糖皮质激素调控家禽采食的机制
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摘要
为探讨应激条件下肉仔鸡能量食欲调控的机制,本研究从应激发展的两个阶段——急性,长期过程着手,利用人工导入外源糖皮质激素的方法模拟应激,分别分析了下丘脑代谢变化和相关基因表达量的改变,为建立应激状态下下丘脑能量食欲调控模式,以改善生产中应激引起的能量浪费提供依据。
第一部分中,试验一包括三个类似的小试验,处理基本一致。Trial 1选取体重相近的Arbor Acres(AA)雄性肉鸡64只,随机分为四个组:应激及日粮自由选择组(DEX,高,低能日粮,n=8),对照组(生理盐水,高,低能日粮,n=8),高能日粮组(代谢能,15.1KJ/Kg;粗蛋白,20%)(n=8),低能日粮组(代谢能,10.9KJ/Kg;粗蛋白,20%)(n=8),单笼饲养,每个笼均设红绿两个饲槽。Trial 2选取体重相近的AA雄性肉鸡96只,在trial 1的基础上多加两个饲槽无颜色处理组(DEX,高,低能日粮;对照组);Trial 3选取选取体重相近的AA雄性肉鸡32只,随机分为两组,处理同trial 2中饲槽无颜色处理组。每个试验5日龄起每日早8:00,应激组以2.6mg/kg体重的剂量注射DEX,对照及高低能日粮组均注射相应剂量的生理盐水。每日统计采食量。10日龄早8:00采血。试验结果表明,本试验成功引发应激,应激组对高能日粮的采食显著升高,提示应激改变了肉鸡对日粮能量的采食偏好;饲槽颜色对肉仔鸡采食量无显著影响。试验二选取体重相近的AA雄性肉鸡96只,随机分为六个处理组:应激及高能日粮组(DEX,高能日粮),高能日粮对照组(生理盐水,高能日粮),应激低能日粮组(DEX,低能日粮),低能日粮对照组(生理盐水,低能日粮),应激及日粮自由选择组(DEX,高,低能日粮),日粮自由选择对照组(生理盐水,高,低能日粮)。每组8个重复,每个重复两只鸡,单笼饲养。应激处理及采食量统计同试验一,10日龄早8:00采样。试验鸡心脏采血,断头后于30s内取出下丘脑,液氮速冻。试验结果表明:应激显著降低了肉仔鸡生产性能,高能日粮能缓解应激对采食的影响;应激及日粮能量水平对下丘脑葡萄糖含量没有显著影响;下丘脑各采食调控基因mRNA水平的变化提示肉仔鸡在长期应激状态下,通过对能量摄入量的控制,维持了下丘脑采食调控的平衡。试验三分为两个小试验,trial 1将体重相近的AA雄性肉仔鸡80只,随机分为两个处理组:应激组,对照组。试验在单饲笼内进行,每组设10个重复,每个重复4只鸡。11日龄起每天06:0(0空腹3h),应激组按2.6 mg/kg BW的剂量皮下注射DEX,对照组同剂量皮下注射生理盐水,连续注射三天,13日龄的08:00(应激2h,空腹5h)和09:00(应激3h,恢复采食1h)两个时间点分别采样(血浆,下丘脑)。Trial 2分组同trial 1,11日龄起注射时间为每天10:00(空腹1h), 13日龄的12:00(应激2h,空腹3h)采样,样品采集同trial 1。试验主要结果显示:禁食状态下,应激显著升高了肉仔鸡下丘脑NPY,AgRP的表达量,这两个基因水平的上调随着采食的恢复消失。
第二部分中,试验一选取体重相近的AA肉仔鸡32只随机分为四组,早6:00禁食3h后,以2.6mg/kgBW剂量注射DEX,同时灌注色氨酸(生理盐水溶解,500mg/kgBW),两个处理分别设一个生理盐水对照组,3h后取血浆及下丘脑。下丘脑用高氯酸萃取后进行NMR研究。通过1H NMR谱,发现应激改变了肉鸡下丘脑乳酸,葡萄糖,ATP,肌醇,3-羟基丁酸的相对含量;未检测到色氨酸信号的改变。试验二选取体重相近的AA肉仔鸡32只,随机分为四个组。早6:30禁食,3小时后A、B组按2.6mg/kg体重注射DEX,饲喂高能日粮。C、D组注射相应剂量的生理盐水,饲喂低能日粮。11:30所有试验鸡心脏采血。取下丘脑液氮速冻。试验结果显示:皮下注射地塞米松成功引发肉仔鸡急性应激;对下丘脑采食调控基因的检测结果主要提示,急性应激可能通过上调下丘脑NPY的表达量导致肉仔鸡短期内的采食量的升高。
To investigate the effects and mechanisms of stress on appetite of broilers(Gallus gallus domesticus), exogenous glucocorticoids was used to mimick stress on two phases of stress development of broilers. Changes of metabolism and mRNA expression level of important genes in appetite regulation in hypothalamus were measured and analyzed, in order to providing countermeasures for reforming energy waste in broiler chicken production.
