Response Of UCP1-KI Pigs To Chronic Cold Stimulation

Fat deposition significantly affects pig production efficiency and meat quality.For many years,reducing fat deposition and improving lean meat rate have been one of the main objectives of pig breeding.Although traditional breeding has greatly improved the lean meat rate of pigs,few genes that can significantly affect pig fat deposition have been excavated.A large number of studies have shown that uncoupled protein 1(UCP1)can not only promote heat production and regulate energy metabolism,but also reduce fat deposition.But the functional UCP1 was lost during the evolution of pigs.Therefore,in previous studies,we collaboratively prepared UCP1 knock-in pig(UCP1-KI)by CRISPR/Cas9.Further studies showed that UCP1-KI pigs were able to maintain body temperature better during acute cold stimulation(4℃ for 4 hours).The fat deposition of UCP1-KI pigs at 6 months was significantly reduced.In the actual livestock production process,animals may be faced with relatively long-term chronic cold stimulation.Therefore,this study was aimed to explore whether the UCP1-KI pigs can improve the cold adaptability under the condition of chronic cold stimulation,and whether it can lead to the beige of white adipose tissue.In addition,previous lipidomic analysis showed that the content of sphingomyelin in the subcutaneous adipose tissue in the inguinal area of UCP1-KI pigs increased significantly.Since sphingomyelins play a regulatory role in cell proliferation,differentiation,apoptosis and migration,this study used mouse C2C12 cells to explore the effect of sphingomyelin on myogenic differentiation.Heterozygous UCP1-KI pigs were used as parents to get progeny of experimental pigs,and their genotypes were identified by PCR.Pigs of the similar age and body weight were selected for chronic cold stimulation,and 3-day-old newborn piglets and 3-month-old piglets were treated with chronic cold environment at 22℃ for 4 weeks and 1℃ for 1 week,respectively.Anal temperature and body weight were measured during cold stimulation.The results showed that body temperature maintenance ability of UCP1-KI pigs was significantly improved under chronic cold stimulation(P<0.05).Body weight measurement of 3-day-old newborn boars showed the weaning weight of KI pigs was significantly higher than that of wild-type pigs(P<0.05).Statistics showed that the growth rate and growth intensity of UCP1-KI boars were about 1.88 times and 1.34 times that of wild-type pigs(P<0.05).After chronic cold stimulation,paraffin section and H&E staining were performed on subcutaneous adipose tissue of dorsal and groin of growing pigs.It was found that the adipose cells in KI pigs was significantly smaller than that of wild-type pigs.In addition,the expression level of beige related marker genes did not change significantly in the back subcutaneous adipose tissue,inguinal subcutaneous adipose tissue and perirenal adipose tissue of KI pigs after chronic cold stimulation,becauseThen,ELISA showed that the content of total sphingomyelin in the back muscle tissue of UCP1-KI pigs was significantly higher than that of wild-type pigs.C2C12 cells were treated with different concentrations of sphingomyelin(0,5,20,50,100 μg/m L)in myogenic differentiation medium.After 6days,there were more muscle tubes in the treatment group supplemented with 20 μg/m L sphingomyelin.Myogenin positive model was enhanced,and the myotube fusion index was significantly increased.The expression levels of Myogenin and Mrf4 genes were significantly higher than those in the control group(P<0.05).The gene expression levels of cyclins(Cyclin D1,Cyclin E,CDK2,CDK4)were further detected at different stages of myogenic differentiation(2,4,6 day).After 2 days of differentiation,It was found that the gene expression levels of Cyclin D1,CDK4 in the sphingomyelin-treated group were significantly higher than those in the control group(P<0.05).And the cell viability of the treatment group was significantly higher than that of the control group(P<0.05).After differentiation for 6 days,the gene expression levels of Cyclin D1,Cyclin E,CDK2 and CDK4 in the sphingomyelin-treated group were significantly lower than those in the control group.These results suggest that sphingomyelin treatment can promote the myogenic differentiation.In conclusion,this study shows that UCP1 gene knock-in can make pigs better adapt to chronic cold stimulation,and increase weaning weight.In addition,the content of sphingomyelin in the back muscle tissue of UCP1-KI pigs significantly increased.And the addition of sphingomyelin can improve the efficiency of myogenic differentiation.This study not only adds new data for analyzing the effects of UCP1 knock-in on pig temperature regulation and fat deposition and other phenotypes,but also provides ideas for further analyzing the interaction between fat and muscle.