Rumen-Protected Glucose Supplementation Enhances Yak Calf Growth Through Gut Microbiota–Metabolic Interactions

TL;DR

Adding rumen-protected glucose to yak mothers' diets improves calf growth by enhancing milk quality and gut microbiota.

Contribution

This study reveals transgenerational benefits of low-dose rumen-protected glucose supplementation in yaks through microbiota-metabolite interactions.

Findings

Low-dose RPG supplementation increased milk nutrients and calf weight gain by 21.74%.

Calves showed improved antioxidant and immune functions with higher SOD, CAT, and lower IL-6, TNF-α.

RPG enriched beneficial gut bacteria like Akkermansia muciniphila and upregulated key metabolites.

Abstract

This study investigated the effects of rumen-protected glucose (RPG) supplementation on calf health in periparturient yaks. The findings revealed that daily supplementation with 150 g of low-dose RPG (L-RPG) significantly increased protein, fat, lactose, and total energy content in dam milk, while concurrently promoting calf body weight gain and activating growth axis-related hormones. Furthermore, calves in the L-RPG group exhibited enhanced antioxidant capacity and improved immunomodulatory function. Multi-omics analysis further revealed that L-RPG could optimize the hindgut microbiota structure of calves, enrich beneficial bacteria such as Akkermansia muciniphila, and upregulate serum levels of metabolites associated with microbial metabolism. In summary, maternal supplementation with low-dose RPG synergistically promotes offspring growth and development by improving milk quality, modulating calf gut microbiota and host metabolism, thereby providing theoretical basis and practical guidance for integrated dam–calf health management in yak farming.

Alleviating negative energy balance in perinatal yaks is a critical challenge for safeguarding the health of both dams and calves and achieving sustainable development of yak husbandry. While RPG supplementation represents an effective nutritional strategy, its transgenerational benefits, particularly the long-term effects on offspring calf development through maternal intervention, remain largely unexplored. In the present study, low-dose RPG (150 g d−1, L-RPG, n = 6) markedly improved milk composition, elevating protein, fat, lactose, and gross energy contents (p < 0.05), and enhance calf weight gain (21.74%) with activation of the somatotropic axis (increased GH, IGF-1, etc.). Calves in this group also exhibited enhanced antioxidant capacity (higher SOD, CAT, and T-AOC) and improved immune regulation (lower IL-6, TNF-α). Multi-omics analyses revealed that L-RPG enriched beneficial taxa such as Solbacillus, Citricoccus, and Akkermansia muciniphila, optimized the hindgut microbiome, and upregulated serum metabolites including di-O-methylfraxetin and phenylalanyl-histidine. Integrated microbiota–metabolite profiling demonstrated significant cross-talk between the altered bacteria and metabolites. Collectively, supplementing dams with 150 g d−1 RPG improves milk quality, modulates the calf gut microbiota, and reshapes host metabolism, thereby synergistically promoting offspring growth. Our findings provide a comprehensive “maternal nutrition–microbiota–metabolism” framework for understanding RPG’s mode of action and furnish both theoretical insights and practical guidance for dam–calf health management in yak production systems.

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1. Introduction

The yak (Bos grunniens) is an indispensable genetic and economic resource for the Qinghai-Tibet Plateau [1,2]. However, yak husbandry is still constrained by low production efficiency and marked seasonal nutrient imbalances. Among these challenges, the health management of periparturient female yak is a critical bottleneck. During this period, female yaks experience physiological stressors such as parturition and the onset of lactation, which cause a sharp rise in energy demand. At the same time, dry-matter intake declines, resulting in energy intake that falls far below requirements and pushing the animals into negative energy balance (NEB) [3]. Persistent negative energy balance not only precipitates metabolic disorders such as ketosis and fatty liver, thereby compromising female yak health and fertility, but also impairs colostrum quality and milk composition, exerting long-lasting detrimental effects on calf growth, immune competence, and lifetime performance [4,5]. Consequently, developing effective nutritional strategies to alleviate periparturient NEB and to safeguard the dam–calf unit has become a central and urgent issue for intensive and sustainable yak production systems.

