【作者】 王丽；

【导师】 蒋宗勇；

【作者基本信息】 东北农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 本课题主要探讨二丁酰环腺苷酸(dbcAMP)对肥育猪胴体品质和肉质的影响及作用机理,主要包括动物饲养试验、脂肪前体细胞培养试验和骨骼肌卫星细胞培养试验三部分。动物饲养试验:选用72头平均体重约60kg的杜×大×长肥育阉公猪,随机分为3个处理,每个处理6个重复,每个重复4头猪。对照组饲喂基础日粮,试验组在基础日粮中分别添加10mg/kg和20mg/kg dbcAMP(纯度98%),自由采食和饮水,饲养至90kg左右结束试验,每重复随机选出1头猪屠宰,研究dbcAMP对肥育猪生产性能、胴体品质和肌肉品质的影响。结果表明:1)饲粮添加dbcAMP未显著影响肥育猪平均日增重、日采食量和料重比(P>0.05)。2)添加10mg/kg dbcAMP显著降低肥育猪宰后花板油所占比例和第一肋骨处的背膘厚(P<0.05),第十肋骨处的背膘厚略有降低的趋势(P=0.10);显著提高瘦肉率(P<0.05),无脂瘦肉日增重有升高的趋势(P=0.10),眼肌面积也提高了13.92%(P=0.10)。3)添加10mg/kg dbcAMP显著提高了腹部肌肉所占比例(P<0.05),添加dbcAMP臀部肌肉所占比例呈线性上升的趋势变化(P=0.06),背部肌肉所占比例提高了1.71%~1.27%(P>0.05),而前腿和后腿肌肉所占比例有所下降(P>0.05)。4)随着dbcAMP添加量的增加,肥育猪背部脂肪细胞直径分别降低了4.37%和7.68%(P<0.05),肌纤维直径有线性上升的趋势(P=0.06)。5)饲粮添加dbcAMP显著增加了血浆环腺苷酸(cAMP)、总蛋白(TP)、甲状腺素(T4)的含量,肾上腺素含量有线性增加趋势(P=0.06),未显著影响血浆胆固醇(CHO)、甘油三酯(TG)、生长激素(GH)、胰岛素(INS)、胰岛素样生长因子(IGF-1)和瘦素(Leptin)的含量(P>0.05)。6)饲粮添加dbcAMP对肥育猪背最长肌、股二头肌和半腱肌的pH值、肉色、大理石纹、嫩度和肌内脂肪均无显著影响(P>0.05),添加10mg/kg dbcAMP显著降低了股二头肌宰后24h的滴水损失值(P<0.05),处理组背最长肌和半腱肌宰后24h、48h的滴水损失值均低于对照组(P>0.05)。7)添加10mg/kg dbcAMP显著提高了背脂中激素敏感脂酶(HSL)活性、β肾上腺素受体(β-AR)、生长激素受体(GHR)mRNA表达量(P<0.05),而添加20mg/kg dbcAMP显著降低了过氧化物酶体增殖物激活受体(PPARγ2)和脂肪型脂肪酸结合蛋白(A-FABP)mRNA表达量(P<0.05);添加10mg/kg和20mg/kg dbcAMP均显著提高腹脂中G蛋白偶联受体(GPCR)mRNA表达量(P<0.05)和降低胰岛素受体(INSR)mRNA表达量(P<0.05),同时10mg/kg dbcAMP显著提高了β-AR、GHR mRNA表达量(P<0.05),20mg/kg dbcAMP显著提高了cAMP反应元件结合蛋白(CREB)、IGF-1 mRNA表达量(P<0.05);添加10mg/kg和20mg/kg dbcAMP均显著抑制肾周脂中的INSR、CCAAT/增强子结合蛋白α(C/EBPα)、A-FABP mRNA表达量(P<0.05),而20mg/kg dbcAMP显著提高了HSL活性、GPCR、CREB、IGF-1 mRNA表达量以及降低脂肪酸合成酶(FAS)、脂蛋白脂酶(LPL)活性(P<0.05)。8)添加10mg/kg和20mg/kg dbcAMP均显著提高了背最长肌中腺苷酸环化酶(AC)活性、生肌决定因子(MyoD)、α-肌动蛋白(α-actin)mRNA表达量(P<0.05),同时10mg/kg dbcAMP显著提高了GPCR、IGF-1、肌球蛋白重链(MHC)mRNA表达量(P<0.05);添加10mg/kg和20mg/kg dbcAMP均显著提高了腹肌中cAMP依赖性蛋白激酶(PKA)活性、GPCR、MHC mRNA表达量(P<0.05),同时10mg/kg dbcAMP显著提高了腹肌中的cAMP含量、CREB、MyoD mRNA表达量(P<0.05);添加10mg/kg和20mg/kg dbcAMP均显著促进股二头肌中IGF-1、MyoD、MHC mRNA表达量(P<0.05),同时10mg/kg dbcAMP显著提高了GPCR、CREB、a-actin mRNA表达量(P<0.05);添加dbcAMP显著促进半腱肌中的MyoD mRNA表达量以及降低肌抑素(Myostatin)mRNA表达量(P<0.05)。综合上述结果提示,饲粮添加dbcAMP可以改善胴体品质,促进蛋白质沉积而减少脂肪沉积,且10mg/kg组效果较好。脂肪前体细胞培养试验:选用7日龄杜×大×长三元杂交仔猪,无菌条件下分离背部皮下脂肪进行脂肪前体细胞培养。使用0、0.001、0.01、0.1、1、10、100、1000μmol/LdbcAMP处理细胞1、2、3、4、5、6天,研究dbcAMP对脂肪前体细胞增殖的影响;处理细胞2、4、6、8、10、12天,研究dbcAMP对脂肪前体细胞分化的影响;使用0、0.001、0.1、10、1000μmol/LdbcAMP处理细胞4天,研究dbcAMP对脂肪细胞内与脂肪代谢沉积相关的酶活和基因表达的影响。结果表明:1)添加0.001~1000μmol/LdbcAMP均有抑制脂肪前体细胞增殖的趋势,其中处理前期dbcAMP浓度大于0.01μmol/L即有显著的抑制效果(P<0.05),而处理后期浓度增大至100~1000μmol/L的抑制增殖作用最大(P<0.05)。2)dbcAMP对脂肪前体细胞分化的影响也存在添加浓度和处理时间效应,短期处理(2~6天)时细胞的分化呈先下降再上升的二次曲线变化(P<0.05),且0.1~1μmol/L的抑制分化作用最大(P<0.05),随着处理时间的延长(10~12天),0.1~1000μmol/L均显著抑制脂肪前体细胞分化(P<0.05),其中最大抑制分化浓度增大到100~1000μmol/L(P<0.05)。3)向脂肪前体细胞培养液中添加dbcAMP显著提高了细胞内cAMP含量、PKA活性(P<0.05)和GPCR、β-AR、GHR和CREB mRNA表达量(P<0.05),而显著降低C/EBPα、PPARγ2和A-FABP mRNA表达量(P<0.05)。以上结果提示,添加适量的dbcAMP可抑制脂肪前体细胞增殖和分化,并有抑制脂肪合成的作用。骨骼肌卫星细胞培养试验:选用7日龄杜×大×长三元杂交仔猪,无菌条件下分离背最长肌进行骨骼肌卫星细胞培养。使用0、0.001、0.01、0.1、1、10、100、1000μmol/LdbcAMP处理细胞1、2、3、4、5、6天,研究dbcAMP对骨骼肌卫星细胞增殖的影响;使用0、0.001、0.1、10、1000μmol/LdbcAMP处理细胞4天,研究dbcAMP对骨骼肌细胞内与蛋白质代谢沉积相关的酶活和基因表达的影响。结果表明:1)随着dbcAMP浓度的增加骨骼肌卫星细胞的增殖呈先升高后下降的二次曲线变化(P<0.0001),除第1天0.1μmol/LdbcAMP显著促进骨骼肌卫星细胞增殖(P<0.05)外,2~6天时均为0.001~0.1μmol/LdbcAMP有促进细胞增殖的趋势,且0.1μmol/L组效果最好(P>0.05),1~1000μmol/L时有抑制细胞增殖的趋势,且1000μmol/L组的抑制增殖作用最大(P<0.05)。