Effect Of Classical Music On Mood And Its Molecular Mechanism Of Weaned Piglets

Music is often played as environmental enrichment which has been shown to improve mood and emotional wellbeing of livestock.This study was designed to testify the effect of music stimulation on piglets' behavior and mood by means of the open field test.Transcriptome analysis was performed in the hypothalamus to identify genes that influence piglet behavior,mood,and brain development,and supply the existing knowledge base for the effect of music on piglets.A total of 72 healthy LDM(hybrids of Large White × Duroc × Minpig)piglets,about 6-7 weeks of age at the onset of the experiment,were used in this study and divided equally into three environmental control chambers,4 replicates in each group and 6 piglets in each repeat.Sound treatments were duplicated and consisted of no music at all(Control group,C),the transmission of Mozart K.448(60-70 dB,Mozart group,A),and background noise(80-85 dB,Noise group,B)at 10:00-16:00 every day.On the 14th day of experiment,two female piglets were randomly selected,from each repetition for the open field test.Day 29 of the experiment,one female piglet was randomly selected,from each repetition as focal animals to collect hippocampus,hypothalamus,prefrontal cortex,amygdala,and nucleus accumbens tissues.Then observe the microscopical and ultrastructural tissue structures of the hippocampus.The hypothalamus is used for transcriptome sequencing analysis,screening differential genes,and using methods such as gene ontology(GO),cluster analysis,and KEGG enrichment analysis to identify pathways affected by music and noise stimulation.At last,real-time quantitative PCR was used to verify some different genes in different brain regions.In the open field test,piglets from groups A and B were more active,spent more time exploring floor and less time exploring wall,heart rate lower than control pigs(p<0.05).In addition,compared to group C,piglets from group B vocalized less than control pigs(p<0.05),piglets from group A have a tendency to decrease the frequency of vocalization,but the difference was not significant(p=0.18).Compared to group A,the results of microscopical observation in group C and B showed more cell damage(such as nuclear membrane shrinkage,linear crest was reduced,fractured and even disappeared,axonal tortuous fracture)and fewer synapses.However,group B has a more complete microstructure than A.Analysis of the differentially expressed(DE)genes showed that A vs.C,B vs.C,and A vs.B had 115,99,and 84 DE genes,respectively(|log2FoldChange|>1,p<0.05),and the genes were mainly involved in cell components,receptor binding and response to hormone.The results of principal components analysis and cluster analysis showed that a sample was mixed with each other from music and noise groups,indicating a similarity function of the two sounds.However,the genes involved in each function were different,such as in terms of the nervous system,the DE genes were mainly concentrated in the structural and functional pathways of synapses and neurons of music group,while the noise-related DE genes were mainly concentrated in the pathways of the nervous system development.Consistent expression results were found for 15 selected genes by quantitative real-time PCR.Thirty-six and thirty-four interesting DE genes were found respectively from GO term annotation of group music and noise,and those genes were related to behavior,hormonal response,and nervous system development.Moreover,the transcripts of receptor protein,including dopamine receptor D1(DRD1)and hypocretin receptor 2(HCRTR2),were significantly altered in response to music stimulation.KEGG enrichment analysis indicates that music and noise stimulation have a larger influence on the Neuroactive ligand-receptor interaction pathways.Nine different genes which participate in behavioral response,emotion,growth regulation,stress response,and ion pathway were detected,including relaxin family peptide receptor 2(RXFP2)?F2R like trypsin receptor 1(F2RL1),somatostatin receptor 2 and 3(SSTR2,3),cholinergic receptor nicotinic alpha 2 subunit(CHRNA2),adrenoceptor alpha 2C(ADRA2C),tachykinin precursor 1(TAC1),cholinergic receptor muscarinic 4(CHRM4),and HCRTR2.The qRT-PCR results of hypothalamus,prefrontal cortex,nucleus accumbens,hippocampus,and amygdala showed that,compared to group C,the expression levels of TAC1 in the multi-brain region of piglets of music group and the noise group both upregulated and decreased,with different trends,but significant differences(p<0.05),the gene expression levels of SSTR3,SSTR2,CHRM4,and HCRTR2 were all up-regulated(p<0.05),the expression of CHRNA2 showed a decreasing trend(p<0.05).In addition,F2RL1 and ADRA2C genes were characteristic up-regulated and down-regulated gene of piglets in the music group,respectively(p<0.05),RXFP2 was a specific down-regulated gene of piglets in the noise group(p<0.05).This suggests that both music and background noise can regulate piglets' emotional responses through Neuroactive ligand-receptor interaction pathways,but the key genes involved in the cortical-limbic system are not identical.In conclusion,music as an environmental enrichment method can be used to improve the emotional state of piglets,promote the expression of genes related to behavior,regulate hormone levels and promote the development of the nervous system of piglets.The neuroendocrine system is highly malleable to noise stimulation,which causes the stress response of piglets in the short term,but also promotes the adaptability of piglets to the noisy environment.The key genes of Neuroactive ligand-receptor interaction pathways in hypothalamus,hippocampus,amygdala,nucleus accumbens,and prefrontal cortex regulate the behavioral expression and emotional response of piglets.