Rumen-protected glucose (RPG) is a precision-nutrition strategy based on encapsulation technology. By physically or chemically shielding glucose from microbial fermentation in the rumen, RPG allows the molecule to reach the abomasum and duodenum intact, where it is rapidly absorbed into the bloodstream, promptly elevating blood glucose and supplying the animal with an immediate source of energy [6]. In dairy cows, RPG supplementation has been shown to effectively ameliorate periparturient negative energy balance by reducing blood ketone bodies, alleviating oxidative stress and immune suppression, and ultimately enhancing productivity [6,7]. However, existing research has primarily focused on dairy cows themselves, with emphasis on their production performance and metabolic health, and has yet to systematically elucidate whether RPG can confer transgenerational health benefits to offspring calves via maternal intervention [8,9]. This mechanism is particularly noteworthy in yaks, as their physiological metabolism, rearing environment, and nutritional patterns are fundamentally distinct from those of intensively raised Holstein dairy cows. Yaks have long adapted to high-altitude, cold, and hypoxic environments, exhibiting unique energy metabolism and nitrogen utilization efficiency [10]. Moreover, their feeding regimen is primarily based on natural grazing, with nutrient supply subject to marked seasonal fluctuations [11]. Consequently, directly extrapolating findings from dairy cow studies may not be applicable, and research on RPG supplementation in yaks remains scarce. Critically, existing RPG research has primarily relied on traditional production performance and blood biochemical indices, which are insufficient to reveal the underlying physiological mechanisms influencing calf development. Early life represents a critical window for gut microbiota colonization and establishment of host metabolic programming, whereby maternal nutrition can modulate offspring microbiota structure to shape long-term growth and health trajectories [12]. However, whether and how RPG modulates the structure and function of the gut microbiota in yak calves, and further promotes their health through the microbe–host metabolic interaction network, currently lacks systematic elucidation [13,14]. Integrating multi-omics approaches such as 16S rRNA sequencing and untargeted metabolomics offers a powerful means to uncover these mechanisms from the perspective of microbe–host co-metabolism [15,16].

Building on the above knowledge gaps, the present study used periparturient female yaks and their calves to systematically evaluate the dose-dependent effects of RPG on calf growth, serum metabolism, antioxidant capacity, immune indices, and related hormone levels. By integrating 16S rRNA sequencing with untargeted metabolomics, we further explored, from a gut microbiota–host metabolic interaction perspective, the underlying mechanisms through which RPG benefits calf health.

2. Materials and Methods

2.1. Experimental Animals and Management

Maiwa yaks used in the trial were supplied by the Yak Research Farm of the Hongyuan Academy of Grassland Science, Sichuan Province, China. RPG was provided by Zhejiang Yaofei Biotechnology Co., Ltd. (Huzhou, China). This product employs microencapsulation technology with a rumen bypass efficiency exceeding 80% and is used according to the manufacturer’s recommended dosage. Sixty healthy periparturient female yaks, aged 3.5 years, parity 2–3, weighing 250 ± 20 kg, with normal clinical examination, physiological indices, and feeding/drinking behavior, were selected for this study. Throughout the trial, the female yaks were housed and allowed ad libitum access to pasture (nutrient composition shown in Table 1). They were randomly divided into three groups (n = 20 per group): control group (NC, without RPG supplementation), low-dose RPG group (L-RPG, 150 g/day), and high-dose RPG group (H-RPG, 300 g/day). After a 5-day adaptation period, the formal trial lasted 38 days (10 days prepartum to 28 days postpartum). After parturition, six healthy female calves with similar birth weight and good health status were selected from each group and allowed ad libitum access to maternal milk and water.

Blood samples were collected from newborn yak calves on days 7, 14, 21, and 28 after birth. Before morning feeding, approximately 8 mL of blood was withdrawn from the jugular vein into non-anticoagulant vacuum tubes, kept at room temperature for 1 h, centrifuged at 3500 r min^−1^ for 10 min, and the serum was harvested and stored at −20 °C until analysis. Mature milk samples (~50 mL) were collected from dams on postpartum days 14, 21, and 28 into sterile containers, stored at −20 °C, and subjected to three-point composite sampling prior to analysis.