In part 1, there are 3 trials in experiment 1. In trial 1, 64 male broilers (Arbor Acres) with similar body weight were divided into 4 groups randomly, rearing in separate cage in which there were two containers with the color of red and green relatively. In group1, animals were stressed and diets were be at choice (DEX, high and low energy diet, n=8). In group 2, treats were the same as in group 1 except that dexamethasone was displaced by saline. In group 3, diets were all high energy(ME, 15.1KJ/kg; CP, 20%) (n=8), while diets were low energy (ME, 10.9KJ/kg; CP, 20%) in group 4(n=8); In trial 2, 96 AA broilers with similar body weight were divided into 6 groups randomly. On the basis of trial 1, there were two more groups in which the containers were noncolored (DEX, high, low energy diet; control). In trail 3, 32 AA broilers with similar body weight were divided into 2 groups. The treatments were the same as the container-noncolored groups in trail 2. All the broilers in this experiment were stressed by subcutaneous injection of DEX with a dose of 2.6mg/kg BW at 8:00 from 5 to 10 days old. Saline was injected with a suitable dose in the control group. During the trial, food intake was noted every day. At the day of 10, blood was obtained. The results showed that, stress was induced successfully and it increased the high energy diet ingestion, however, the body weigh was decreased significantly. All of these suggest that stress alters the appetite of broilers; there was no significant effect of color on food intake. In experiment 2, 96 AA broilers with similar body weight were divided into 6 groups: A (DEX, high energy diet), B (Saline, high energy diet), C (DEX, low energy diet), D (saline, low energy diet), E (DEX, diets at choice), F (saline, diets at choice). The stress treatment and food intake data record was the same as in experiment 1. At the day of 10, blood and hypothalamus were obtained. The results show that, stress reduced the production performance of broilers significantly, while high energy diet blunted the effect of stress on food intake; stress and diet energy level have no significant effect on glucose content of hypothalamus; changes of appetite regulated genes’mRNA level in hypothalamus suggested that in the state of chronic stress, broiler can maintain the balance of food intake regulation in hypothalamus via the controll of the energy intake. In experiment 3, there were two similar trails. In trial 1, 80 AA broilers were divided into two groups: stress and contro group. There were 10 repetitions in every group with 4 broilers in every repetition. From 11 days old to 13 days old, broilers were injected with the same dose of DEX and saline in experiment 1 at 6:00 (fasting for 3h), respectively. At the day of 13, samples (blood and hypothalamus) were obtained at 8:00 (stress for 2h, fasting for 5h) and 9:00 (stress for 3h, refeed for 1h). In trial 2, group dividing and the treatment were the same as which in trail 1, except that the time of injection was 10:00 (fasting for 1h), and samples were obtained at 12:00 (stress for 2h, fasting for 3h). The results primarily showed that, in the state of fasting, stress up-regulated the expressions of NPY and AgRP in hypothalamus significantly, while refeed vanished the effect.
In part 2, trail 1, 32 chickens of 10-day-old was divided into 4 groups randomly, DEX (2.6mg/kg BW) and tryptophan (0.5g/kg BW) were adminstrated after 3h fasting at 9:00, and saline control was set up respectively. Blood and hypothalamus was obtained and hypothalamus extraction was used for NMR study. The ~1H NMR spectra shows that, the relative concentration of lactate, glucose, ATP, myo-Inositol and 3-Hydroxybutyrate was changed by stress, while tryptophan signal was unchangeable. In experiment 2, 32 AA broilers were divided into 4 groups. Broilers were fasted at 6:30, and 3 hours later, two groups were injected DEX and refeeded high energy diet. Another two groups were injected saline, and refeeded low energy level. At 11:30, all the blood samples were obtained, and hypothalamus were quick-freezed in liquid nitrogen. The results showed that, the detection of food intake regulated genes in hypothalamus showed primarly that, acute stress possibly induced the temporary increase of food intake via up regulated the expression of hypothalamic NPY.
