Milk fat synthesis in cows mainly includes the synthesis of short- and medium-chain fatty acids, the uptake, transport, and activation of long-chain fatty acids (LCFAs), the synthesis of triglycerides, and the synthesis of the genes, transcription factors, and signaling pathways involved. Although the various stages of milk fat synthesis have been outlined in previous research, only partial processes have been revealed. CLA consists of an aggregation of positional and geometric isomers of linoleic fatty acid, and the accumulated evidence suggests that the two isomers of the active forms of CLA (cis-9, trans-11 conjugated linoleic acid and trans-10, cis-12 conjugated linoleic acid, abbreviated as c9, t11-CLA and t10, c12-CLA) can reduce the fat content in milk by regulating lipogenesis, fatty acid (FA) uptake, oxidation, and fat synthesis. However, the mechanism through which CLA inhibits milk fat synthesis is unique, with most studies focusing only on the effects of CLA on one of the genes, transcription factors, or signaling pathways involved. In this study, we summarized the structure and function of classic genes and pathways (mTOR, SREBP, AMPK, and PPARG) and new genes or pathways (THRSP, METTL3, ELOVL, and LPIN1) involved in each stage of milk fat synthesis and demonstrated the interactions between genes and pathways. We also examined the effects of other substances (melanin, nicotinic acid, SA, etc.). Furthermore, we evaluated the influence of β-sitosterol, sodium butyrate, Met arginine, and Camellia oleifera Abel on milk fat synthesis to improve the mechanism of milk fat synthesis in cows and provide a mechanistic reference for the use of CLA in inhibiting milk fat biosynthesis.

Dairy cows experiencing negative energy balance (NEB) are prone to metabolic and inflammatory disorders, including ketosis, fatty liver, mastitis, endometritis, and hypocalcemia, which impair productive and reproductive performance. NEB elevates non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA), leading to disrupted lipid metabolism characterized by increased fatty acid synthesis (via SREBP-1c, ACC, FASN), impaired lipid export (downregulated MTTP, ApoB100, ACAT2), and reduced oxidation (suppressed SIRT1–PPARα–CPT1A/2 pathway), resulting in triacylglycerol (TAG) accumulation and ketosis. Excess reactive oxygen species (ROS) trigger oxidative and endoplasmic reticulum (ER) stress and apoptosis through JNK, p53/Nrf2, and PERK–eIF2α signaling, while HIF-2α–mediated hypoxia aggravates hepatic damage. Elevated NEFA/BHBA impair polymorphonuclear neutrophil (PMN) chemotaxis and phagocytosis, promoting mastitis and endometritis, and hypocalcemia further weakens immune defense. Rumen-protected choline (RPC) improves lipid metabolism by enhancing VLDL assembly and TAG export (upregulating MTTP, ApoB100, ATG3; inhibiting SREBF1, DGAT2), stimulating fatty acid oxidation (activating AMPK–PPARα–CPT1α), and reducing oxidative stress (suppressing ROS–ERN1). Moreover, RPC decreases IL-6 and TNF-α levels and enhances antioxidant capacity and PMN function. Overall, RPC alleviates NEB-induced metabolic and inflammatory diseases, supporting its inclusion in periparturient management to mitigate NEB and associated disorders.