2.2. Determination of Yak Milk Composition

Prior to analysis, frozen yak-milk samples were thawed in a 40 °C water bath and gently mixed to achieve uniform temperature. Crude protein was determined with an automatic Kjeldahl nitrogen analyzer (VAP 450, C. Gerhardt, Königswinter, Germany), fat with a milk-fat analyzer (HM-RF2, Shandong Hengmei Electronic Technology Co., Ltd., Weifang, China), ash using a muffle furnace (SX2-8-10, YeTuo, Shanghai, China), lactose by high-performance liquid chromatography (Ultimate 3000, Thermo Fisher, Waltham, MA, USA), and moisture with a forced-air drying oven (101-3AB, Chuanyu Experimental Instrument Co., Ltd., Shanghai, China).

2.3. Growth-Performance Measurements

Calves were weighed and measured on days 1, 7, 14, 21 and 28 before the morning feeding. Body weight was recorded after overnight fasting. Body length was taken as the distance from the midpoint of the line connecting the two ears to the base of the tail while the calf stood naturally. Height at withers was the vertical distance from the highest point of the scapula to the ground. Chest girth was the circumference of the thorax immediately behind the posterior border of the scapula with the animal standing horizontally.

2.4. Determination of Serum Routine Biochemical Indices

After the serum samples had returned to room temperature and were mixed by gentle inversion, the concentrations of glucose (GLU), β-hydroxybutyric acid (BHBA), total cholesterol (TC), triglyceride (TG), albumin (ALB), total protein (TP), and alanine aminotransferase (ALT) were measured using a Catalyst One blood chemistry analyzer (IDEXX, Westbrook, ME, USA).

2.5. Determination of Growth-Related Hormone Levels

The concentrations of insulin (INS, JM-00553B1), growth hormone (GH, JM-00569B1), somatostatin (SST, JM-08472B1), insulin-like growth factor 1 (IGF-1, JM-00482B1), insulin-like growth factor binding protein 2 (IGFBP-2, JLC_Y9323), insulin-like growth factor binding protein 3 (IGFBP-3, JLC_Y9324), glucagon-like peptide-2 (GLP-2, JM-08713B1), and growth hormone releasing hormone (GHRH, JM-08543B1) in calf serum were measured using the corresponding ELISA kits (Jingmei Biological Engineering Co., Ltd., Yancheng, China) according to the manufacturer’s instructions.

2.6. Immune-Index Assay

Following the manufacturer’s instructions (Jingmei Biological Engineering Co., Ltd.), serum concentrations of bovine immunoglobulin G (IgG, JM-00513B1), bovine interleukin-6 (IL-6, JM-00561B1), bovine interleukin-10 (IL-10, JM-00568B1), and bovine tumor necrosis factor-α (TNF-α, JM-08321B1) were determined with the corresponding ELISA kits.

2.7. Oxidative- and Antioxidant-Related Indices

According to the kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), serum superoxide dismutase (SOD, A001-3-2), catalase (CAT, A007-2-1), total antioxidant capacity (T-AOC, A015-2-1) and malondialdehyde (MDA, A003-2-2) were measured in calves.

2.8. DNA Extraction and 16S rRNA Sequencing

Total fecal microbial genomic DNA was extracted using a CTAB/SDS protocol [17] and checked for purity on 1% agarose gels. DNA samples were diluted with sterile water to 1 ng µL^−1^. The V3-V4 and V4 hypervariable regions of the 16S rRNA gene were amplified by PCR with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), respectively. The PCR reaction mixture (20 μL total) consisted of 4 μL 5× TransStart FastPfu buffer, 2 μL 2.5 mM dNTPs, 0.8 μL forward primer (5 μM), 0.8 μL reverse primer (5 μM), 0.4 μL TransStart FastPfu DNA polymerase, and 10 ng template DNA, adjusted to a final volume of 20 μL. The amplification program was as follows: initial denaturation at 95 °C for 3 min, followed by 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min, and a final hold at 4 °C. PCR was performed using an ABI GeneAmp^®^ 9700 thermal cycler (Thermo Scientific, Waltham, MA, USA). Amplicons (≈400–450 bp) were generated with Phusion^®^ High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions, verified on 2% agarose gels, and purified using the GeneJET DNA Purification Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol prior to Illumina sequencing.

