Studies On Effects And Mechanisms Of Dbcamp On Carcass Traits And Pork Quality In Finishing Pigs

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.