第一部分中,试验一包括三个类似的小试验,处理基本一致。Trial 1选取体重相近的Arbor Acres(AA)雄性肉鸡64只,随机分为四个组:应激及日粮自由选择组(DEX,高,低能日粮,n=8),对照组(生理盐水,高,低能日粮,n=8),高能日粮组(代谢能,15.1KJ/Kg;粗蛋白,20%)(n=8),低能日粮组(代谢能,10.9KJ/Kg;粗蛋白,20%)(n=8),单笼饲养,每个笼均设红绿两个饲槽。Trial 2选取体重相近的AA雄性肉鸡96只,在trial 1的基础上多加两个饲槽无颜色处理组(DEX,高,低能日粮;对照组);Trial 3选取选取体重相近的AA雄性肉鸡32只,随机分为两组,处理同trial 2中饲槽无颜色处理组。每个试验5日龄起每日早8:00,应激组以2.6mg/kg体重的剂量注射DEX,对照及高低能日粮组均注射相应剂量的生理盐水。每日统计采食量。10日龄早8:00采血。试验结果表明,本试验成功引发应激,应激组对高能日粮的采食显著升高,提示应激改变了肉鸡对日粮能量的采食偏好;饲槽颜色对肉仔鸡采食量无显著影响。试验二选取体重相近的AA雄性肉鸡96只,随机分为六个处理组:应激及高能日粮组(DEX,高能日粮),高能日粮对照组(生理盐水,高能日粮),应激低能日粮组(DEX,低能日粮),低能日粮对照组(生理盐水,低能日粮),应激及日粮自由选择组(DEX,高,低能日粮),日粮自由选择对照组(生理盐水,高,低能日粮)。每组8个重复,每个重复两只鸡,单笼饲养。应激处理及采食量统计同试验一,10日龄早8:00采样。试验鸡心脏采血,断头后于30s内取出下丘脑,液氮速冻。试验结果表明:应激显著降低了肉仔鸡生产性能,高能日粮能缓解应激对采食的影响;应激及日粮能量水平对下丘脑葡萄糖含量没有显著影响;下丘脑各采食调控基因mRNA水平的变化提示肉仔鸡在长期应激状态下,通过对能量摄入量的控制,维持了下丘脑采食调控的平衡。试验三分为两个小试验,trial 1将体重相近的AA雄性肉仔鸡80只,随机分为两个处理组:应激组,对照组。试验在单饲笼内进行,每组设10个重复,每个重复4只鸡。11日龄起每天06:0(0空腹3h),应激组按2.6 mg/kg BW的剂量皮下注射DEX,对照组同剂量皮下注射生理盐水,连续注射三天,13日龄的08:00(应激2h,空腹5h)和09:00(应激3h,恢复采食1h)两个时间点分别采样(血浆,下丘脑)。Trial 2分组同trial 1,11日龄起注射时间为每天10:00(空腹1h), 13日龄的12:00(应激2h,空腹3h)采样,样品采集同trial 1。试验主要结果显示:禁食状态下,应激显著升高了肉仔鸡下丘脑NPY,AgRP的表达量,这两个基因水平的上调随着采食的恢复消失。
第二部分中,试验一选取体重相近的AA肉仔鸡32只随机分为四组,早6:00禁食3h后,以2.6mg/kgBW剂量注射DEX,同时灌注色氨酸(生理盐水溶解,500mg/kgBW),两个处理分别设一个生理盐水对照组,3h后取血浆及下丘脑。下丘脑用高氯酸萃取后进行NMR研究。通过1H NMR谱,发现应激改变了肉鸡下丘脑乳酸,葡萄糖,ATP,肌醇,3-羟基丁酸的相对含量;未检测到色氨酸信号的改变。试验二选取体重相近的AA肉仔鸡32只,随机分为四个组。早6:30禁食,3小时后A、B组按2.6mg/kg体重注射DEX,饲喂高能日粮。C、D组注射相应剂量的生理盐水,饲喂低能日粮。11:30所有试验鸡心脏采血。取下丘脑液氮速冻。试验结果显示:皮下注射地塞米松成功引发肉仔鸡急性应激;对下丘脑采食调控基因的检测结果主要提示,急性应激可能通过上调下丘脑NPY的表达量导致肉仔鸡短期内的采食量的升高。
To investigate the effects and mechanisms of stress on appetite of broilers(Gallus gallus domesticus), exogenous glucocorticoids was used to mimick stress on two phases of stress development of broilers. Changes of metabolism and mRNA expression level of important genes in appetite regulation in hypothalamus were measured and analyzed, in order to providing countermeasures for reforming energy waste in broiler chicken production.