2.9. Untargeted Metabolomics Analysis

Thawed serum was mixed with 4 volumes of ice-cold methanol–water (4:1, v/v), vortexed at −10 °C, held at -20 °C for 30 min, and centrifuged (16,000 g, 4 °C, 20 min). An aliquot of supernatant was injected into a SHIMADZU-LC30 UHPLC (Shimadzu Corporation, Kyoto, Japan) system equipped with an ACQUITY UPLC^®^ HSS T3 column (2.1 × 100 mm, 1.8 µm; Waters, Milford, MA, USA) maintained at 40 °C and 0.3 mL min^−1^ flow rate (injection volume 18 µL). Each sample was analyzed in both positive- and negative-ion modes by electrospray ionization (ESI). Following UPLC separation, metabolites were detected on a Q Exactive Plus mass spectrometer (Thermo Scientific, USA) using a heated electrospray ionization source.

2.10. 16S rRNA and Metabolomics Data Analysis and Visualization

Bioinformatics analysis of gut microbiota and serum metabolites was performed using the Majorbio Cloud platform (https://cloud.majorbio.com). Details are as follows: taxonomic abundance tables were generated with QIIME 1.9.1 (http://qiime.org/), and microbial phenotypes were predicted using BugBase (http://bugbase.cs.umn.edu/). Inter-group comparisons were carried out with the stats package in R v3.3.1 and scipy v1.0.0 in Python v3.x. KEGG compound annotation and functional pathway enrichment were performed against the KEGG COMPOUND database (version keg v20230830). Differential metabolites were selected when VIP ≥ 1 from the OPLS-DA model and two-tailed p < 0.05. O2PLS modeling was implemented in R v3.3.1 with the ropls package v2.0.2, and Mantel-test network heat-maps were drawn with the vegan package v2.6-4 in R v4.1.3.

2.11. Statistical Analysis

All longitudinal data (e.g., body weight, blood metabolites) were analyzed using two-way repeated measures mixed models with the PROC MIXED procedure in SAS 9.4. In the model, treatment, time, and their interaction (Treatment × Time) were included as fixed effects, while individual animal ID was set as a random intercept to account for correlations among repeated measurements from the same animal. Model residuals were modeled using a compound symmetry structure, which was determined to be the most appropriate for these data based on Akaike information criterion comparisons. Post hoc multiple comparisons were performed using the Tukey–Kramer method when fixed main effects or interactions were significant. The significance level was set at p < 0.05. Results are expressed as mean ± standard error of the mean (SEM). Significance was set at p < 0.05. Statistical graphs were created using GraphPad Prism 9. In the figures, different lowercase letters denote significant differences between groups at p < 0.05, while identical letters or absence of letters indicate no significant difference.

3. Results

3.1. RPG Supplementation Mitigates Postpartum Body-Weight Loss and Elevates Milk Nutritional Value in Female Yaks

As shown in Table 2, all cows exhibited a marked postpartum decline in body weight, consistent with the physiological pattern of negative energy balance during the periparturient period. Across every postpartum time point, mean body weight was higher in both RPG-supplemented groups (L-RPG and H-RPG) than in the NC controls, and their average daily weight change (i.e., the rate of weight loss) was attenuated, with the H-RPG group showing the most pronounced effect. At day 28, body weight in the L-RPG group was 21.74% higher than that in the NC group. These data suggest a potential trend toward reduced weight loss.

Regarding milk composition, the L-RPG group displayed significantly higher concentrations of milk protein, fat, lactose, and gross energy compared with both the NC and H-RPG groups (p < 0.01), demonstrating that a low dose of RPG effectively promotes the synthesis of organic milk nutrients. The high dose was less effective than the low dose, suggesting the existence of an optimal dose–response window for RPG. No significant differences were observed among groups for moisture, ash, or solids-not-fat (Table 3), indicating that RPG primarily enhances organic nutrient fractions rather than basal milk constituents.

3.2. Supplementing Periparturient Female Yaks with RPG Effectively Promotes Calf Growth

As shown in Table 4, from day 1 through day 28, calves in the L-RPG group were significantly heavier at every time point than those in the NC and H-RPG groups (p < 0.05). Body length, withers height and chest girth did not differ among the three groups.

