Inhibition of KU70 and KU80 by CRISPR interference,not NgAgo interference,increases the efficiency of homologous recombination in pig fetal fibroblasts

Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.

Dietary supplementation with pioglitazone hydrochloride and L-carnosine improves the growth performance,muscle fatty acid profiles and shelf life of yellow-feathered broiler chickens

The present study aimed to investigate the effects of dietary pioglitazone hydrochloride(PGZ)and L-carnosine(LC)supplementation on the growth performance,meat quality,antioxidant status,and meat shelf life of yellow-feathered broiler chickens.Five hundred broiler chickens were randomly assigned into 4 experimental diets using a 2×2 factorial arrangement with 2 PGZ supplemental levels(0 and 15 mg/kg)and 2 LC supplemental levels(0 and 400 mg/kg)in basal diets for 28 d.The feed-to-gain ratio decreased whereas the average daily gain increased with PGZ supplementation.Greater dressing per-centages,contents of intramuscular fat(IMF)in breast and thigh muscles,C18:3n-6,C18:1n-9 and monounsaturated fatty acid(MUFA)percentages of thigh muscle were observed with PGZ addition.Additionally,significant synergistic effects between PGZ and LC on the C18:1n-9 and MUFA contents were found.Supplementation with LC decreased drip loss,cooking loss and total volatile basic nitrogen,and increased the redness(a*)value,the superoxide dismutase and glutathione peroxidase activities in thigh muscles.Moreover,the malondialdehyde content decreased when diets were supplemented with LC,and there was a synergistic effect between PGZ and LC.Additionally,the mRNA abundance of lipogenesis-related genes,such as peroxisome proliferator-activated receptor y(PPARγ),PPARγco-activator 1αand fatty acid-binding protein 3,increased with PGZ supplementation,and relevant anti-oxidation genes,such as nuclear factor erythroid-2-related factor 2 and superoxide dismutase 1,were enhanced with LC supplementation.In conclusion,the results indicated that the supplementation of PGZ and LC could improve the growth performance,antioxidant ability,IMF content,and meat shelf life of yellow-feathered broiler chickens.

Aminopeptidase N Knockout Pigs Are Not Resistant to Porcine Epidemic Diarrhea Virus Infection

Dear Editor,Porcine epidemic diarrhea virus(PEDV)is the etiologic agent of porcine epidemic diarrhea(PED),which is an acute,highly contagious,and devastating enteric viral disease in pigs(Lee 2015).PEDV is a coronavirus that mainly infects and replicates in villous enterocytes of the small intestine in pigs(Li et al.2016).PEDV can infect pigs in all age groups,with symptoms of varying degrees,such as watery diarrhea,dehydration,vomiting,and growth retardation.Particularly,PEDV is most detrimental to neonatal pigs,and the mortality rates can reach 100%(Stevenson et al.2013;Lee 2015).PEDV can persist in pigs after infection and become endemic in the farm.PED outbreaks occur periodically in many countries and have resulted in great economic losses to the pig industry worldwide.Effective infection prevention and control of PEDV can only be achieved through the use of vaccines.

Genome-wide association analysis reveals genetic loci and candidate genes associated with intramuscular fat in Duroc pigs

Intramuscular fat(IMF) is a major meat-quality trait in pigs. The content of IMF is directly associated with the taste and flavor of pork. As a complex trait, there could be multiple genes affecting IMF content in pork. Genomewide association study is a powerful tool to detect genomic regions associated with phenotypic variations. The objectives of the present study were to identify or refine the positions of genomic regions affecting IMF, and to characterize candidate genes and pathways that may influence this trait. Of note, we identified a significant region in longissium dorsi muscle in a Duroc pig population for IMF content with Porcine SNP60 v2 Bead Chip. This region spans 1.24 Mb on chromosome 8 and had been identified as a quantitative trait locus for IMF in Pietrain, Large White, Landrace, and Leicoma pigs. In this region, eight SNPs were significantly associated with IMF content. Three genes proximal to these significant SNPs were considered candidate genes, including ZDHHC16, LOC102162218 and PCDH7. Our results confirm several previous findings and highlight several genes that may contribute to IMF variation in Duroc pigs.

Effects of enucleation method on in vitro and in vivo development rate of cloned pig embryos

Enucleation is a crucial procedure for mammalian somatic cell nuclear transfer(SCNT), especially for domestic animal cloning. Oocytes of domestic animals such as pigs and cattle contain dark lipid droplets that hinder localization and removal of the nucleus. Using an oocyte enucleation technique that can obtain a high enucleation rate but has minimal negative effects on the reprogramming potential of oocyte for cloning is bene?cial for enhancing the outcome of SCNT. In this study, we compared the pig cloning ef?ciency resulting from blind aspiration-based(BA-B) enucleation and spindle imaging system-assisted(SIS-A) enucleation, and compared the pig SCNT success rate associated with BA-B enucleation and blind aspiration plus post-enucleation staining-based(BAPPS-B) enucleation. SIS-A enucleation achieved a signi?cantly higher oocyte enucleation success rate and tended to obtain a higher in vivo full term development rate of SCNT embryos than BA-B enucleation. BAPPS-B enucleation also obtained signi?cantly higher in vitro as well as in vivo full term development ef?ciency of cloned porcine embryos than BA-B enucleation. These data indicate that SIS-A and BAPPS-B enucleation are better approaches for pig SCNT than BA-B enucleation.