| Duck(Anas platyrhynchos)is one of the main poultry species that produce animal protein(meat and eggs)worldwide,occupying a particularly important position in Asia.China is an important producer and consumer of meat ducks,and duck meat production accounts for more than half of the world’s total duck meat production.In recent years,the breeding and improvement of meat duck growth speed and feed conversion rate have significantly accelerated the growth speed and yield of muscle.The physiological functions of duck embryos have been fully exploited and can be used to study the molecular regulatory mechanisms of early muscle growth and development.As a simple and easy technology,embryo egg injection(IOI)is widely used to study how substances with different functional regulatory effects(such as amino acids,carbohydrates,and probiotics)affect the growth and metabolism of duck embryos,and its specific molecular mechanisms are a recent research hotspot.Branched Chain Amino Acids(BCAA)are essential to the growth,development and health of poultry.Dietary imbalance of leucine,isoleucine and valine(oversupply or undersupply of a certain BCAA)is one of the main causes of negative effects on poultry growth rates.The imbalance of BCAA will lead to the antagonism of BCAA and affect the immune function of the body.A deficiency of valine inhibits growth performance and leads to deformities in feathers and legs.A series of experiments have proved that BCAA affects growth performance,especially muscle yield of broilers.Muscle growth and development is mainly regulated through the AKT/m TOR pathway.However,the detailed molecular mechanism of excess valine on liver and muscle growth remains unclear.The purpose of this study was to:(1)compare the effects of different concentrations of valine on the growth and lipid deposition of duck embryos;(2)The transcriptome and metabolome sequencing of duck embryo pectoral muscle were conducted to screen key genes and marker metabolites and explore their molecular mechanisms.1.Compare the effects of different valine additions on the growth,development,and lipid deposition of duck embryosThe effects of different concentrations of valine on the weight or length of important tissues were compared through egg incubation,injection,and tissue collection experiments.The results showed that the weight of pectoral muscle,the weight of male pancreas,and the length of female gonads(ovaries)were significantly increased in both sexes.There was no significant difference in the weight of liver,leg muscles,heart,spleen,female pancreas,abdominal fat,and the length of duodenum,jejunum,ileum,and male gonads.According to the statistics of hatching time,it was observed that the valine injection group significantly shortened the incubation time of duck embryos,and the incubation time of the valine injection group with a concentration of 2% was the shortest.After the completion of the injection experiment,blood was collected and serum was separated from each group of duck embryos every three days.It was found that the serum IGF-1 levels were different between male and female embryos,and different valine concentrations had a significant impact on them.At the same time,injection of higher concentrations of valine may increase serum IGF-1 levels at different embryo ages.According to the previous experimental results,the liver and pectoral muscle tissues of female and male duck embryos in the E22,E25,and E28 embryo age2% valine groups and the control group were selected for paraffin sections and HE staining,and quantitative analysis was conducted on the stained sections.The results showed that supplementing the liver with 2% valine significantly increased the number of fat droplets in ducklings at three time points,while valine had no effect on the average area of fat droplets,For chest muscles,2% valine treatment can reduce the proportion of muscle fibers,but increase the proportion of connective tissue,especially E25.Quantitative analysis found that the diameter of muscle fibers significantly increased,but the number of muscle fibers significantly decreased.In order to further study the effect of valine on growth and lipid deposition and its regulatory mechanism,IGF1 related genes were selected and their expression in the liver and pectoral muscle tissues of duck embryos on the first day of hatching was observed using real-time fluorescence quantitative analysis technology.Experiments have shown that intraegg injection of valine can inhibit the growth and development of duck pectoral muscles,increase the number of liver fat droplets,and reduce the number and diameter of pectoral muscle fibers.In addition,muscle inhibin(MSTN)significantly increased its expression level in male chest muscles.2.Sequence analysis of specific molecular mechanisms of pectoral muscle transcriptome and metabolomeThe breast muscle tissues of the same batch of incubated E22 male duck embryos were randomly selected,including six in the 2% valine group and six in the control group,and sequenced in the transcriptome and metabolome,respectively.Using transcriptome and metabolome sequencing to screen differential genes and identify marker differential metabolites,and through joint analysis to explore the molecular signaling pathways and mechanisms involved in the regulation of valine on the growth and development of duck embryo pectoral muscles.After comparing the transcriptome sequencing data between Val and Con groups,statistical screening revealed a total of267 genes with significant differences in expression,including 137 up-regulated genes and 130 down-regulated genes.In order to explore the biological function of DEGs,GO function enrichment analysis was conducted on all differentially expressed genes in the two treatment groups,and it was found that they were significantly enriched in biological processes related to fat synthesis,autophagy regulation,and so on;Analysis of the KEEG pathway revealed that DEGs participate in signal pathways such as m TOR,AMPK,and insulin to regulate the growth and development of duck embryos.In order to better determine the reliability of RNA-seq data,11 genes with significant characteristics were screened for q PCR validation on the same batch of duck pectoral muscles.The results were found to be basically the same as the previous transcriptome sequencing data,demonstrating the reliability of RNA-seq results.A total of 14 differential metabolites were found in the metabolic group sequencing of Val and Con groups,including 5 upregulated differential metabolites and 9 downregulated differential metabolites.Among these differential metabolites,there are not only organic acids and related derivatives,but also amino acids,nucleotides,and other substances.Through experimental analysis using metabolic pathways,it also includes amino acid metabolism,alkaloid synthesis,fatty acid metabolism,phosphotransferase,and other substances,which also play a regulatory role in duck pectoral muscles.Two marker differential metabolites related to muscle growth and development,aminoadipic acid and 1-methylhistidine,were validated by enzyme linked immunosorbent assay.Statistical calculations showed that the results were consistent with the sequencing conclusion,indicating that the metabolomic sequencing results were accurate and reliable.In addition,group comparison was also conducted to analyze the correlation between the differentially expressed genes and metabolites of Val and Con,and construct a correlation clustering heat map.The study found that there was a high correlation between the differential metabolites and the first 45 differentially expressed genes in these two groups,which also reflected that the changes in the differential metabolites that reduced the weight of the breast muscle of the duck embryo were related to the changes in the differentially expressed genes.In summary,this study found that intraegg injection of valine can inhibit the growth and development of duck pectoral muscles,shorten incubation time,and affect the number of lipid droplets in the liver as well as the number and diameter of muscle fibers in the pectoral muscles.Excessive addition of valine can affect insulin and autophagy related signaling pathways in duck embryo breast muscle tissue,as well as important metabolic pathways that jointly regulate duck embryo liver and muscle tissue.The results laid a foundation for further exploring the mechanism of excessive valine on the growth and development of duck embryos,and provided a theoretical basis for further research on molecular breeding of duck embryos,as well as new insights into the impact of BCAA imbalance on poultry production. |
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