Downregulation of ALAS1 by nicarbazin treatment underlies the reduced synthesis of protoporphyrin IX in shell gland of laying hens

Abstract

Shell colour is an important trait for eggs and an understanding of pigment deposition will assist potential management of egg shell colour loss. We demonstrated that nicarbazin feeding down-regulated ALAS1 and reduced protoporphyrin IX (PP IX) in both shell gland and eggshell, indicating the role of nicarbazin in inhibiting the synthesis of PP IX. Additionally, the expression levels of the genes did not show sequential upregulation in the same order of diurnal time-points (TP) during egg formation. The gene SLC25A38, responsible for transporting glycine from cytoplasm to mitochondria, and the gene ALAS1, encoding rate-limiting enzyme (delta-aminolevulinic acid synthase 1), had higher expression at 15 hr, as compared with 2, 5 and 23.5 hrs postoviposition. Interestingly, ABCB6, a gene encoding an enzyme responsible for transporting coproporphyrinogen III, showed higher expression level at 2 and 5 hrs. However, the expression of CPOX that converts coproporphyrinogen III to protoporphyrinogen III, and ABCG2 that transports PP IX out from mitochondria did not alter. Nevertheless, mitochondrial count per cell did not show consistent change in response to time-points postoviposition and nicarbazin feeding. The information obtained in the study sheds light on how nicarbazin disrupts the synthesis of PP IX.

Genes involved in mitochondrial biogenesis and function may not show synchronised responses to mitochondria in shell gland of laying chickens under infectious bronchitis virus challenge

“…For standard curve construction, eight different serial dilutions (10 − 2 ~10 − 9 ) of the recombinant plasmid DNA were prepared and qPCR amplified. To construct the standard curves for GAPDH and ND4 , quantification cycles (Cq) were plotted against log 10 copy number of plasmids calculated according to the molecular weight of recombinant plasmids [71]. The amplified PCR products were sequenced by the Australian Genome Research Facility, Australia for confirmation of GAPDH and ND4 fragment inserts into the plasmid vector.…”

“…For mitochondrial count per cell enumeration, mtDNA copies in a cell were normalised with genomic DNA copies [71, 72]. The SYBR green method of the SensiFAST SYBR No-ROX Kit (Bioline, Australia) was followed for qPCR as per the manufacturer’s protocol.…”

“…Fluorescent data were acquired at the end of each annealing/extension step during qPCR cycles (40). The mtDNA copy number per cell in the magnum, isthmus and shell gland was calculated according to the equation: (mtDNA copies)/(gDNA copies/2) [71].…”

“…The cloned plasmid DNA with insert was calculated as number of copies per μL in six different dilutions for standard curve construction. Plasmid copy number was calculated based on the concentration of plasmid DNA and its molecular weight [71]. Individual sample amplicons from all the three segments of the oviduct were run in the Bioanalyzer as described earlier, to assess the specificity and size of the virus nucleic acid fragment.…”

Isolation of blue-green eggshell pigmentation-related genes from Putian duck through RNA-seq

“…ALAS1 catalyses the rate-limiting step in the condensation of succinyl coenzyme and glycine to delta-aminolevulinic acid (ALA) [18]. Following its synthesis, ALA is exported to the cytosol and converted to coproporphyrinogen III [19]. ABCB6 belongs to the ATP-binding cassette family that transports coproporphyrinogen III back to mitochondria [20].…”