Dietary energy enhances conception in Holstein heifers via interactions with rumen microbiota

Objective: This study aimed to elucidate how dietary energy levels regulate the hypothalamic-pituitary-gonadal axis in Holstein heifers during initial breeding, with a focus on rumen microbiota-host interactions. Methods: Forty-four pubertal heifers (398.96±6.56 kg BW, 12.72±0.02 months) were stratified by body condition score and estrous cyclicity, then randomly allocated to control (CON, 8.64 MJ/kg DM NE_L) or high-energy (HE, 9.50 MJ/kg DM NE_L) diets (n = 22/group). Although practical constraints limited pen replication, we implemented rigorous matching procedures: Pens were matched for surface area (120 m²), feed bunk space (0.8 m/head) and growth performance, serum biochemical/immune/antioxidant markers, reproductive hormones, rumen fermentation parameters, microbiota, and metabolome profiles were analyzed. Results: The HE group exhibited elevated gonadotropins (follicle-stimulating hormone, luteinizing hormone [LH]) and prolactin (PRL), indicating enhanced hypothalamic-pituitary activity. Serum triglycerides increased, while immune markers showed the altered state of immunoregulation characterized by significant increases in interleukin (IL)-2 and IL-6, reductions in IL-4, and decreases in tumor necrosis factor-α and interferon-γ. Antioxidant capacity improved with lower malondialdehyde levels. Rumen pH decreased, accompanied by elevated total volatile fatty acid, bacterial crude protein, acetic acid, propionic acid, butyric acid, and valeric acid concentrations. Microbial shifts included Treponema and Prevotellaceae_UCG_003 showing positive correlations with PRL and LH, while Ruminococcus was associated with acetyl-CoA precursors through enriched pyruvate metabolism. Conclusion: HE diets (9.50 MJ/kg NE_L) enhance hypothalamic-pituitary signaling and rumen fermentation efficiency, advancing first-service conception rates by 15% (55% vs. 70%) in pasture-based systems. This strategy optimizes reproductive management in intensive dairy operations through microbiota-driven metabolic modulation.