To clarify how maternal RPG affects offspring growth, serum concentrations of growth-related hormones were measured (Figure 1). Overall, L-RPG calves exhibited higher IGF-1, IGFBP-3, insulin and GLP-2, but lower GH, SST and IGFBP-2, than NC and H-RPG calves. GHRH did not differ among groups at any of the four sampling times.

3.3. Effects of Maternal RPG Supplementation on Calf Serum Biochemistry and Energy Metabolism

As shown in Table 5, GLU concentrations in the L-RPG group were markedly higher than those in both the NC and H-RPG groups at 14, 21 and 28 d postpartum (p < 0.01). Conversely, BHBA levels in the L-RPG calves remained significantly lower than in the NC group at the same time points (p < 0.01). TG exhibited a complex pattern: L-RPG calves had lower values at 7 d, but concentrations tended to rise thereafter and surpassed those of the NC group by 28 d. TC in the L-RPG group was either significantly elevated or similar to the NC group at 7 and 14 d, whereas no inter-group differences were detected at 21 and 28 d. Serum TP did not differ among treatments. ALB concentrations in the L-RPG group were significantly higher than in the other two groups at 21 d. Additionally, ALT activity in the L-RPG calves was consistently lower than in the NC group at all sampling times.

3.4. Maternal RPG Supplementation Enhances Calf Antioxidant and Immune Status

Antioxidant indices (CAT, MDA, SOD, and T-AOC) and immune-related parameters (IgG, IL-6, IL-10, and TNF-α) are summarized in Table 6. In terms of antioxidant indicators, T-AOC and SOD activities in the L-RPG group were significantly higher than those in the NC and H-RPG groups at day 28 (p < 0.05). CAT activity was consistently elevated in the L-RPG group compared with the other two groups at all sampling points (p < 0.05). MDA concentrations were significantly greater in the H-RPG group than in the NC and L-RPG groups at every time point, whereas no significant difference was observed between the latter two groups.

With respect to immune indices, IgG concentrations did not differ among the three groups at most time points; however, on day 28, the NC group exhibited significantly higher IgG levels than the L-RPG group, while the H-RPG group showed intermediate values. At all sampling times, TNF-α and IL-6 concentrations in the L-RPG group were markedly lower than those in both the NC and H-RPG groups (p < 0.05). IL-10 levels did not differ significantly among the three groups at any of the four time points.

3.5. Maternal RPG Supplementation Improves the Gut-Bacterial Architecture of Calves

Figure 2A shows that rarefaction curves for NC, L-RPG and H-RPG rose rapidly and plateaued as sequencing depth increased, indicating that the data adequately captured the microbial diversity of each sample and are therefore reliable for further analysis. A core of 53 OTUs was shared among the three groups, whereas 61 OTUs were unique to L-RPG, fewer than the 126 and 113 unique OTUs found in NC and H-RPG, respectively (Figure 2B). Alpha-diversity indices revealed that ACE and Shannon values were highest in L-RPG and significantly greater than in NC and H-RPG (p < 0.01), demonstrating elevated richness and evenness. Chao1, coverage and Sobs indices did not differ among groups. Simpson index was significantly lower in L-RPG than in the other two groups (p < 0.01), further confirming higher community uniformity (Table 7).

At the genus level, the dominant taxa across the three groups included Bacteroides, Lactobacillus, Escherichia-Shigella, norank-o-Clostridia-UCG-014, Butyricicoccus, Faecalibacterium, Fusobacterium, Subdoligranulum, UCG-005, and norank-o-RF39 (Figure 2C). Genera that differed significantly among groups were Solbacillus, Citricoccus, Phocea, norank-f-67-14, and Oxalobacter (Figure 2D). At the species level, the core community comprised Bacteroides fragilis, unclassified-g-Bacteroides, Escherichia coli, Bacteroides fluxus, unclassified-g-Butyricicoccus, Faecalibacterium prausnitzi, unclassified-g-Fusobacterium, uncultured-organism-g-Subdoligranulum, Lactobacillus reuteri, and unclassified-g-norank-o-RF39 (Figure 2E). Differentially abundant species included Clostridium aldenense, uncultured_bacterium_g_Paenibacillus, Citricoccus alkalitolerans, uncultured_bacterium-g-Phocea, Arthrobacter citreus, metagenome-f-67-14, and Oxalobacter formigenes (Figure 2F).

