Absolute expressions of hypoxia-inducible factor-1 alpha (HIF1A) transcript and the associated genes in chicken skeletal muscle with white striping and wooden breast myopathies

Abstract

Development of white striping (WS) and wooden breast (WB) in broiler breast meat have been linked to hypoxia, but their etiologies are not fully understood. This study aimed at investigating absolute expression of hypoxia-inducible factor-1 alpha subunit (HIF1A) and genes involved in stress responses and muscle repair using a droplet digital polymerase chain reaction. Total RNA was isolated from pectoralis major collected from male 6-week-old medium (carcass weight ≤ 2.5 kg) and heavy (carcass weight > 2.5 kg) broilers. Samples were classified as "non-defective" (n = 4), "medium-WS" (n = 6), "heavy-WS" (n = 7) and "heavy-WS+WB" (n = 3) based on abnormality scores. The HIF1A transcript was up-regulated in all of the abnormal groups. Transcript abundances of genes encoding 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), lactate dehydrogenase-A (LDHA), and phosphorylase kinase beta subunit (PHKB) were increased in heavy-WS but decreased in heavy-WS+WB. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-regulated in non-defective samples. The muscle-specific mu-2 isoform of glutathione S-transferases (GSTM2) was up-regulated in the abnormal samples, particularly in the heavy groups. The genes encoding myogenic differentiation (MYOD1) and myosin light chain kinase (MYLK) exhibited similar expression pattern, of which medium-WS and heavy-WS significantly increased compared to non-defective whereas expression in heavy-WS+WB was not different from either non-defective or WS-affected group. The greatest and the lowest levels of calpain-3 (CAPN3) and delta-sarcoglycan (SCGD) were observed in heavy-WS and heavy-WS+WB, respectively. Based on micrographs, the abnormal muscles primarily comprised fibers with cross-sectional areas ranging from 2,000 to 3,000 μm2. Despite induced glycolysis at the transcriptional level, lower stored glycogen in the abnormal muscles corresponded with the reduced lactate and higher pH within their meats. The findings support hypoxia within the abnormal breasts, potentially associated with oversized muscle fibers. Between WS and WB, divergent glucose metabolism, cellular detoxification and myoregeneration at the transcriptional level could be anticipated.

Fig 1. Characteristics of muscle fibers of breast meat.

The samples collected from 6-week-old male Ross 308 broilers classified into four groups based on the degree of white striping (WS) and wooden breast (WB) abnormalities combined with carcass weight; “non-defective” = non-defective (n = 4), “medium-WS” = WS-affected samples with carcass weight ≤ 2.5 kg (n = 7), “heavy-WS” = WS-affected samples with carcass weight ≥ 2.5 kg (n = 6), and “heavy-WS+WB” = WS and WB affected samples with carcass weight ≥ 2.5 kg (n = 3). (a) Microscopic images of cross-sectional pectoralis major muscle fibers (300× magnification) displayed under field-emission scanning electron microscope using an acceleration voltage of 10 kV. Scale bar = 100 μm. (b) Bars illustrate size distribution (%) of muscle fiber regarding cross-sectional area within each sample group.

Fig 2. Absolute expression of hypoxia-inducible factor 1 alpha subunit (HIF1A) gene.

The absolute transcript abundance was determined using an EVAGREEN-based droplet digital polymerase chain reaction. (a) One-dimensional plots between fluorescent intensity and droplet events. Positive (high amplitude) and negative (low amplitude) droplets are manually separated by threshold line. (b) Absolute expression level of the gene evaluated in 6-week-old male Ross 308 broilers classified into four groups based on the degree of white striping (WS) and wooden breast (WB) abnormalities combined with carcass weight; “non-defective” = non-defective (n = 4), “medium-WS” = WS-affected samples with carcass weight ≤ 2.5 kg (n = 7), “heavy-WS” = WS-affected samples with carcass weight ≥ 2.5 kg (n = 6), and “heavy-WS+WB” = WS and WB affected samples with carcass weight ≥ 2.5 kg (n = 3). Bars represent mean ± standard error in copies per nanogram cDNA template. Asterisks above each bracket indicate statistical difference between each pair. *p<0.05, **p<0.01, ***p<0.001, NTC = no template control.

Fig 3. Absolute transcript abundances of genes associated with glucose utilization.

The genes include (a) 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), (b) lactate dehydrogenase kinase subunit A (LDHA), (c) phosphorylase kinase beta subunit (PHKB), and (d) glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The absolute expression level of the target genes was quantified in 6-week-old male Ross 308 broilers classified into four groups based on the degree of white striping (WS) and wooden breast (WB) abnormalities combined with carcass weight; “non-defective” = non-defective (n = 4), “medium-WS” = WS-affected samples with carcass weight ≤ 2.5 kg (n = 7), “heavy-WS” = WS-affected samples with carcass weight ≥ 2.5 kg (n = 6), and “heavy-WS+WB” = WS and WB affected samples with carcass weight ≥ 2.5 kg (n = 3). Bars represent mean ± standard error in copies per nanogram cDNA template. Asterisks above each bracket indicate statistical difference between each pair. *p<0.05, **p<0.01, ***p<0.001.

Fig 4. Absolute transcription levels of genes encoding glutathione S-transferases.

The absolute expression level of the target genes, including (a) glutathione S-transferase alpha 4 (GSTA4) and (b) glutathione S-transferase mu 2 (GSTM2), was quantified in 6-week-old male Ross 308 broilers classified into four groups based on the degree of white striping (WS) and wooden breast (WB) abnormalities combined with carcass weight; “non-defective” = non-defective (n = 4), “medium-WS” = WS-affected samples with carcass weight ≤ 2.5 kg (n = 7), “heavy-WS” = WS-affected samples with carcass weight ≥ 2.5 kg (n = 6), and “heavy-WS+WB” = WS and WB affected samples with carcass weight ≥ 2.5 kg (n = 3). Bars represent mean ± standard error in copies per nanogram cDNA template. Asterisks above each bracket indicate statistical difference between each pair. *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Absolute transcription levels of genes associated with muscle regeneration.

The absolute expression level of the target genes, including (a) myogenic differentiation 1 (MYOD1), (b) sarcoglycan delta subunit (SCGD), (c) calpain 3 (CAPN3) and (d) myosin light chain kinase (MYLK), was quantified in 6-week-old male Ross 308 broilers classified into four groups based on the degree of white striping (WS) and wooden breast (WB) abnormalities combined with carcass weight; “non-defective” = non-defective (n = 4), “medium-WS” = WS-affected samples with carcass weight ≤ 2.5 kg (n = 7), “heavy-WS” = WS-affected samples with carcass weight ≥ 2.5 kg (n = 6), and “heavy-WS+WB” = WS and WB affected samples with carcass weight ≥ 2.5 kg (n = 3). Bars represent mean ± standard error in copies per nanogram cDNA template. Asterisks above each bracket indicate statistical difference between each pair. *p<0.05, **p<0.01, ***p<0.001.