Heat stress (HS) is directly correlated with mammary gland dysfunction and the hypothalamic-pituitary-mammary gland (HPM) axis is involved in regulating stress responses and lactation in dairy cows. Circular RNAs (circRNAs) play major roles in regulating transcription and post-transcription but their expression in the HPM axis of dairy cows under HS is still unclear. In the present study, we performed RNA sequencing to identify diferentially expressed (DE) circRNAs, DE microRNAs(miRNAs) and DEmRNAs, and performed bioinformatics analysis on those in HPM axis-related tissues of heat-stressed and normal cows. A total of 1680, 1112 and 521 DEcircRNAs, 120, 493 and 108 DEmiRNAs, 274, 6475 and 3134 DEmRNAs were identified in the hypothalamic, pituitary, and mammary gland tissues, respectively. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses indicated that the MAPK signaling pathway is potentially a key pathway. Competitive endogenous RNA (ceRNA) networks related to HS response and lactation regulation were established in three tissues. In conclusion, our results indicate that HS induces differential circRNA expression profiles in HPM axis-related tissues, and the predicted ceRNA network provides a molecular basis for regulating the stress response and lactation regulation in heat-stressed dairy cows.

Heat stress (HS) is directly correlated to mammary gland dysfunction in dairy cows, especially in summer. The hypothalamic−pituitary−mammary gland axis (HPM axis) plays an important role in the regulation of stress response and lactation physiology in heat−stressed dairy cows. The aim of this study was to explore the lncRNA profile, and the competitive endogenous RNA (ceRNA) regulatory network in hypothalamus, pituitary, and mammary gland tissues of heat−stressed and normal dairy cows. We performed RNA sequencing (RNA−seq) to identify differentially expressed (DE) lncRNAs, and the ceRNA regulatory network was established in HPM−axis−related tissues. Our results showed that a total of 13, 702 and 202 DE lncRNAs were identified in hypothalamus, pituitary, and mammary glands, respectively. Of lncRNAs, 8, 209 and 45 were up−regulated, and 5, 493 and 157 lncRNAs were down−regulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that DE lncRNAs target genes that might play a role in hormone synthesis, secretion and action, apoptosis, mitogen−activated protein kinase (MAPK), AMP−activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR) signaling pathway. Moreover, the ceRNA regulatory network associated with the MAPK signaling pathway in HPM−axis−related tissues contains 3286 lncRNA–mRNA pairs. Furthermore, the ceRNA regulatory network associated with apoptosis, prolactin, AMPK, and mTOR signaling pathway in the mammary gland contains 772 lncRNA–mRNA pairs. Thus, some lncRNAs may be involved in the regulation of stress response and the physiological process of lactation. The changes in lncRNA expression profiles and ceRNAs (lncRNA–miRNA–mRNA) in HPM−axis−related tissues are the key to affect the stress response and lactation physiology of dairy cows under HS, which provide a theoretical basis for the molecular mechanism in the stress response of HPM−axis−related tissues in dairy cows under HS.

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).

High-yielding dairy cows are highly susceptible to lactational oxidative stress, which compromises mammary barrier integrity and elevates mastitis risk. This study investigated the potential biological mechanisms by which dietary hydroxy-selenomethionine (HMSeBA) alleviates oxidative stress and improves health in dairy cows. Forty Holstein cows were assigned to a basal control group (0.32 mg Se/kg DM) or an HMSeBA-supplemented group (0.64 mg Se/kg DM) for 105 days. HMSeBA significantly enhanced selenium bioavailability in both milk and blood, comprehensively strengthening antioxidant defenses (increased glutathione peroxidase activity, decreased malondialdehyde) and elevated serum immunoglobulins (IgA, IgM, IgG), accompanied by a reduction in milk somatic cell count, without significantly affecting milk yield, feed intake, or milk production efficiency. Multi-omics analysis revealed that HMSeBA supplementation altered the rumen microenvironment by enriching fiber-degrading genera (Prevotellaceae_Ga6A1_group, Xylanibacter, Segatella) and shifting metabolites, including feed flavonoids, peptides, 1-deoxy-D-xylulose-5-phosphate, and 3-OH-C6-HSL. The positive correlation of ruminal 3-OH-C6-HSL with both blood selenium and these enriched taxa suggests a potential link between microbial activity and host selenium status. These findings indicate that HMSeBA supplementation improves the antioxidant and immune status of dairy cows, accompanied by exploratory, hypothesis-generating shifts in the ruminal microbiome and metabolome. Collectively, these findings highlight HMSeBA as a promising nutritional strategy to produce selenium-enriched milk while safeguarding udder health.

