Dietary energy and protein gradients drive metabolic adaptation in growing-finishing yaks on the Qinghai-Tibet plateau

Abstract

The study investigated the metabolic responses of yaks to dietary net energy for gain (NEg) and metabolizable protein (MP), and explored potential biomarkers for average daily gain (ADG) in serum metabolites. A total of 40 three-year-old yaks (initial body weight = 255 ± 8.04 kg) were assigned to a 2 × 2 factorial treatment arrangement based on dietary NEg (4.18 vs 4.81 MJ/kg DM; low NEg [LE] vs high NEg [HE]) and MP (72.0 vs 82.8 g/kg DM; low MP [LP] vs high MP [HP]), comprising 4 treatments with 10 replicates each (1 yak per replicate). The yaks were fed these diets for 55 days including a 15-day adaptation period. Yaks on HP diets had greater ADG (P = 0.013) and a reduced feed-to-gain ratio (P = 0.019) compared to those on the LP diet, and those on the LE diet had greater dry matter intake (P < 0.001) but unaffected ADG compared to those on the HE diet (P > 0.05). Dietary NEg interacted with MP in relation to the apparent digestibility of dry matter (P = 0.028) and crude protein (P < 0.001). The apparent digestibility of gross energy (GE), organic matter, and neutral detergent fiber in yaks on the HE diet was greater than those in yaks on the LE diet (P < 0.05). The digestibility of GE in yaks on the HP diet was lower than those on the LP diet (P = 0.011). An significant interaction between dietary NEg level and MP level was observed on serum total bilirubin concentration, catalase concentration, and superoxide dismutase concentration (P < 0.05). Yaks fed HE diets exhibited higher serum glutathione peroxidase protein concentration (P < 0.001), but lower serum triglyceride concentration (P = 0.012) compared to those fed LE diets. Serum level of alanine transaminase was higher in yaks on the HP diet compared to those on the LP diet (P = 0.038). Untargeted metabolomics identified novel biomarkers linked to dietary NEg and MP levels, revealing that the HE diet enhanced adenosine triphosphate production through acetyl-CoA synthesis and affected amino acid, fat, and carbohydrate pathways (P < 0.05). The HP diet altered the synthesis of aromatic amino acids and vitamins (P < 0.05). Additionally, random forest analysis identified N-(9-oxodecyl) acetamide and biliverdin, as biomarkers for predicting ADG. These findings provide a theoretical foundation for the effective feeding and nutritional management of yaks.

Keywords: Metabolizable protein; Net energy for gain; Serum metabolite; Yak.

Fig. 1

Multivariate statistical analysis of serum metabolites of different dietary NEg and MP Levels. (A) The score plot of PCA of four treatments in the negative ion modes. (B) The score plot of OPLS-DA of four treatments in the negative ion modes. (C) The OPLS-DA response permutation test of four treatments in the negative ion modes. (D) The score plot of PCA of four treatments in the positive ion modes. (E) The score plot of OPLS-DA of four treatments in the positive ion modes. (F) The OPLS-DA response permutation test of four treatments in the positive ion modes. PCA = principal component analysis; OPLS-DA = orthogonal partial least squares discriminant analysis; LELP = low NEg and low MP; LEHP = low NEg and high MP; HELP = high NEg and low MP; HEHP = high NEg and high MP; NEg = net energy for gain; MP = metabolizable protein.

Fig. 2

Differential serum metabolite analysis of dietary NEg and MP levels. (A) K-means cluster analysis divided 168 differential metabolites from the four groups into 6 clusters. (B) Heat map of differential metabolite content in each group corresponding to 6 clusters. (C) Differential metabolite KEGG classification donut chart. LELP = low NEg and low MP; LEHP = low NEg and high MP; HELP = high NEg and low MP; HEHP = high NEg and high MP; NEg = net energy for gain; MP = metabolizable protein.

Fig. 3

Effects of dietary NEg and MP levels on serum metabolites. (A) Heatmap of the most significant 50 differential metabolites analyzed by the hierarchical clustering analysis of serum from yaks fed with different dietary NEg levels. (B) Differential metabolite KEGG classification donut chart of dietary NEg levels. (C) The heatmap of the most significant 50 differential metabolites analyzed by the hierarchical clustering analysis of serum from yaks fed with different dietary MP levels. The horizontal axis represents samples and the vertical axis represents metabolites. Red and green represent increased and reduced concentrations of metabolites in serum of yaks fed with HE diets compared to yaks fed with LE diets (A), and yaks fed with LP diets compared to yaks fed with HP diets (C). (D) Differential metabolite KEGG classification donut chart of dietary MP levels. (E) The diagrams for the most significant KEGG pathways enrichment degree of differential metabolites of serum from yaks fed with different dietary NEg levels. (F) The diagrams for the most significant KEGG pathways enrichment degree of differential metabolites of serum from yaks fed with different dietary MP levels. The P-value of each term is represented by the color depth. The impact of the pathway was indicated by the size of the circle. NEg = net energy for gain; LE = low NEg; HE = high NEg; MP = metabolizable protein; LP = low MP, HP = high MP; VIP = variable importance in projection; BH = Benjamini-Hochberg; M = metabolism; E = environmental information processing; C = cellular processes; O = organismal systems; H = human diseases. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Fig. 4

Analysis of metabolites with interactive effects between dietary NEg and MP. (A) The 3D (interactive) PCA plots clustering patterns of differential metabolites under the two factors of MP and NEg levels. (B) Two-way ANOVA analysis explored differential metabolites in the correlation between dietary NEg and MP levels. (C) The ANOVA simultaneous component analysis of the main patterns of dietary NEg and MP factors and their interactions. (D) The heatmap of the interaction effect among NEg and MP 21 differential metabolites analyzed by the hierarchical clustering analysis of serum from yaks. The horizontal axis represents samples and the vertical axis represents metabolites. Red and green represent increased and reduced concentrations of metabolites in serum. E. The interaction KEGG pathways between dietary NEg and MP. NEg = net energy for gain; LE = low NEg; HE = high NEg; MP = metabolizable protein; LP = low MP, HP = high MP; VIP = Aariable importance in projection; SPE = squared prediction error. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Fig. 5

The main KEGG enrichment pathway diagram of differential metabolites under different dietary NEg and MP levels. The red metabolites denote differential metabolites, with an upward arrow indicating upregulation and a downward arrow indicating downregulation of the said differential metabolites. NEg = net energy for gain; LE = low NEg; HE = high NEg; MP = metabolizable protein; LP = low MP, HP = high MP; Trp = tryptophan; Phe = phenylalanine; Tyr = tyrosine; Gln = glutamine; Pro = proline; Arg = arginine; Asn = aspartate; Met = methionine; Thr = threonine; Lys = lysine; Ala = alanine; Val = valine; Leu = leucine; Ile = isoleucine; ATP = adenosine triphosphate.

Fig. 6

Analysis of serum metabolic biomarkers based on random forest model. AUC = area under curve.

Fig. 7

Correlation analysis of serum biochemical indicators and blood differential metabolites. TG = triglyceride.