| This experiment in vivo was conducted on broilers to evaluate the effects of circular heat stress (CHS) on growth performance, slaughter performance, carcass characteristic, serum biochemical parameter, meat quality, skeletal metabolism of Glutamine (Gln) and gamma-aminobutyric acid (GABA), heat shock protein (HSP70) and relative indexes in AMP-activated protein kinase (AMPK) signal path. The effects and interactions of dietary Gln and GABA on the broilers under CHS and the possible mechanism were also be evaluated. Moreover, the correlations between some skeletal indexes and meat quality indexes were analyzed. Another experiment in vitro was also conducted to evaluate regular relationship between HSP70and AMPK in poultry skeletal cell under heat stress (HS) and intervention of exogenous Gln and GABA on these indexes. Resolution of these issues will provide theoretical foundation for applying Gln and GABA against HS.1. Experiment in vivoThe experiment was a2×2plus positive group (PC) of the treatments with two levels each of Gln (0and5g/kg Gln of diet) and GABA (0and100mg/kg GABA of diet). A total of36010-day-old healthy male Arbor Acres broiler chicks were procured and were housed from day10to day21in thermostatically controlled cages and were fed with same basic compound daily diet. On day22, after overnight (12h) feed withdrawal, chicks were wing-banded and randomly assigned to five treatment groups (6replicates of12birds per cage). The chicks of the four treatment groups (2×2) were housed in a circular high temperature controlled room (from9:00to14:00at30-34℃, then from14:00to18:00at34-30℃, and rest of the time at average23℃) while the PC group was housed in another optimal temperature controlled room (average23℃). Growth performance for each group was recorded from22to42days of age. At28.35,42days of age, three broilers from each cage with a body weight (BW) close to the group average was selected to analysis slaughter performance, carcass characteristics, serum biochemical parameters, meat quality, skeletal metabolism of Gin and GABA, HSP70and relative indexes in AMPK signal path, and to evaluate the correlations between some skeletal indexes and meat quality indexes. Results as below:1.1CHS decreased weight gain (WG), feed intake (FI) of broiler from28d to42d old, carcass weight (CW), half-eviscerated weight (HEW), eviscerated weight (EW), breast muscle weight (BMW), breast muscle yield (BMY), leg muscle weight (LMW), abdominal fat weight (AFW), abdominal fat yield (AFY), intermuscle fat width (IFW)&subcutaneous fat thick (SFT) of42d old broiler, while increased feed-to-gain ratio (F/G)&leg muscle yield (LMY). The diet supplemented with5g/kg Gln increased WG, FI, CW, HEW, EW, BMW, BMY, LMW, AFW, AFY, IFW&SFT, while decreased F/G and had decreasing tendency for LMY of28-42d old broilers under CHS. The addition of100mg/kg GABA increased WG, FI, EW, BMW, BMY, AFW, AFY&SFT, while had increasing tendency for CW, HEW&IFW of28-42d old broilers under CHS. Different extent synergistic effects were found between Gln and GABA for WG, FI, F/G, EW, eviscerated yield (EY), BMW, BMY, AFW, AFY, IFW&SFT. With regard to the effects of Gln and GABA on growth performance,Gln had persistent action, while GABA had gradually weakened action.1.2CHS decreased concentrations of TP, Glucose, triglyceride, Gln, Glu and T4, activities of ALP in serum of28,35,42d old broilers, and GABA content of28,35d old broilers, while increased concentrations of CS&GN, activities of GOT, GPT, LDH, CK&NOS in serum of28,35,42d old broilers. The diet supplemented with5g/kg Gln increased concentrations of TP, Gln&Ins, while decreased concentrations of triglyceride, CS&GN, activities of GOT, CK&NOS in serum of28,35,42d old broilers under CHS. The addition of100mg/kg GABA increased concentrations of TP, Gln, GABA&T3, ALP activities, while had obvious time-specifically effects on concentrations of triglyceride, Glu, T4, CS, Ins&GN, activities of GOT, GPT, LDH, CK&NOS in serum of28-42d old broilers under CHS. Different extent synergistic effects were found between Gln and GABA for serum concentrations of TP, triglyceride, GABA, Glu, T3, T4, Ins&GN, activities of ALP, LDH&CK, and GOT, GPT&NOS were found only in1st week under HS.1.3CHS increased pH, L*, moisture, CP, CF, CA, TL, SF&muscle fiber density (MFDS) of