Holstein (H) cows and first-generation Montbéliarde × Holstein (MH) crossbred cows were compared for lactation performance, culling reasons, and cull price under the same intensive dairy production system. Data were obtained from a single commercial dairy farm in Jiangsu Province, China, including lactation records from 504 H cows and 367 MH cows and culling records from 345 H cows and 178 MH cows born between 2013 and 2022. Lactation traits from the first 3 parities were analyzed using linear mixed models, and culling reasons were evaluated using chi-squared tests and multinomial logistic regression, with low milk yield as the reference outcome. Overall milk yield and milk component yields did not differ between breeds across lactations (differences <1.6%). However, H cows exhibited a higher peak milk yield (48.3 vs. 46.9 kg), whereas days to peak milk yield were similar between breeds, with only a small numerical difference. The distribution of culling reasons differed markedly between breeds, with body condition abnormalities accounting for a larger proportion of culling in MH cows (41.6% vs. 15.4%) and a higher proportion of culling events due to death in H cows (20.6% vs. 10.7%), and overall removal risk was higher in H cows than in MH cows when expressed per 100 cow-yr at risk. Multinomial regression showed that calving season, parity, and age (modeled within parity) were significant predictors of multiple culling outcomes, whereas inclusion of BW in sensitivity analyses did not materially alter breed-related effects. Cull price was positively associated with BW but did not differ between breeds. These results indicate that although H and MH cows achieve comparable cumulative milk production, breed differences are primarily reflected in lactation curve characteristics and culling structure rather than total productivity.

Nitrogen use efficiency (NUE) is commonly calculated as the ratio of milk nitrogen output to nitrogen intake in dairy cows. However, because milk nitrogen output is intrinsically determined by milk production and nitrogen intake is largely driven by dry matter intake, conventional NUE is mathematically dependent on production level and feed intake. This dependency makes it difficult to distinguish apparent efficiency caused by higher milk yield from intrinsic biological efficiency in nitrogen utilization. Therefore, this study aimed to evaluate NUE in dairy cows using a combined mathematical and metabolic framework based on residual NUE (rNUE) analysis. A total of 126 early-lactation Chinese Holstein cows were screened, and 16 high-NUE and 16 low-NUE cows were selected after matching for parity, days in milk, and dry matter intake. High-NUE cows had greater milk yield, milk nitrogen output, and NUE than low-NUE cows, despite similar nitrogen intake. They also exhibited higher ruminal microbial crude protein and ammonia nitrogen concentrations, a lower acetate-to-propionate ratio, and reduced circulating essential and total amino acid concentrations. Multiple regression analysis showed that energy-corrected milk and dry matter intake explained 71.4% of the variation in NUE. The residuals from this model were defined as rNUE, which was independent of milk production. After removing production-related effects, rNUE remained positively associated with ruminal microbial crude protein and ammonia nitrogen concentrations, and negatively associated with the acetate-to-propionate ratio and circulating amino acid pools. These findings indicate that conventional NUE in dairy cows is largely driven by production level, whereas residual-based modeling can identify a production-independent component associated with rumen nitrogen metabolism and amino acid utilization. The residual NUE approach provides a useful mathematical and metabolic framework for evaluating intrinsic NUE in dairy cows beyond milk production level.