Identification of differentially expressed genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia

Figures & data

Figure 1.  Histological characteristics of the different zones of tibia growth plate 4 days after feeding the thiram-containing diet. (Bar=200 µm.) P, proliferative zone; PH, prehypertrophic zone; H, hypertrophic zone. The growth plate of the thiram diet-fed chicken was thickened significantly.

Table 1.  Primers used in real-time PCR analysis

Gene	Primers	Size (base pairs)	Annealing temperature (°C)
Col X	Forward: 5′-cca cct gga cct cct gga cct aaa g-3′	181	60
	Reverse: 5′-gtt gcc cca ggg tag gct ttt gag-3′
NADH dehydrogenase	Forward: 5′-caa ggc aac gag ttt tta cga tac-3′	230	56
	Reverse: 5′-tca tag tta ttt tca ggc ggg tc-3′
Pro-alpha-1 Col I	Forward: 5′-gag cga cgg ctt cca gtt tga g-3′	237	60
	Reverse: 5′-cca tag gtg aag cgg ctg ttt cct t-3′
Hsp90	Forward: 5′-cct cct cca tac gtg atg tgt ca-3′	108	60
	Reverse: 5′-gcc tgg gca ttg atg aag atg-3′
Cyt c oxidase	Forward: 5′-caa ggg ggt tta ggg gtt tga ctc-3′	217	60
	Reverse: 5′-caa tga tga cga gac gt agt ccg ag-3′
CA2	Forward: 5′-cca ttc aaa cca agg gga aac at-3′	121	60
	Reverse: 5′-cat tca tgc agt ggt gga gta gtc a-3′
Col IX	Forward: 5′-gga gaa aca caa cct tgc acc ca-3′	219	60
	Reverse: 5′-ggg taa aag tat gtt cag ggc aac t-3′
EON1	Forward: 5′-ctc cct gtg ggt gct gac aac tt-3′	131	60
	Reverse: 5′-gca aag cca ccc tca tca cc-3′
Matrilin-3	Forward: 5′-cac act ttt act ctg cct tgc cat ttc-3′	296	60
	Reverse: 5′-gcg cag ctt aca gaa gag cag ttg g-3′
Chondromodulin-I	Forward: 5′-cat gca gaa tgc taa aca gcg tat c-3′	153	60
	Reverse: 5′-tcc agt tgg agt att tca aag caa g-3′
β-actin	Forward: 5′-tgg gtat gga gtc ctg tgg t-3′	160	60
	Reverse: 5′-agg gct gtg atc tcc ttc tg-3′

Table 2.  Up-regulated genes and down-regulated genes isolated by SSH and dot blot analysis^a

Clone number	Homologous gene	Genbank accession number
BTDF2A1	Chicken type X collagen	M13496
BTDF3G2	Gallus gallus NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5	NM_001006553
BTDF2B3	Messenger fragment for chicken pro-alpha-1 (I) collagen	V00401 J00836
BTDF2C11	Chicken mRNA for 90 kDa heat shock protein	X07265
BTDF 8B11	Gallus domesticus cytochrome C oxidase subunit III	X53434
BTDF 8B9	Gallus gallus carbonic anhydrase II (CA2)	NM_205317
BTDF 6G3	Gallus gallus similar to alpha (IX) collagen-proteoglycan	XM_419889
BTDF 6G5	Gallus gallus enolase 1, (alpha) (ENO1)	NM_205120
BCR8D5	Gallus gallus matrilin 3 (MATN3)	NM_205072
BCR8D6	Gallus gallus leukocyte cell derived chemotaxin 1 (LECT1)	NM_204810
^aNumber for BTDF contains up-regulated genes and number for BCR includes down-regulated genes.

Figure 2.  Analysis of the genes differentially expressed in growth plates. The levels of mRNA transcripts for 10 genes were determined by real-time PCR. Relative quantification of gene amplification was performed using the Ct values. Statistical differences were analysed by Student t test. *P<0.05, significant; **P<0.01, very significant.

Figure 3.  Expression changes of Col I in control and thiram-fed chicken growth plates at different stages. (Bar=100 µm.) Col I mainly lies in the prehypertrophic and hypertrophic zones with brown colour, but increased Col I was detected in the thiram-fed chicken growth plate on days 4, 9, 16 and 23 or around the TD lesion at the 16th and 23rd days. C, control; T, thiram-fed; PH, prehypertrophic zone; H, hypertrophic zone.

Figure 4.  Expression changes of Hsp90 in control and thiram-fed chicken growth plates at different stages. (Bar=100 µm.) Hsp90 mainly lies in the prehypertrophic and hypertrophic zones with brown colour, but increased Hsp90 was detected in the thiram-fed chicken growth plate on days 4 and 9, and especially at days 16 and 23 or around the TD lesion at the 16th and 23rd days. C, control; T, thiram fed; PH, prehypertrophic zone; H, hypertrophic zone.