2)向骨骼肌卫星细胞培养液中添加dbcAMP显著提高了cAMP含量、AC活性(P<0.05)以及GPCR、MHC mRNA表达量(P<0.05);显著降低了calpain活性(P<0.05)以及Myostatin mRNA表达量(P<0.05),而有升高calpastatin活性的趋势(P=0.07);此外,0.1μmol/LdbcAMP均有提高GHR、CREB、IGF-1、MyoD、α-actin mRNA表达量的趋势(P>0.05)。上述结果提示:一定浓度的dbcAMP可促进骨骼肌卫星细胞增殖,并有促进蛋白质合成和抑制蛋白质降解的作用。综合三个试验的结果得出,在本试验条件下,细胞中最佳dbcAMP添加浓度为0.1μmol/L,而动物饲养试验中的最佳剂量为10mg/kg,同时得出dbcAMP调控脂肪沉积的可能机理是:适量添加dbcAMP能促进脂肪中β-AR mRNA表达量的增加,dbcAMP与β-AR结合,激活Gs蛋白,进而活化AC,促使细胞内cAMP含量增加,而后激活PKA,活化的PKA释放催化亚基去催化细胞内其它蛋白质(如CREB或HSL)的磷酸化,HSL浓度的提高可使线粒体进入解偶联呼吸状态,底物氧化耗能增加,最终使脂肪分解代谢增强;也可通过降低FAS、LPL的活性和PPARγ2、A-FABP mRNA的表达,抑制脂肪细胞分化,降低脂肪沉积;还可通过降低INSR数量,减少INS与受体结合的机会,抑制INS对体脂沉积的促进作用。得出dbcAMP调控蛋白质沉积的可能机理是:通过GPCR-AC-cAMP-PKA途径,调节转录因子CREB的活性,加速了基因的转录和表达,从而使蛋白质合成增多;通过增加骨骼肌内结构蛋白α-actin和MHC的mRNA表达量以及生肌决定因子MyoD的表达,提高蛋白质的合成量,同时降低肌抑素Myostatin mRNA表达,减少蛋白质的降解量;还可通过GH/IGF-1途径,IGF-1 mRNA表达量的提高,能直接诱导骨骼肌细胞分化,促进DNA合成,从而导致蛋白质合成增加。更多还原

【Abstract】 This study was conducted to investigate the effects and mechanisms of dbcAMP (N6, 2’-O-dibutyryl adenosine 3’, 5’cyclic monophosphate) on carcass traits and pork quality in finishing pigs by feeding experiment, preadipocytes culture and skeletal muscle satellite cells culture experiment.Feeding experiment: seventy-two Duroc×(Landrace×Large White) crossed barrows (57.3±0.6 kg) were randomly allotted to 3 treatments with 6 replicate pens (4 pigs per pen). The pigs were fed containing 0 (control), 10 and 20 mg/kg dbcAMP (purity, 98%) diets, respectively, and allowed ad libitum access to feed and water, until final slaughter weight of about 90 kg to determine the effects of dietary dbcAMP supplementation on growth performance, carcass traits and pork quality in finishing pigs. Results showed that there were no significant differences in average daily gain (ADG), average daily feed intake (ADFI) and gain to feed ratio (G:F) between treatments (P>0.05). Leaf fat percentage and the first rib backfat thickness of pigs fed 10 mg/kg dbcAMP were significantly reduced (P<0.05) and the tenth rib backfat thickness slightly decreased (P=0.10). Lean percentage was obviously increased (P<0.05) in the pigs treated with 10 mg/kg dbcAMP compared with the control and longissimus muscle area (LMA) was slightly increased by 13.92% (P=0.10). Growth rate of free-fat lean from pigs fed dbcAMP had a trend to increase (P=0.09). Dietary dbcAMP improved significantly muscle of abdomen percentage (P<0.05), increased the ratio of croup muscle to the whole body linearly (P=0.06), and elevated the ratio of back muscle to the whole body by 1.71%~1.27% (P<0.05). Dietary dbcAMP decreased significantly adipocytes diameter of back subcutancous fat by 4.37%~7.68% (P<0.05). Fiber diameter of LM had an augmented tendency (P=0.06) with increasing level of dbcAMP, however, there was no difference in fiber density of LM (P>0.05) among all treatments. The content of total protein, T4 and cAMP (3’,5’-cyclic adenosine monophosphate) in serum were increased significantly (P<0.05), and adrenaline content was increased linearly with increasing level of dbcAMP (P=0.06). Dietary dbcAMP had no significant effects