| During the final days of incubation, a large amount of energy was required for development of small intestine and frequent activity of late-term poultry embryos, which finally drive to mobilize the skeletal muscle to provide the substrates for glyconeogenesis and fuel for small intestine. Therefore, the skeletal muscle development posthatch was delayed by the removal of muscle during the final days of incubation. Based on the duck embryos model, the objective of present study was to:disclose the relationship between energy status and protein metabolism in skeletal muscle and to explore the effect of nutrient manipulation on the small intestine and skeletal muscle by in ovo injecting disaccharide, glutamine andβ-hydroxy-β-methylbutyrate to late-term duck embryos. The present study was composed of 4 parts.The first experiment was conducted to evaluate the developmental changes in energy status, small intestine growth and protein metabolism in pectoral muscle, aiming to discuss the relationship between energy status, small intestine development and skeletal muscle growth, At 22 days (22E),25 days (25E) of incubation, hatch (0d),3 days (3d),7 days (7d) posthatch, ten ducks at each time point were selected for pectoral muscle, liver, blood (n=10) and jejunum sampling (n=8). Experiment was conducted for determination of plasma glucose concentration, hepatic and pectoral glycogen content (iodine colorimetic assay method), cross section area of muscle fiber bundle (paraffin section and HE staining), Atrgoin-1 and FoxO1 gene expression by using qPCR method and p-AMPK, p-FoxO1 and p-S6K1 protein expression by using Western Blot method. The results of this experiment showed that:There was a sharp decrease in glycogen content from 22E to Od, which reached 58% for hepatic glycogen content and 85% for pectoral glycogen content, respectively (P<0.0001); glycogen in both liver and muscle increased quickly after hatch. Plasma glucose level increased by 135% from 22E to hatch (P<0.05), which showed increasing tendency posthatch. From 22E to hatch, the weight and length of small intestine increased by 352%(P<0.0001) and 48%(P<0.05), respectively. At 7d posthatch, weight and length of small intestine are 500% and 107%, respectively. Similarly, the jejunal villus length and maltase activity was 1.5 folds and 12 folds higher than that of hatch, respectively. After hatch, villus length, maltase and sucrase activity in jejunum increased markedly with the age of ducklings posthatch. Pectoral mass decreased by 45% from 22E to hatch (P<0.05). From 22E to hatch, the cross section area of muscle fiber bundles and myofiber decreased by 54% and 50%, respectively (P<0.05), while there was a sharp increase in cross section area of muscle fiber bundles and myofiber posthatch. Atrogin-1 mRNA expression abundance was elevated by 74-fold from 22E to hatch (P<0.0001), whereas, reached a very low level at 7d posthatch. Similarly, FoxO1 mRNA expression abundance at hatch was 5 folds higher than that at 22E (P<0.0001), and reached a very low level. From 22E to hatch, p-AMPK/AMPK and in pectoral muscle decreased by 77.5%(P<0.05), and the value of p-AMPK/AMPK at low level sustained to d 7. From 22E to hatch, p-S6K1/S6K1 decreased by 71.4%(P<0.05) and increased markedly after hatch. The results of present experiment demonstrated that energy store removed markedly and small intestine develop at high rate in late-term duck embryo, which probably retard the muscle growth by stimulating ubuiquitin mediated protein degradation and inhibiting protein synthesis.The second experiment was carried out to evaluate the effect of sugar and amino acid administration to late-term duck embryos on the energy status of duck. At 21 days of incubation (21E),520 eggs containing viable embryos were allocated into 4 groups. At 23 days of incubation (23E), ducks in 3 treatments received in ovo injection as follows:1) sucrose and maltose (DS); 2) alanyl-glutamine (Ala-Gln); 3) sucrose, maltose and alanyl-glutamine (DS+Ala-Gln), the non-injected duck eggs served as control. Ducklings in each treatment were randomly divided into 10 replicates with pen for each replicate. Commercial diet in pellet form was provided to the four groups. At 25 days of incubation (25E), hatch (0d),3 days (3d) and 7 days (7d) posthatch, the liver, pectoral muscle and blood within each group were sampled for determination of glycogen content, plasma glucose level and hepatic glucose-6-phosphatase enzyme activity(n=10). The results of this experiment showed that:DS and DS+Ala-Gln in ovo injected ducks had higher hepatic glycogen content at 25E and hatch in comparison to control (P<0.05). At 3d posthatch, the hepatic glycogen content in control and DS treatment was significantly higher than Ala-Gln and DS+Ala-Gln treatment (P<0.05). The pectoral glycogen content in Ala-Gln and DS+Ala-Gln treated ducks were significantly higher than control (P<0.05) at 25 days of incubation (25E). Compared with control, in ovo injecting DS and Ala-Gln increased plasma glucose level at Od and 3d (P<0.05). At 25 days of incubation (25E), the glucose-6-phosphatase enzyme activity was supressed by DS, Ala-Gln and DS+Ala-Gln in ovo injection (P<0.0001), whereas, DS and DS+Ala-Gln treated ducks had lower glucose-6-phosphatase enzyme activity than that of control. Based on the correlation analysis, it was shown that the glycogen content was highly and positively correlated with body weight (r=0.809, P<0.001). The results of present