Melatonin improves ovum pick-up efficiency and oocyte quality in dairy cows by modulating follicular steroidogenesis and mitigating oxidative stress

Results: This study investigated the effects of MT on the developmental competence of oocytes collected by OPU from Holstein cows, integrating oocyte quality assessment, follicular fluid metabolomics, and blastocyst transcriptomics. Subcutaneous administration of 80 mg/cow MT prior to OPU significantly increased the proportion of high-quality Grade A oocytes (3.40 ± 0.37 vs 1.40 ± 0.27, P < 0.05) and reduced the proportion of Grade D oocytes (2.80 ± 0.42 vs 8.80 ± 1.26, P < 0.05).

Mechanistically, MT alleviated systemic oxidative stress, enhanced glutathione metabolism, and prevented premature luteinization by reducing serum progesterone. Untargeted metabolomics further revealed that MT reshaped the follicular fluid microenvironment through the upregulation of estrogen derivatives and glutathione-related pathways. Transcriptomic analysis of ovarian cells showed significant upregulation of the rate-limiting genes steroidogenic acute regulatory protein (STAR) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR).

In vitro, supplementation with 10^-7 mol/L MT in oocyte maturation media significantly improved cleavage (87.43 ± 1.59 vs 73.19 ±2.77, P < 0.05) and blastocyst rates (46.99 ± 5.67 vs 32.97 ± 2.38, P < 0.05), scavenged intracellular reactive oxygen species (ROS), and increased total blastocyst cell numbers. Smart-seq2 analysis demonstrated that MT reprogrammed the embryonic transcriptome by activating the AMPK, FoxO, and autophagy pathways while inhibiting endoplasmic reticulum stress and cellular senescence.

Conclusions MT effectively optimizes OPU efficiency by dually improving the follicular microenvironment and protecting embryos from oxidative damage. These findings provide the first in vivo evidence of MT's multi-layered action in a repeated OPU model and establish a strong basis for its clinical application in commercial dairy breeding to mitigate the competence-limiting stress of frequent follicular aspiration.