on triglyceride, total cholesterol, urea nitrogen, insulin, leptin, growth hormone (GH) and insulin-like growth factor 1 (IGF-1) in serum (P>0.05). Compared with the control, supplementation with 10 mg/kg dbcAMP reduced notably drip loss of biceps femoris muscle (BFM) at 24h post-slaughter (P<0.05), decreased slightly drip loss of longissimus dorsi muscle (LDM) and semitendinous muscle (STM) at 24h and 48h post-slaughter (P>0.05), but did not affect significantly pH value, meat color, marbling score, tenderness, intramuscular fat of LDM, BFM and STM (P>0.05). Supplementation with 10 mg/kg dbcAMP enhanced significantly hormone-sensitive lipase (HSL) activity,β-adrenergic receptor (β-AR) and growth hormone receptor (GHR) mRNA expression (P<0.05), however, supplementation with 20 mg/kg dbcAMP depressed notably peroxisome proliferators-activated receptor (PPARγ2) and adipocyte fatty acid binding protein (A-FABP) mRNA expression in backfat (P<0.05). G-protein couple receptor (GPCR) mRNA expression were enhanced significantly and insulin receptor (INSR) mRNA expression was decreased in abdominal fat by dbcAMP supplementation (P<0.05), meanwhile, in contrast to the control, adding 10 mg/kg dbcAMP in the diet increasedβ-AR, GHR mRNA expression (P<0.05) and adding 20 mg/kg dbcAMP promoted cAMP response element binding protein (CREB), IGF-1 mRNA expression in abdominal fat (P<0.05). Dietary dbcAMP decreased significantly INSR, A-FABP and CCAAT/enhancer binding proteinα(C/EBPα) mRNA expression in perirenal fat (P<0.05), however, supplementation with 20 mg/kg dbcAMP increased notably HSL activity and GPCR, CREB, IGF-1, PPARγ2 mRNA expression (P<0.05), as well as decreased significantly fatty acid synthetase (FAS) and lipoprotein lipase (LPL) activity (P<0.05). Compared with the control, supplementation with dbcAMP elevated significantly adenylate cyclase (AC) activity, and myogenic determinative factor (MyoD),α-actin mRNA expression in LDM (P<0.05), furthermore, adding 10 mg/kg dbcAMP in the diet promoted obviously GPCR, IGF-1 and myosin heavy chain (MHC) mRNA expression (P<0.05). Supplementation with 10 and 20 mg/kg dbcAMP enhanced significantly cAMP dependent protein kinase A (PKA) activity and GPCR, MHC mRNA expression in abdominal muscle (P<0.05), and supplementation with 10 mg/kg dbcAMP elevated cAMP content and CREB, MyoD mRNA expression (P<0.05). Dietary dbcAMP promoted notably IGF-1, MyoD, MHC mRNA expression in BFM (P<0.05), and adding 10 mg/kg dbcAMP in the diet increased obviously GPCR, CREB,α-actin mRNA expression (P<0.05). MyoD mRNA expression in STM was promoted (P<0.05) and myostatin mRNA expression was depressed significantly by dbcAMP supplementation (P<0.05). These results indicated that supplementation with dbcAMP in the diet could improve carcass traits, promote protein deposition and reduce fat deposition in finishing pig, moreover, 10 mg/kg dbcAMP was advantageous.Preadipocytes culture experiment: seven day old Duroc×Landrace×Large White three-crossed piglets were killed to separate back subcutaneous fat for preadipocytes culture under the sterile situation. The cells grown at humidified atmosphere of 