3.6. Impact of Maternal RPG Supplementation on the Calf Serum Metabolome

To elucidate how RPG reshapes the metabolic network of calves at the molecular level, non-targeted metabolomics was performed on NC, L-RPG and H-RPG sera. Compared with NC, L-RPG yielded 111 differential metabolites—more than H-RPG vs. NC (50) or L-RPG vs. H-RPG (97) (Figure 3A). The altered compounds were dominated by fatty acids, nucleosides, amino acids and bases (Figure 3B). Pathway mapping revealed that these metabolites were mainly enriched in nucleotide metabolism, ABC transporters, tryptophan metabolism, pyrimidine/purine metabolism, linoleic acid metabolism, cofactor biosynthesis and unsaturated-fatty-acid biosynthesis (Figure 3C). Further enrichment analysis revealed significant activation of folate biosynthesis, cholesterol metabolism, α-linolenic acid metabolism, TRP-channel regulation by inflammatory mediators, pyrimidine/purine metabolism, ABC transporters, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, and nucleotide metabolism, indicating that RPG intervention may modulate calf physiological functions via these metabolic pathways (Figure 3D).

3.7. Integrated Analysis of Calf Gut Microbiota and Differential Metabolites

As illustrated in Figure 4A, genera such as Bacteroides, Lactobacillus, Butyricicoccus, norank-o-RF39, Faecalibacterium, and Clostridia-UCG-014 were the main contributors to inter-group variation, implying their potential importance in shaping host phenotype. Comparison between the L-RPG and NC groups revealed upregulation of canrenoate, polyanethole sulfate, 11β,13-dihydrolactucopicrin, carnosic acid, neoporrigenin B, and 3-hydroxynona-4,6-dienoylcarnitine, whereas 5′-deoxy-5-fluorocytidine, arabinosylhypoxanthine, benzamide, isoguanosine, gibberellin A75, guanosine, and dobesilic acid were downregulated (Figure 4B). Correlation analysis further demonstrated a significant inter-relationship pattern among these differential metabolites (Figure 4C). Pathway-enrichment analysis indicated that these metabolites are mainly involved in nucleotide metabolism, linoleic acid metabolism, and biosynthesis of unsaturated fatty acids (Figure 4D).

Integration via the O2PLS model further revealed that Akkermansia muciniphila, Mycetocola-sp-449, and Sanguibacter-keddieii-DSM-10542 were the microbial drivers most responsible for inter-group separation, while zedoarol, phenylalanyl-histidine, glycyl-isoleucine, and (2S)-2-(diaminomethylideneamino)-3-phenylpropanoic acid were among the metabolites predicted to exert positive regulatory effects (Figure 4E). To screen key metabolites, a Mantel-test network heat-map was constructed for the L-RPG group, showing strong correlations between L-RPG intervention and zedoarol, 4-hydroxy-6-methyl-2-pyrone, Fa(12:1+1O), 11β,13-dihydrolactucopicrin, di-O-methylfraxetin, and phenylalanyl-histidine (Figure 4F). Thus, beneficial microbes such as Akkermansia muciniphila may improve host metabolism by modulating the levels of these key metabolites.

