Exploring The Mitochondrial DNA Effect By Bovine Cybrid Models

In bovine animals,mitochondrial DNA polymorphisms and the correlation to economic traits had been widely reported across breeds and individuals.However,the experimental evidence is still in absence.The present study aims to identifiy the mtDNA effect in bovine animals by constructing transmitochondrial models.First of all,inter-and intra-species cybrids were deployed to investigate the mtDNA effect across bovine animals.Transmitochondrial cybrids were generated.The nucleus was from MAC-T cell line derived from a Holstein dairy cow,and mitochondria from either primary cell line derived from a Luxi cattle or Datong yak.The energy metabolic characters for each cell model were analyszed,including mitogenome sequencing,mtDNA copy number,oxygen consumption(OCR),extracellular acidification(ECAR),and expression levels of nuclear-encoded mitochondrial genes.The results indicated that the mitogenomes harbored 1037 divergent nucleotides between Datong yak and MAC-T cell line,while Luxi cattle had closer maternal relationship to Holstein cow with 250 SNPs across.The Datong yak primary cell had a higher metabolic level than that of Luxi cattle cell,including proton leak,spare respiratory capacity,basal glycolysis and glycolytic reserve.However,a universal drop-in among all metabolic parameters in yak cybrid was observed,along with a mito-nuclear imbalance.On the other hand,Luxi cattle cybrid was generally higher on rates of OCRs and ECARs,alongside with lower mRNA expression levels of mitochondrial biogenesis related genes(PPARGC1A,TFAM,NRF1 and NRF2)and fat synthesis related genes(GPAM and ACSL1),reflecting the active energy metabolism and cellular stress resistance.Furthermore,in an attempt to verify the mtDNA effect on fat percentage trait in dairy cows,the transmitochondrial cybrids were generated,which the nucleus was from MAC-T,and mitochondria from different maternal dairy cows representing high,medium and low milk fat percentages.The mitogenomes harboured 10,7 and 9 SNPs between high/low,medium/high and medium/low dairy cows,respectively.Low fat percentage cow exhibited higher rates of ECARs and lower rates of OCRs.In cybrids,the low fat percentage cow cybrid showed the lowest oxidative phosphorylation(OXPHOS)and lowest mitochondrial substrate utilization capacity,while high and medium fat percentage cow cybrids had similar capacity on fat percentage traits.The low fat percentage cow cybrid also exhibited the highest expression levels in OXPHOS related genes,which illustrated a compensation of decreased mitochondrial functions.