5% in CO2 at 37℃for 2 days, then the cells were exposed to the medium supplemented with different concentrations (0, 0.001, 0.01, 0.1, 1, 10, 100, 1000μmol/L) of dbcAMP and incubated for 1, 2, 3, 4, 5 and 6 days, respectively, to investigate the effects of dbcAMP on proliferation of porcine preadipocytes, and incubated for 2, 4, 6, 8, 10 and 12 days, respectively, to investigate the effects of dbcAMP on differentiation of porcine preadipocytes. The different concentrations (0, 0.001, 0.1, 10, 1000μmol/L) of dbcAMP was supplemented to the cell culture medium for 4 days, to explore the effects of dbcAMP on enzyme activities and gene expressions that related to fat metabolism of porcine adipocytes. Results showed that, supplementation with 0.001~1000μmol/L dbcAMP had a consistent tendency to inhibit proliferation of porcine preadipocytes all through the time, and in early stage, exceeding 0.01μmol/L dbcAMP had significant inhibition on proliferation of porcine preadipocytes (P<0.05), but in later stage, when porcine preadipocytes were incubation with 100 or 1000μmol/L dbcAMP, the inhibiting effect on proliferation reached maximum (P<0.05). Effect of dbcAMP on differentiation of porcine preadipocytes depended on its concentration and duration time, the differentiation of porcine preadipocytes changed quadratically with increasing concentration of dbcAMP at short duration time (2 to 6 days), and the differentiation of 0.1 or 1μmol/L group reached minimum (P<0.05), with lasting incubation time (10 to 12 days), the differentiation of porcine preadipocytes were inhibited significantly treated with 0.1 to 1000μmol/L dbcAMP (P<0.05), and the differentiation of the cells treated with 100 or 1000μmol/L dbcAMP reached minimum. In contrast to the control, PKA activity and cAMP content of porcine preadipocytes were increased significantly (P<0.05), and GPCR,β-AR, GHR, CREB mRNA expression were promoted notably (P<0.05), moreover, C/EBPα, PPARγ2 and A-FABP mRNA expression were decreased significantly (P<0.05) by supplementation with dbcAMP in the medium. Above-mentioned results indicated the suitable dosage of dbcAMP could inhibite proliferation and differentiation of porcine preadipocytes.Skeletal muscle satellite cells culture experiment: seven day old Duroc×Landrace×Large White three-crossed piglets were killed to separate LDM for skeletal muscle satellite cells (SMSC) culture under the sterile situation. The cells grown at humidified atmosphere of 5% in CO2 at 37℃for 2 days, then the cells were exposed to the medium supplemented with different concentrations (0, 0.001, 0.01, 0.1, 1, 10, 100, 1000μmol/L) of dbcAMP and incubated for 1, 2, 3, 4, 5 and 6 days, respectively, to investigate the effects of dbcAMP on proliferation of porcine SMSC. The different concentrations (0, 0.001, 0.1, 10, 1000μmol/L) of dbcAMP was supplemented to the cell culture medium for 4 days, to explore the effects of dbcAMP on enzyme activities and gene expressions that related to protein metabolism of porcine SMSC. Results showed that, the proliferation of porcine SMSC changed quadratically with increasing concentration of dbcAMP all through the time (P<0.0001). The proliferation of porcine SMSC adding 0.001 to 0.1μmol/L dbcAMP in the medium were numerically greater than those of the control (P>0.05) except that the proliferation of the cells treated with 0.1μmol/L group of incubated for 1 day was promoted significantly (P<0.05), while supplementation with 1 to 1000 dbcAMP had a tendency to inhibit proliferation of porcine SMSC (P>0.05), and the inhibiting effect of 1000μmol/L group reached maximum (P<0.05). The content of AC and cAMP of porcine SMSC were enhanced significantly (P<0.05) and calpastatin activity had some elevation (P=0.07), GPCR, MHC mRNA expression were promoted notably (P<0.05), furthermore, calpain activity and myostatin mRNA expression were decreased significantly (P<0.05). In addition, relative to the control, GHR, CREB, IGF-1, MyoD andα-actin mRNA expression had increased numerically (P>0.05). The results indicated the suitable dosage of dbcAMP could promote proliferation of porcine SMSC, increased protein synthesis and suppressed protein degradation. In conclusion, under this experimental condition, the optimal dosage was 0.1μmol/L in cell culture experimental and 10 mg/kg in feeding experimental. Simultaneously, possible mechanism of dbcAMP regulating fat deposion were that appropriate dosage of supplementation with dbcAMP could promoteβ-AR mRNA expression in porcine fat tissue, thenβ-AR on the membrane is coupled to Gs-protein, they transmit an activation signal to AC increasing cAMP production, then PKA is activated by the increasing intracellular cAMP levels and this finally leads to phosphorylation and activation of HSL, furthermore, HSL breaks the triglycerides stored in adipocytes thus producing glycerol and FFA. On the other hand, other signalling pathways are involved in the lipolytic response of fat. One of these pathways involves that appropriate dosage of supplementation with dbcAMP could reduce FAS and LPL activities and PPARγ2, A-FABP mRNA expression to suppress differentiation of preadipocytes, thus to reduce fat deposition. Another pathway is that appropriate dosage of supplementation with dbcAMP could reduce INSR amount, decrease the opportunity of INS binding to INSR, inhibit the promotion of fat deposition of INS. From the results of muscle tissues and cells, possible mechanism of dbcAMP mediating protein deposition involved several pathways, and the GPCR-AC-cAMP-PKA pathway plays important roles. The activity of transcription factor CREB is regulated by the activation of PKA, and accelerates the transcription and expression of genes, thus increases protein synthesis. In addition to cAMP pathway, one of other signalling pathways involves that appropriate dosage of supplementation with dbcAMP could stimulateα-actin, MHC and MyoD mRNA expression of skeletal muscle to elevate protein synthesis, simultaneously, suppress myostatin mRNA expression to decrease protein degradation. The other possible mechanisms of regulating protein deposition implies GH/IGF-1 pathway, appropriate amount of supplementation with dbcAMP could promote IGF-1 mRNA expression, induce directly differentiation of SMSC to elevate protein synthesis.更多还原