In part 1, there are 3 trials in experiment 1. In trial 1, 64 male broilers (Arbor Acres) with similar body weight were divided into 4 groups randomly, rearing in separate cage in which there were two containers with the color of red and green relatively. In group1, animals were stressed and diets were be at choice (DEX, high and low energy diet, n=8). In group 2, treats were the same as in group 1 except that dexamethasone was displaced by saline. In group 3, diets were all high energy(ME, 15.1KJ/kg; CP, 20%) (n=8), while diets were low energy (ME, 10.9KJ/kg; CP, 20%) in group 4(n=8); In trial 2, 96 AA broilers with similar body weight were divided into 6 groups randomly. On the basis of trial 1, there were two more groups in which the containers were noncolored (DEX, high, low energy diet; control). In trail 3, 32 AA broilers with similar body weight were divided into 2 groups. The treatments were the same as the container-noncolored groups in trail 2. All the broilers in this experiment were stressed by subcutaneous injection of DEX with a dose of 2.6mg/kg BW at 8:00 from 5 to 10 days old. Saline was injected with a suitable dose in the control group. During the trial, food intake was noted every day. At the day of 10, blood was obtained. The results showed that, stress was induced successfully and it increased the high energy diet ingestion, however, the body weigh was decreased significantly. All of these suggest that stress alters the appetite of broilers; there was no significant effect of color on food intake. In experiment 2, 96 AA broilers with similar body weight were divided into 6 groups: A (DEX, high energy diet), B (Saline, high energy diet), C (DEX, low energy diet), D (saline, low energy diet), E (DEX, diets at choice), F (saline, diets at choice). The stress treatment and food intake data record was the same as in experiment 1. At the day of 10, blood and hypothalamus were obtained. The results show that, stress reduced the production performance of broilers significantly, while high energy diet blunted the effect of stress on food intake; stress and diet energy level have no significant effect on glucose content of hypothalamus; changes of appetite regulated genes’mRNA level in hypothalamus suggested that in the state of chronic stress, broiler can maintain the balance of food intake regulation in hypothalamus via the controll of the energy intake. In experiment 3, there were two similar trails. In trial 1, 80 AA broilers were divided into two groups: stress and contro group. There were 10 repetitions in every group with 4 broilers in every repetition. From 11 days old to 13 days old, broilers were injected with the same dose of DEX and saline in experiment 1 at 6:00 (fasting for 3h), respectively. At the day of 13, samples (blood and hypothalamus) were obtained at 8:00 (stress for 2h, fasting for 5h) and 9:00 (stress for 3h, refeed for 1h). In trial 2, group dividing and the treatment were the same as which in trail 1, except that the time of injection was 10:00 (fasting for 1h), and samples were obtained at 12:00 (stress for 2h, fasting for 3h). The results primarily showed that, in the state of fasting, stress up-regulated the expressions of NPY and AgRP in hypothalamus significantly, while refeed vanished the effect.
In part 2, trail 1, 32 chickens of 10-day-old was divided into 4 groups randomly, DEX (2.6mg/kg BW) and tryptophan (0.5g/kg BW) were adminstrated after 3h fasting at 9:00, and saline control was set up respectively. Blood and hypothalamus was obtained and hypothalamus extraction was used for NMR study. The ~1H NMR spectra shows that, the relative concentration of lactate, glucose, ATP, myo-Inositol and 3-Hydroxybutyrate was changed by stress, while tryptophan signal was unchangeable. In experiment 2, 32 AA broilers were divided into 4 groups. Broilers were fasted at 6:30, and 3 hours later, two groups were injected DEX and refeeded high energy diet. Another two groups were injected saline, and refeeded low energy level. At 11:30, all the blood samples were obtained, and hypothalamus were quick-freezed in liquid nitrogen. The results showed that, the detection of food intake regulated genes in hypothalamus showed primarly that, acute stress possibly induced the temporary increase of food intake via up regulated the expression of hypothalamic NPY.
引文
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