breast muscle, pH, L*, CA, DL, SF&MFDS of leg muscle, while decreased DL, CL&muscle fiber diameter (MFDM) of breast muscle, a*, b*, CP, CF, CL, TL, CL&MFDM of leg muscle of28,35,42d old broilers. The diet supplemented with5g/kg Gln decreased L*, SF&MFDS of breast muscle, L*, DL, SF&MFDS of leg muscle, while increased CP, CF, CA, TL, DL&MFDM of breast muscle, a*, CP, CF, moisture CL&MFDM of leg muscle of broilers under CHS. The addition of100mg/kg GABA decreased pH, TL&SF breast muscle, pH, DL&SF of leg muscle, while increased a*, moisture, CP&CF of breast muscle, a*&CF of leg muscle of broilers under CHS. Different extent interactions were found between Gln and GABA for pH, b*, moisture, CP, WHC, TL&SF of breast muscle, pH, L*, a*, moisture, CP, WHC, TL&SF of leg muscle. Moreover, dietary Gln and GABA could improve color stability of breast and leg muscle during cold-storage.1.4CHS decreased concentrations of Gln, Glu&GABA, activities of Glnase&GAD, while increased activities of GS&GABA-T in breast and leg muscle of28,35,42d old broilers. The diet supplemented with5g/kg Gin increased concentrations of Gln&Glu, activities of Glnase&GAD in breast muscle, concentrations of Gln, Glu&GABA, activities of GAD in leg muscle, while decreased GS activates of breast and leg muscle, and had different effects on GABA concentration and GABA-T activity in breast muscle, Glnase, GABA-T activities in leg muscle of broilers under CHS. The addition of100mg/kg GABA increased concentrations of Gln&Glu, activities of Glnase&GAD in breast muscle, Gln concentration and GAD activity in leg muscle, while decreased GABA-T activity in breast muscle and GS activity in leg muscle of broilers under CHS. Significant synergistic effects between dietary Gln and GABA on Gln content, GABA content, Glnase activity, GS activity&GABA-T activity were found. There were significant correlations among indexes of Gln content, GABA content, Glnase activity&GS activity and indexes of L*, CP, CF, CL, MFDS&MFDM in breast&leg muscle of42d broilers.1.5CHS decreased concentrations of glycogen, Glucose&lactic acid, while increased AMP/ATP, AMPK content, AMPK activity, HSP70content, expression of AMPK a2mRNA and HSP70mRNA in breast and leg muscle of28,35,42d old broilers. The diet supplemented with5g/kg Gin increased HSP70content, HSP70mRNA expression in breast and leg muscle, glycogen content in breast muscle, Glucose content in leg muscle, while decreased AMP/ATP, AMPK content, AMPK activity in leg muscle of broilers under CHS. The addition of100mg/kg GABA increased glycogen and glucose contents in breast muscle, glucose and lactic acid contents in leg muscle, while decreased AMP/ATP and AMPK activity in breast and leg muscle, HSP70content, HSP70mRNA expression in leg muscle of broilers under CHS. Significant interactions between dietary Gln and GABA on glucose and lactic acid contents in breast muscle, glycogen and lactic acid in leg muscle, AMP/ATP, AMPK activity, HSP70mRNA expression in breast and leg muscle were found. There were significant correlations among indexes of glycogen, glucose&lactic acid and indexes of moisture, CP&CF, indexes of AMP/ATP, AMPK content&K activity and indexes of L*, moisture, CP, CF, WHC, DL, CL, MFDS&MFDM in breast&leg muscle. The significant correlations among HSP70and WHC, TL&DL were found.2. Experiment in vitroThis experiment in vitro was conducted to evaluate the relationship between HSP70and AMPK in poultry skeletal cell under heat stress (HS), and the intervention of exogenous Gin and GAB A. The skeletal cell with density of1-2×106/ml were used in the experiments. After experimental treatment, the skeletal cell was repaired in37℃water bath for3h, and then was collected for analysis.2.1Analysis on time difference of HSP70and AMPK under HSA total of five treatment groups (6replicates of per group) were used in this experiment. HS (42℃and45℃,0-120min) was conducted on skeletal cell after culture in basic medium for3h. The results showed that HS increased HSP70expression and AMPK activity and the HS with45℃had more quickly and heavily increasing on the two indexes The increasing of AMPK activity was proceeding to HSP70expression.2.2Analysis on regulation relationship between HSP70expression and AMPK activity(1) Experiment about Quercetin (QD) inhibits HSP70expression:A total of seven treatment groups (6replicates of per group) were used in this experiment. HS (42℃and45℃,30min) was conducted on skeletal cell after culture in basic medium with supplementation of0-50umol/L QD for3h. The results showed that inhibition of QD on HSP70expression showed obvious temperature-specificity, and the inhibition only displayed under45℃condition. Meanwhile, the increasing of AMPK activity under45℃showed that HSP70could inhibit AMPK activity to a certain extent.