Liver mitochondrial content is reduced in response to dietary crude protein and phosphorus deficiencies in wethers

Grazing ruminants consuming CP- and phosphorus (P)-deficient pastures experience reduced voluntary feed intake and liveweight gain, indicating systemic metabolic changes. Because mitochondrial abundance reflects oxidative capacity and metabolic investment, altered mitochondrial content may provide insight into how sheep adapt to nutrient-deficiency-induced reduction of feed intake. This study aimed to determine whether reduced intake caused by CP and P deficiency, or by restriction of an otherwise adequate diet, alters tissue mitochondrial content in young sheep. Forty Merino wethers (7 months old, 23.7 ± 1.4 kg liveweight) underwent a 63-day feeding trial where they were fed one of five nutritional treatments (n = 8/treatment). Four treatment diets were fed ad libitum, with combinations of either high or low CP (110 and 55 g/kg DM) with high or low P (2.5 and 0.7 g/kg DM). Another treatment (Restricted) restricted intake of the High CP, High P diet to model hunger. Mitochondrial DNA copy number (mtDNA-CN) was quantified from rumen, duodenum, liver, heart, M. semitendinosus and M. soleus samples using a newly developed quantitative PCR test for sheep, as a high-throughput proxy for tissue mitochondrial content. This assay was validated against transmission electron microscopy (TEM), and mtDNA-CN correlated with TEM-derived mitochondrial content (ρ = 0.67, P < 0.01). Duodenum and rumen mtDNA-CN were also positively correlated (ρ = 0.41, P = 0.01), indicating functional metabolic relationships among these tissues. Liver mtDNA-CN was significantly higher in the wethers fed the High CP, High P diet than those fed nutrient-deficient and restricted diets (P < 0.02), with as much as a two-fold difference between this group and those fed the dual-deficient diet (High CP, High P v. Low CP, Low P). Wethers fed the High CP, High P diet also had 45% greater mitochondrial content in the M. semitendinosus compared to wethers fed the High CP, Low P and Low CP, High P diets (P < 0.03). Across individuals, liver mtDNA-CN was positively associated with liveweight gain after accounting for DM intake and diet (β = 34.7 ± 12.2 g/day, P < 0.01; ΔR² = 0.017). These results show that hepatic mitochondrial content declines under both feed restriction and nutrient-deficiency-induced intake reduction, and that higher liver mitochondrial content is positively associated with growth. Together, the findings support the liver as a metabolically responsive tissue under nutritional constraint and suggest that reduced mitochondrial abundance may form part of the adaptive response to chronic energy deficit.