4. Discussion

Although RPG supplementation, especially the low-dose L-RPG regimen, did not markedly accelerate maternal body-weight recovery during the peripartum period, it significantly improved elevating milk protein, fat, lactose, and gross energy contents. This suggests that RPG, as a readily available energy precursor, is preferentially allocated to the mammary gland for synthesis of milk protein, lactose and other key milk solids rather than being used for female yak body-condition restoration. This “high-quality milk” effect was subsequently transmitted to the offspring: calves in the L-RPG group gained significantly more body weight during the first 28 days of life than their counterparts in the other two groups. The absence of differences in body-frame measurements among treatments is likely because, in early life, nutrient partitioning first favors accretion of soft tissues (muscle and fat), whereas measurable effects on skeletal dimensions require a longer observation window [18,19]. The marked increase in calf body weight provides compelling evidence that RPG intervention is acutely effective in promoting early growth. The fact that calf performance improved despite the absence of significant maternal body-condition recovery indicates that yaks possess a distinctive physiological adaptation in nutrient-partitioning strategy during the periparturient period. Under sustained negative energy balance, exogenous glucose is preferentially directed toward milk synthesis rather than toward restoring the female yak’s own reserves, reflecting a survival strategy that prioritizes offspring nutrition in harsh environments with limited resources [20]. This is consistent with findings in bighorn sheep, with both species demonstrating an “offspring-first” strategy in nutrient allocation [21]. This adaptive mechanism ensures that calves receive adequate nutrients during the critical early period and establishes a solid foundation for their subsequent healthy development. Furthermore, the effectiveness of H-RPG in this study was lower than that of the low-dose group, suggesting a non-linear dose-effect window for this supplement. The beneficial effects of the low dose may be attributed to its closer approximation to the natural, gradual glucose supply pattern under physiological conditions. Conversely, H-RPG intervention may disrupt the balance of key metabolic hormones and exert negative effects on parturition and postpartum recovery. This “optimal dose” phenomenon is not unique in ruminant nutrition research; Miranda et al. also reported similar dose-dependent effects in their study on rumen-protected fat in sheep [22].

We next conducted a comprehensive panel of key hormones and biochemical indices to dissect, from multiple angles, the physiological mechanisms underlying the L-RPG-induced growth acceleration. The results demonstrate that the growth-promoting effect is not mediated by a single pathway, but rather arises from the concerted modulation of the endocrine axis, optimization of energy metabolism, alleviation of oxidative stress, and balancing of the immune-inflammatory network. Regarding endocrine regulation, L-RPG shaped a growth-favorable hormonal milieu by markedly elevating IGF-1 and its principal binding protein IGFBP-3 while reducing the inhibitory binding protein IGFBP-2 and somatostatin, thereby establishing a highly efficient “high IGF-1 activity/low inhibitory tone” pro-growth profile. Concurrent increases in insulin and GLP-2 further synergized to enhance anabolism and intestinal function [23,24,25]. At the metabolic level, elevated serum glucose, reduced BHBA concentrations, and improved hepatic indices (e.g., lower ALT) furnished a robust metabolic foundation for protein synthesis, as reflected by increased albumin-and for rapid calf growth. Within the internal milieu, enhanced T-AOC and CAT activities, combined with consistently low MDA levels, indicated effective control of lipid peroxidation. Simultaneously, pro-inflammatory cytokines (TNF-α, IL-6) declined markedly, whereas the anti-inflammatory mediator IL-10 tended to increase, revealing that L-RPG alleviated oxidative stress and re-balanced immune homeostasis. This “low-oxidative-damage, low-inflammatory-waste” physiological environment reduced the metabolic cost of maintenance, thereby channeling nutrients more efficiently into growth processes and providing the intrinsic basis for the superior growth performance observed in L-RPG calves.

16S rRNA sequencing revealed that L-RPG markedly altered the calf gut microbial community, and the enrichment of several key functional taxa was closely linked to improved host metabolism. For instance, the genus Solbacillus possesses strong phosphate-solubilizing activity, releasing soluble phosphate that supplies critical phosphorus to the microbial community and is essential for intestinal epithelial health and overall metabolism [26]. Citricoccus, belonging to the phylum Actinobacteria, is known for its potent capacity to degrade organic matter-especially cellulose and chitin, suggesting that Citricoccus may play an important role in the cooperative decomposition network within the gut microbiota [27,28]. Additionally, the increased abundance of Oxalobacter formigenes facilitates the degradation of dietary oxalate, thereby reducing the risk of calcium-oxalate urolithiasis, while Citricoccus alkalitolerans and Arthrobacter citreus both possessing strong organic-matter-degrading capabilities are likely involved in the elimination of endogenous or exogenous toxic compounds within the gut, alleviating hepatic metabolic burden [29,30,31]. The synergistic activities of these functional microbes may therefore play a pivotal role in RPG-mediated regulation of nutrient metabolism and improvement of intestinal health in calves.