(2) Experiment about AICAR activates AMPK:A total of seven treatment groups (6replicates of per group) were used in this experiment. Skeletal cell was cultured in basic medium with supplementation of0-0.5mmol/L AICAT for3h under thermoneutrality. The results showed that AICAR could activate AMPK, but HSP70expression was not effected by AICAR.2.3Effect of Gln and GABA on HSP70and AMPKTwo totals of five treatment groups (6replicates of per group) were used respectively in this experiment. HS (45℃,30min) was conducted on skeletal cell after cultured in basic medium supplemented with0-20mmol/L Gln or0-0.8mmol/L GABA for3h. The results showed that Gln could increase HSP70expression, while inhibit AMPK activity, and GABA could inhibit AMPK activity to a certain extent, but had no effect on HSP70expression in skeletal cell under HS.2.4Effect of Gln and GABA on survival rate of skeletal cell under extremely HSTwo totals of three treatment groups (6replicates of per group) were used respectively in this experiment. HS (48℃,3h and6h) was conducted on skeletal cell after cultured in basic medium supplemented with0,10,20mmol/L Gln or0,0.4,0.8mmol/L GABA for3h. The results showed that10,20mmol/L Gln could increase survival rate of skeletal cell under HS for3h,6h (P<0.05or0.01), but0.4,0.8mmol/L GABA had no effect.As stated above, the conclusions are as follows:(1) The CHS (30-34℃) had obviously negative effects on growth performance, slaughter performance, carcass characteristics and serum biochemical parameters (main metabolites, hormones, metabolic enzymes), which also showed adaptability against HS. The diet supplemented with5g/kg Gln and100mg/kg GABA offered a potential nutritional strategy to prevent HS-related depression in these indexes of broiler, and also showed significant synergistic effect and time-specificity.(2) From the material metabolism, CHS affected significantly Gln, Glu and GABA contents in serum and skeletal muscle and metabolic enzyme among them. Dietary Gln (5g/kg) and GABA (100mg/kg) improved metabolic state of Gln and GABA in skeletal muscle of broiler under CHS, and also showed a certain synergistic effect and skeletal tissue-variability.(3) CHS obviously formed low-energy state of organism, activated AMPK signal path, and increased HSP70expression in skeletal muscle of broiler. Dietary Gln (5g/kg) and GABA (100mg/kg) improved energy state, inhibited AMPK signal path in skeletal muscle, and also showed a certain interactions between this two amino acids. The results also indicated that the positive action of Gln might be relevant to promoting HSP70expression. But the improvement of GABA was unrelated to HSP70expression, which may be involved in function of GABA in nervous system and physiological regulation.(4) CHS had significant negative effects on chemical compositions and quality of breast and leg meat. Dietary Gln (5g/kg) and GABA (100mg/kg) offered a potential nutritional strategy to prevent HS-related depression in these indexes of broiler, and also showed a certain interaction and skeletal tissue-variability. The impossible mechanism of CHS affects meat quality is related to metabolic change of Gln, activated state of AMPK signal path, and variation of HSP70expression to agreater extent(5) By test in vitro proved that HS activated AMPK signal path, and increased HSP70expression in skeletal muscle cell. Elevated HSP70expression could inhibit the activation of AMPK signal path in skeletal cell under HS and thus protect skeletal cell from HS-relative damage. The activation of AMPK could not affect HSP70expression in skeletal cell under thermoneutrality. Exogenous Gln and GABA could both inhibit activation of AMPK, but only Gln had effective protection to skeletal cell under HS, which was related to increasing HSP70expression. |
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