomatic index (HSI), hepatopancreatic protein content (HPC), intestine weight (IW), intestosomatic index (ISI) and intestinal protein content (IPC) increased with increasing dietary threonine levels up to 1.58% and thereafter decreased (P<0.05). The activities of chymotrypsin, trypsin, amylase and lipase rose as dietary threonine levels increased up to 1.58% threonine level (P<0.05); while these activities decreased in most case after 1.58% threonine except for chymotrypsin and trypsin in the hepatopancreas(plateau between 1.58 and 2.08% threonine). The relative gene expressions of chymotrypsin,trypsin, amylase, lipase, TOR and insulin-like growth factor I (IGF-I) were up-regulated,and the highest values were observed with 1.58% threonine or 1.58 and 2.08% threonine;whereas the relative gene expression levels of 4E-BP2 gradually (P<0.10) decreased as dietary threonine levels increased up to 1.58% and thereafter significantly (P<0.05)increased, which could explain that about 1.58% threonine could improve the growth and development of digestive organ and activities of digestive enzymes of blunt snout bream.4. Threonine influences the absorption capacity and brush-border enzymes gene expression in the intestine of juvenile blunt snout bream (Megalobrama amblycephala)This study was carried-out to investigate the effects of graded dietary threonine levels(0.58 - 2.58%) on the enzyme activities, plasma ammonia content and intestinal brush-border enzymes gene expressions in juvenile blunt snout bream. The results indicated that the activities of alkaline phosphatase (AKP), sodium/potassium-ATPase transporter(Na+/K+-ATPase) and γ-giutamyl transpeptidase (γ-GT) in the three intestinal segments rose as dietary threonine levels increased and reached a pick values at 1.58% dietary threonine and thereafter decreased. The highest glutamate-oxaloacetate transaminase (GOT) activity in hepatopancreas was found in the group fed with high (2.58%) threonine diet (P<0.05).Glutamate-pyruvate transaminase (GPT) activity in hepatopancreas and plasma ammonia content (PAC) exhibited significant (P<0.05) "V-shaped" trends with increasing dietary threonine levels and the lowest values were found at 1.58% threonine, while plasma alkaline phosphatase (ALP) didn’t significantly respond to threonine supplementation. Dietary threonine regulated the gene expressions of brush-border enzymes (AKP, Na+/K+-ATPase and y-GT) in the intestinal segments, which may explain further that threonine enhanced the absorptive function of fish via regulating brush-border enzymes synthesis and secretion.5. Threonine modulates immune response, antioxidant status and gene expressions of antioxidant enzymes and antioxidant-immune-cytokine-related signaling molecules in juvenile blunt snout bream (Megalobrama amblycephald)This study was conducted to investigate the effects of graded dietary threonine levels(0.58 - 2.58%) on the hematological parameters, immune response, antioxidant status and hepatopancreatic gene expression of antioxidant enzymes and antioxidant-immune-cytokine-related signaling molecules in juvenile blunt snout bream. The results indicated that white blood cell (WBC), red blood cell (RBC) and haemoglobin significantly responded to the graded dietary threonine levels,while hematocrit didn’t significantly respond.Complement components (C3 and C4), total iron-binding capacity (TIBC), immunoglobulin M (IgM), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT)increased with increasing threonine levels up to 1.58-2.08% and thereafter tended to decrease. Threonine regulated the gene expressions of Cu/Zn-SOD, Mn-SOD, CAT, GPx1,glutathione S-transferase mu (GST), nuclear factor erythroid 2-related factor 2 (Nrf2), heat shock protein-70 (Hsp70), tumour necrosis factor-alpha (TNF-a), apolipoprotein A-I(ApoAl), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and fructose-bisphosphate aldolase B (ALDOB). These genes are involved in different functions including antioxidant,immune, cytokine and defense responses, energy metabolism and protein synthesis.The overall results indicate that the optimum dietary threonine level could improve growth performance,and enhance digestive,absorptive,antioxidative and immune functions and protein synthesis of juvenile blunt snout bream. This study could provide a new molecular tool for studies in fish nutrition and shed light on the regulatory mechanisms that dietary threonine improved the digestion, absorption, antioxidant and immune capacities of fish.