Metabolomic profiling revealed 97 differential metabolites between the L-RPG and H-RPG groups, demonstrating that RPG dose markedly alters systemic metabolism and confirming a clear dose–response relationship. These metabolites were predominantly enriched in lipid-related pathways. Beyond serving as an energy source, lipids are essential for membrane construction and signaling-molecule synthesis, implying that RPG effects extend beyond simple energy supply to the modulation of fundamental cellular processes [32]. Calves in the L-RPG group showed significant activation of the “biosynthesis of unsaturated fatty acids” pathway. Unsaturated fatty acids, such as omega-3 and omega-6-exert anti-inflammatory effects, maintain membrane fluidity, and serve as precursors for signaling molecules; their upregulation therefore provides a metabolic explanation for the lower inflammatory status (marked reductions in TNF-α and IL-6) and enhanced antioxidant capacity observed in these animals [33,34,35]. In addition, L-RPG enriched several amino-acid-related pathways, including tryptophan metabolism, arginine biosynthesis, and phenylalanine/tyrosine/tryptophan biosynthesis. Amino acids are not only the building blocks for protein synthesis but also precursors for bioactive signals; their active metabolism is tightly linked to rapid growth [36,37]. Thus, L-RPG appears to coordinate growth promotion and physiological improvement by modulating key pathways such as unsaturated-fatty-acid synthesis and amino-acid metabolism.

Multi-omics integration identified Akkermansia muciniphila as a high-scoring taxon, implying it may be a species mediating RPG’s beneficial effects. This obligate mucin degrader catabolizes mucus-layer glycoproteins, thereby stimulating the host to secrete a thicker, more intact mucus barrier and reinforcing intestinal integrity [38,39,40]. Numerous studies have linked greater abundance of Akkermansia muciniphila with improved glucose homeostasis, enhanced insulin sensitivity, and favorable lipid metabolism [41,42,43]. Metabolomic correlation analysis further revealed that zedoarol, 4-hydroxy-6-methyl-2-pyrone, Fa(12:1+1O), 11β,13-dihydrolactucopicrin, di-O-methylfraxetin and phenylalanyl-histidine were strongly positively associated with L-RPG treatment. Zedoarol and 11β,13-dihydrolactucopicrin have been reported to exhibit antioxidant and anti-inflammatory activities [44,45]; together with Akkermansia muciniphila, they may cooperatively sculpt a beneficial intestinal microenvironment and systemic physiological state in calves. This study focused on yaks and lacks validation in other cattle breeds and farming systems (e.g., grazing vs. semi-intensive).

5. Conclusions

This study demonstrates that low-dose (150 g/day) RPG supplementation in perinatal yaks is most effective in promoting calf growth and health, with mechanisms linked to improved dam milk composition and modulation of offspring gut microbiota–host metabolic interactions. Compared to both the high-dose (300 g/day) and control groups, low-dose RPG demonstrated superior efficacy in enhancing dam milk composition (protein and fat content), promoting calf growth performance (body weight), and improving overall health status (antioxidant capacity and immune state), indicating that 150 g/day represents an effective and practical supplementation dose for yaks. Multi-omics analysis revealed the underlying mechanisms: RPG supplementation (especially at low dose) significantly increased the relative abundance of beneficial bacteria such as Akkermansia muciniphila in the calf gut and upregulated serum levels of metabolites like zedoarol, which are involved in anti-inflammatory and antioxidant pathways. The O2PLS model further confirmed strong associations between microbiome and metabolite changes, suggesting that RPG primarily promotes calf health through gut microbiota–host metabolic interactions. This study provides an economically viable nutritional strategy for yak farming, with significant implications for improving early-life calf vitality and promoting herd sustainability and economic benefits. This study investigated the effects of RPG on yak calf health from multiple perspectives, but it also has certain limitations. The optimal supplementation dose was determined based on only two levels (150 g/day and 300 g/day), and the study population comprised exclusively female yaks; therefore, the applicability of these findings to male individuals requires further validation. Moreover, the causal relationships between gut microbiota and metabolites remain to be established through experiments such as fecal microbiota transplantation and germ-free animal models. Future studies should evaluate the long-term effects of RPG intervention on calf production performance and the economic feasibility of this approach across different production systems.