Conjugated linoleic acid, a long-chain fatty acid, reduces milk fat content in dairy cows, which may alleviate the negative energy balance during the early lactation period by reducing lactation energy output and thereby decreasing the occurrence of ketosis and fatty liver due to a negative energy balance. Although research has revealed the milk fat inhibition mechanism, the mechanisms underlying the metabolic interactions among the liver, subcutaneous fat, and mammary gland tissues during this period remain unclear. In this study, the molecular mechanisms underlying CLA-induced milk fat depression in the liver, subcutaneous white adipose tissue, and mammary gland tissues of dairy cows were investigated. Chinese Holstein dairy cows at 30 d postpartum were fed a control diet (6 cows) or a CLA-supplemented diet (6 cows) for 7 d, and all cows were fed the control diet only for another 7 d. Tissue samples from the liver, subcutaneous white adipose tissue, and mammary gland were collected on d 0 (before CLA supplementation) and d 7 (after supplementation) for RNA sequencing analysis. In addition, blood samples were collected from control and CLA-treated cows on d 0, 4, 7, and 14 (post-treatment) to measure the concentrations of nonesterified fatty acids (NEFA) and β-hydroxybutyric acid (BHBA). Furthermore, from d 0 to 14, milk composition and milk yield were measured. The results of lactation performance indicated that supplementation with CLA led to a significant reduction in milk fat percentage, decreasing from 4.0% to 3.5%, and no other changes in the lactation performance of the CLA group were observed. With respect to the indicators of excessive fat mobilization and circulating ketone bodies, the CLA group presented significantly lower serum NEFA concentrations than did the control group, with levels decreasing from 0.70 to 0.55 mmol/L between one day before CLA supplementation (d 0) and d 7 of CLA supplementation (d 7), whereas the BHBA concentration tended to decrease over this period. Transcriptomic analysis of mammary tissue identified 674 differentially expressed genes (DEG). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the downregulated DEG in the CLA group were involved mainly in lipid biosynthetic processes, fatty acid biosynthesis, the p53 signaling pathway, and steroid biosynthesis. Transcriptomic analysis of subcutaneous white adipose tissue revealed 614 DEG between the CLA and control groups. These DEG were significantly enriched in pathways related to fatty acid metabolism, fatty acid transport, fatty acid β-oxidation, lipid metabolism, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and AMP-activated protein kinase (AMPK) signaling pathway. Transcriptomic analysis of liver tissue identified 479 DEG, which were enriched in pathways related to fatty acid metabolism, the PPAR signaling pathway, lipid transport activity, the AMPK signaling pathway, the phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) signaling pathway, the insulin signaling pathway, and the insulin resistance pathway. Collectively, these findings indicate that CLA successfully induced milk fat depression in postpartum dairy cows. Based on the up- and downregulation of genes within fatty acid metabolism, fatty acid β-oxidation, and lipid metabolism pathways in adipose tissue, CLA may promote lipid synthesis while inhibiting fatty acid oxidation and lipolysis in subcutaneous white adipose tissue. Additionally, gene expression patterns related to lipid transport activity, fatty acid metabolism, PPAR, and AMPK pathways in liver tissue suggest that CLA may increase lipid transport capacity while reducing hepatic lipid accumulation. These findings provide theoretical evidence in support of the regulation of energy balance in early-lactating cows through the inhibition of milk fat synthesis by CLA, which may be used as a strategy for preventing type I ketosis.

This study compared the regulatory effects of dietary supplementation with natural clinoptilolite (CLN) versus a dietary cation-anion difference (DCAD) regimen on calcium homeostasis in dairy cows during the last 21 days prepartum. Results showed that cows in the DCAD group exhibited significantly higher blood ionized calcium (iCa) and parathyroid hormone (PTH) concentrations than those in the CLN group (p < 0.05). Serum PTH concentrations displayed a declining trend in both groups prepartum, which deviates from the classical theory of compensatory PTH secretion, suggesting that alternative compensatory pathways may be involved in the regulation of calcium homeostasis during the periparturient period in dairy cows. Monitoring of calcium homeostasis and related metabolic parameters following postpartum oral calcium bolus administration revealed that the incidence of subclinical hypocalcemia in the DCAD group was 26%, lower than the 62% observed in the CLN group. However, blood iCa concentrations returned to normal levels more rapidly in the CLN group. Additionally, CLN supplementation was associated with more stable blood pH and lower prepartum serum potassium concentrations (p < 0.05) that remained within the physiological range, which may contribute to improved tissue sensitivity to PTH.