Previous studies have identified oxidative stress and inflammatory responses in granulosa cells (GCs) of periparturient dairy cows. However, whether non-esterified fatty acids (NEFA)-induced endoplasmic reticulum (ER) stress is involved in GC apoptosis remains unclear. In this study, treatment with NEFA (0.9 mM, 24 h) activated the ER stress pathway. This was evidenced by increased expression of both CHOP and GRP78. Furthermore, upregulation of pro-apoptotic factors BAX and Caspase-3 and downregulation of the anti-apoptotic factor Bcl-2 were observed. Pretreatment of GCs with 4-phenylbutyric acid (4-PBA, 2.5 mM, 2 h) reversed the ER stress and apoptotic effects. This suggests that NEFA-induced apoptosis is mediated through activation of the PERK pathway of ER stress, and that 4-PBA alleviates this effect. Furthermore, targeted metabolomics revealed disruptions in lipid and hormone metabolism in GCs following NEFA treatment. Analysis revealed an increase in the levels of 26 types of fatty acids, while a decrease was detected in the levels of 3 types of fatty acids. In summary, NEFA induces ER stress and disrupts intracellular fatty acid, ultimately leading to cell apoptosis. Our findings offer valuable insights for developing strategies to regulate follicular development in dairy cows and mitigate the impacts of postpartum negative energy balance (NEB).

This study evaluated the effects of dietary supplementation with a commercial lactoferrin–osteopontin complexes preparation (LF-OPN; LF:OPN mass ratio 8:1; 240 mg/kg body weight) on growth, immune indices, antioxidant status, intestinal barrier-related markers, fecal microbiota, short-chain fatty acids (SCFAs), peripheral lymphocyte subsets, and the whole-blood transcriptome of growing British Shorthair kittens. Sixteen kittens were randomly assigned to either a basal diet group (CON) or a group supplemented with LF-OPN complexes (LFT) for 28 days. LF-OPN supplementation did not significantly affect body weight or average daily gain. On D28, the LFT group had a higher serum immunoglobulin M concentration and a lower tumor necrosis factor-α concentration (p < 0.05). Serum superoxide dismutase and glutathione peroxidase activities and total antioxidant capacity were higher, whereas malondialdehyde was lower (p < 0.05) in the LFT group (p < 0.01). Fecal 16S rRNA gene sequencing revealed no significant difference in alpha diversity; howerer, LEfSe identified selective taxonomic difference, including higher relative abundances of Holdemanella and Erysipelotrichaceae and lower relative abundances of Enterobacteriaceae and Escherichia-Shigella in the LFT group. Fecal short-chain fatty acid concentrations were unchanged. Flow cytometry revealed a higher proportions of peripheral CD4+ lymphocytes and a higher CD4+/CD8+ ratio in the LFT group (p < 0.01), whereas the CD8+ proportion was unchanged. Whole-blood RNA sequencing identified 283 differentially expressed genes (199 upregulated and 84 downregulated), with enrichment of immune-related pathways including hematopoietic cell lineage, antigen processing and presentation, cytokine–cytokine receptor interaction, NF-κB signaling, and IL-17 signaling, but these findings were not independently validated. The tested LF-OPN preparation was well tolerated and was associated with elevated antioxidant status, altered immune indices, and selective modulation of the fecal microbiota in growing kittens. The study does not establish synergy or identify the individual contributions of either protein.