| MSTN, Insulin-like growth factor-I (IGF-I) and Insulin-like growth factor-II (1GF-II) are regulators of skeletal muscle growth. This study investigated the effects of different intake levels on skeletal muscle. The experiment was conducted at the R&D Centre of Haimen Goat of Nanjing Agriculture University (Haimen, Jiangsu, China)(longitude121.28°, latitude31.89°) from May to July2011, using18Dorper sheep and Hu sheep hybrid F1rams of the similar age, body weight (weighed35.83±0.87kg), and body composition. During the experiment, the animals were available to water freely, maintained under natural photoperiod and received two equal allotments of feed at8:00AM and16:00PM. In the beginning of the experiment, animals were pre-fed a2.15×maintenance diet until the25th day (the weight similar to42±0.96kg), then they were randomly assigned to three groups:the maintenance diet group (M), the1.4×maintenance diet group (1.4M) and the2.15×maintenance diet group (2.15M). According to the previous report, all sheep received a total mixed ration diet that the M received a diet of1000g/day per sheep while the1.41M received a diet of1400g/day per sheep and2.15M received a diet of2150g/day per sheep to achieve daily weight gained350g,150g and0g respectively. Till the weight of the sheep in2.15×maintenance diet group similar to50kg (50.01±1.11kg), before the animals were slaughtered blood (5mL) was sampled from the jugular vein by venipuncture, placed immediately in heparinized tubes, maintained on ice, separated by centrifugation at4℃, and stored at-20℃for further measurement; the animals were slaughtered by jugular vein bloodletting. Within5min of death different pieces of skeletal muscle including longissimus dorsi, semitendinosus, semimembranosus, gastrocnemius, soleus and chest muscle were quick-frozen and stored in liquid nitrogen until RNA extraction. Small blocks of tissue (2×1×1cm3in size) were cut off from semitendinosus, semimembranosus, longissimus dorsi, gastrocnemius, soleus and chest muscle, placed in physiological saline and stored in4%paraformaldehyde for further dispose. All experimental procedures were performed according to the guide for animal care and use of laboratory animals of the Institutional Animal Care and use Committee of Nanjing Agricultural University. This study contained the following three parts:Experiment1. Effects of different dietary intake on the mRNA levels of MSTN, IGF-I and IGF-II in skeletal muscle of sheepExpression analysis with real-time fluorescence quantita-tive PCR showed that expressions of IGFs and MSTN in skeletal muscle varied under different intake level. With the improvement of the dietary intake, the mRNA of MSTN and IGF-II decreased and IGF-I increased. The mRNA of MSTN was negatively correlation with IGF-I; MSTN was positively correlation with IGF-I.(1) In soleus, expression of MSTN in the M significant higher than in the2.15M and the1.4M (P<0.01, P<0.05respectively); In chest muscle, MSTN mRNA expressed in the1.4M higher than in the2.15M (P<0.05).(2) The IGF-I mRNA in the2.15M gained the highest expression among the three treatment groups, following by the expression in the M and1.4M:in longissimus dorsi, expression of IGF-I in the1.4M extremely significant lower than in the M (P<0.01), expression of IGF-I in the2.15M extremely significant higher than in the M (P<0.05); in the semitendinosus, expression of IGF-I in the2.15M significant higher than in the1.4M and the M (P<0.01, P <0.05respectively), expression of IGF-I in the1.4M significant lower than in the M(P<0.05); in semimembranosus and soleus, expression of IGF-I in the1.4M significant lower than in the2.15M and the M (P<0.05, P<0.05respectively).(3) The IGF-II mRNA in the M gained the highest expression among the three treatment groups, following by the expression in the2.15M and1.4M:in longissimus dorsi, expression of IGF-II in the1.4M significant lower than in the2.15M and the M (P<0.05, P<0.01respectively); in semitendinosus, expression of IGF-II in the M significant higher than in the2.15M and the1.4M (P<0.05, P<0.01respectively); in semimembranosus, expression of IGF-II in the1.4M extremely significant lower than in the2.15M and the M (P<0.01, P<0.01respectively); in gastrocnemius, expression of IGF-II in the1.4M significant lower than in the2.15M and the M (P<0.05, P<0.01respectively); in soleus, expression of IGF-II in the1.4M extremely significant lower than in the2.15M(P<0.01), in the M significant higher than in the2.15M and the1.4M (P<0.05, P<0.05respectively); in chest muscle, expression of IGF-II in the M significant higher than in the2.15M(P<0.05).Experiment2. Effects of different dietary intake on the plasma and cerebrospinal fluid concentration of sheep This study used spectrophotometer to detect plasma protein metabolism (urea and uric acid), lipid metabolism (HDL-c and LDL-c) and enzyme activity index (ALT and AST), using radioimmunoassay to determine growth hormone and metabolic hormone. With the improvement of the dietary intake, urea in the2.15M extremely significant higher than in the1.4M and the M (P<0.01, P<0.01respectively). Uric acid gained the highest concentration in2.15M. No significant correlation between plasma HDL-c and LDL-c and feeding level. In1.4M, HDL-c and LDL-c content was the lowest. LDL-c in the2.15M significant higher than in the1.4M (P<0.01). With the improvement of the dietary intake, the concentration of plasma AST increased. ALT in the2.15M significant higher than in the1.4M (P<0.01). AST in the M significant lower than in the2.15M and the1.4M (P<0.01, P<0.05respectively). With the improvement of the dietary intake, the insulin and glucagon increased meanwhile the concentration of GH, T3and T4decreased. The insulin in the M was significant lower than that in2.15M (P<0.01) and1.4M (P<0.05). GH in the M was significant higher than that in2.15M (P<0.05). T3in the M was significant higher than that in1.4M (P<0.05). The effects of intake level had similar influence on growth hormone in the cerebrospinal fluid and plasma; the dietary intake improved meanwhile GH concentration decreased. The content of1.4M is slightly higher than the content in M.Experiment3. Effects of different dietary intake on the skeletal muscle fibre diameter and cross-sectional are of sheepIn skeletal muscle, the muscle fibre diameter and cross-sectional area increased significantly when the intake levels improved. The change tend of them was similar to the expression of IGF-I while that were converse to the expression of MSTN and IGF-II. |
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