experiment implied that in ovo injection of DS or Ala-Gln to late-term duck embryos improved the hepatic glycogen status in duck embryos, but lowered the hepatic glycogen store in the early days posthath.The third experiment was conducted to estimate the effect of administrating energy substrates and enteric modulator to late-term duck embryos on the small intestine development posthatch. Five hundred and twenty eggs containing viable embryos were randomly divided into 4 groups. At 23 days of incubation (23E), three treatments were provided in ovo injection solution as follows:1) disaccharide (maltose and sucrose) (DS); 2) disaccharide and glutamine (DS+Gln); 3) disaccharide andβ-hydroxy-β-methylbutyrate (DS+HMB); the remaining eggs served as non-injected control. After hatch, the ducklings within each treatment were randomly allocated into 8 replicates with one pen for each replicate. The BW of ducklings in each pen was recorded weekly (5 weeks). At 25 days of incubation (25E), the day of hatch (0d),3 days (3d) and 7 days (7d) posthatch, eight ducklings were killed for jejunum sampling(n=8). Morphological analysis was conducted after paraffin section and HE stain. Experiment in lab was conducted for determination of sucrose and maltase activity, DNA and protein content in jejunum. The result showed that:In ovo injection of DS+HMB significantly increased the average hatching weight of ducklings (P<0.05), from 4-7d, DS+Gln has higher average daily gain than DS and control (P<0.05), by 7d, the BW in DS+Gln is 8% higher than control and 9% higher than DS, respectively (P<0.05). From 4 to 7d, Feed/Gain in DS+HMB and DS+Gln treated ducks is significantly lower than that of control (P<0.05). DS in ovo injection decreased the relative weight and length of small intestine at Od and 3d(P<0.05), but increased the villous width of 3d and villous surface area of 7d (P<0.05). DS+Gln increased the muscular thickness by 29% at 25E and 48% at 3d, respectively, as compared with control (P<0.05). DS treated ducks had higher jejunal sucrase enzyme activity than control at 25E, hatch, and 7d posthatch (P<0.05). Compared with control, there was higher maltase activity observed in DS+Gln treatment at hatch and DS+HMB treatment at 7d posthatch, respectivly (P<0.05).The results of this experiment indicated that in ovo injecting DS+Gln improved small intestine development and growth performance of duck in the early days posthatch, and that in ovo injection of DS exerted inhibitory effect on the early development of small intestine, the DS+HMB in ovo injection exerted positive but limited effect on the small intestine development and growth of duck posthath.The fourth experiment was conducted to demonstrate the role of administration of DS or Ala-Gln to late-term ducks in affecting the protein metabolism in skeletal muscle. In ovo injection nutrient was provided to duck embryos as described in the second experiment. At 25 days of incubation (25E), hatch (0d),3 days (3d) and 7 days (7d) posthatch, the pectoral muscle were sampled(n=10) for morphological analysis, Atrogin-1 and FoxO1 mRNA expression(qPCR method) and AMPK, FoxO1, S6K1 and their corresponding phosphorylated protein expression(Western Blot method). The results of this experiment showed that:As compared with control, Ala-Gln increased cross section area of muscle fiber bundle at 25E, Od and 3d as compared with control(P<0.05), DS only increased cross section area of muscle fiber bundle at 3d (P<0.05). There was no in ovo injection effect on the cross section area of myofiber. The Atrogin-1 mRNA expression at 25E was significantly down-regulated by in ovo injecting DS and Ala-Gln(P<0.05); by 3d, all the in ovo injected ducks had significantly lower Atrogin-1 mRNA expression abundance than control(P<0.05), whereas, all treatments had higher Atrogin-1 mRNA expression abundance than control by 7d (P<0.05). Similarly, the FoxO1mRNA expression in pectoral muscle in three treatments was higher than that of control at 3d (P<0.05), whereas it was significantly lower than that of control by 7d (P<0.05). The DS in ovo injection decreased p-AMPK protein expression at 25E (P<0.05). At 25E, in ovo injected DS significantly decreased the p-AMPK relative expression (P<0.05), and Ala-Gln increased p-AMPK and p-FoxO1 expression (P<0.05). In addition, Ala-Gln treated ducks had significantly higher p-S6K1 expression than control at 25E and 3d (P<0.05). The results of this experiment demonstrated that in ovo injecting DS or Ala-Gln alone would attenuate the ubiquitin-mediated protein degradation; Ala-ln in ovo injection additionally increased protein synthesis in skeletal muscle, hence improved the early growth of skeletal muscle.In conclusion, energy store in liver and pectoral muscle was markedly removed toward to hatchling and small intestine developed at high rate, which finally leaded to energy crisis in late-term duck embryos. During this period, development of skeletal muscle was characterized by the increased ubiquitin-proteasome mediated protein degradation and decreased protein synthesis. When DS+Gln was provided to late-term ducks embryos by in ovo injection, small intestine development and growth performance in the early days of posthath could be improved. In addition, in ovo injecting DS or Ala-Gln improved the energy status and attenuated the protein degradation in muscle, thereafter improved the early growth of skeletal muscle. |
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