a-protocol-for-size-based-enrichment-lipid-droplets-from-bovine

A Protocol for Size-Based Enrichment of Lipid Droplets from Bovine Mammary Epithelial Cells and Mammary Tissues

This protocol describes a sequential differential centrifugation approach to enrich lipid droplets (LDs) of different sizes from bovine mammary epithelial ...

Henan Agricultural University

Density dependence in reproduction plays an important role in stabilizing population dynamics via immediate negative feedback from population density to reproductive output. Although previous studies have shown that negative density-dependent reproduction is associated with strong spacing behavior and social interaction between individuals, the proximal mechanism for generating negative density-dependent reproduction remains unclear. In this study, we investigated the effects of density-induced stress on reproduction in root voles. Enclosed founder populations were established by introducing 6 (low density) and 30 (high density) adults per sex into per enclosure (four enclosures per density in total) during the breeding season from April to July 2012 and from May to August 2015. Fecal corticosterone metabolite (FCM) levels, reproductive traits (recruitment rate and the proportion of reproductively active individuals), and founder population numbers were measured following repeated live trapping in both years. The number of founders was negatively associated with recruitment rates and the proportion of reproductively active individuals, displaying a negative density-dependent reproduction. FCM level was positively associated with the number of founders. The number of founder females directly affected the proportion of reproductive females, and directly and indirectly through their FCM levels affected the recruitment rate; the effect of the number of male founders on the proportion of reproductive males was mediated by their FCM level. Our results showed that density-induced stress negatively affected reproductive traits and that density-induced stress is one ecological factor generating negative density-dependent reproduction.

Is negative density-dependent reproduction regulated by density-induced stress in root voles? Two field experiments.

In mammary epithelial cells, milk fat is synthesized as lipid droplets and secreted in the form of globules. Milk fat globules (MFGs) are covered by a lipid-protein membrane known as the milk fat globule membrane (MFGM). We randomly divided 12 Holstein cows into control and conjugated linoleic acid (CLA) groups. The control group was fed a basal diet, while the CLA group was fed the basal diet + CLA (15 g per kg DM) for 10 days. Cow performance, milk composition, and MFG size were measured daily. On day 10, we extracted MFGM proteins ( = 3) and identified them  quantitative proteomic analysis. We investigated the effects of the MFGM proteins from control and CLA-treated milk on the lipid droplet formation in MAC-T cells. Compared with the control group, the CLA group had reduced milk fat content (3.39 g/100 mL  2.45 g/100 mL) and MFG size parameters ( of 3.85 μm  3.37 μm;  of 3.24 μm  2.83 μm). The specific surface area (SSA) increased in the CLA group. A total of 361 differentially expressed proteins were identified in the CLA group by iTRAQ quantitative proteomic analysis. Among these proteins, 100 were upregulated and 251 were downregulated ( < 0.05). In MAC-T cells, CLA-MFGM proteins increased the diameter of the lipid droplets to 1.32 μm. CLA-MFGM proteins decreased the proportion of the small lipid droplets (15.33%  47.78%) and increased the proportion of the large lipid droplets (25.04%  11.65%). CLA-MFGM proteins promoted lipid droplet fusion. Therefore, MFGM proteins play an important role in the regulation of the lipid droplet size.

Proteomic analysis of milk fat globule membranes from small-sized milk fat globules and their function in promoting lipid droplet fusion in bovine mammary epithelial cells.

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 μmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 μmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro.

In milk, fat exists in the form of milk fat globules (MFGs). The average size (average fat globules of different particle sizes) is the most common parameter when describing MFG size. There are different views on whether there is a correlation between MFG size and milk fat content. Is the MFG size correlated with milk fat content in ruminants? To address this question, we conducted two experiments. In experiment Ⅰ, dairy cows (n = 40) and dairy goats (n = 30) were each divided into a normal group and a low-fat group according to the milk fat content. In experiment Ⅱ, dairy cows (n = 16) and dairy goats (n = 12) were each divided into a normal group and a conjugated linoleic acid (CLA)-induced low-fat group. The normal groups were fed a basal diet, and the CLA-induced low-fat groups were fed the basal diet + 300 g/d CLA (cows) or the basal diet + 90 g/d CLA (goats). In both experiments, we determined the correlation between MFG size and milk composition and MFG distribution. The results showed that in the normal and low-fat groups of cows and goats, MFG size was not correlated with milk fat, protein, or lactose content or fat-to-protein ratio. Additionally, there was no difference in the distribution of large, medium, and small MFGs (P > 0.05). However, in the CLA-induced low-fat groups, we found a correlation between MFG size and milk fat content and fat-to-protein ratio (R > 0.3). Moreover, there was a significant change in the size distribution of MFGs. Therefore, in natural milk, MFG size was not correlated with milk fat content. Following CLA supplementation, MFG size was correlated with milk fat content. Our findings revealed that CLA and not milk fat affects MFG distribution and size.

Is milk fat globule size correlated with milk fat content in Ruminants?

The literature shows that maternal stress can influence behavior and immune function in F1. Yet, most studies on these are from the laboratory, and replicated studies on the mechanisms by which maternal stress drives individual characteristics are still not fully understood in wild animals. We manipulated high- and low-density parental population density using large-scale field enclosures and examined behavior and immune traits. Within the field enclosures, we assessed anti-keyhole limpet hemocyanin immunoglobulin G (anti-KLH IgG) level, phytohemagglutinin (PHA) responses, hematology, cytokines, the depressive and anxiety-like behaviors and prevalence and intensity of coccidial infection. We then collected brain tissue from juvenile voles born at high or low density, quantified mRNA and protein expression of corticotropin-releasing hormone (CRH) and glucocorticoid receptor gene () and measured DNA methylation at CpG sites in a region that was highly conserved with the prairie vole CRH and  promoter. At high density, we found that the F1 had a lower DNA methylation level of CRH and a higher DNA methylation level of , which resulted in an increase in the expression levels of the CRH mRNA and protein expression and further reduced the expression levels of the  mRNA and protein expression, and ultimately led to have delayed responses to acute immobilization stress. Juvenile voles born at high density also reduced anti-KLH IgG levels and PHA responses, increased cytokines, and depressive and anxiety-like behaviors, and the effects further led to higher coccidial infection. From the perspective of population density inducing the changes in behavior and immunity at the brain level, our results showed a physiological epigenetic mechanism for population self-regulation in voles. Our results indicate that altering the prenatal intrinsic stress environment can fundamentally impact behavior and immunity by DNA methylation of HPA-axis genes and can further drive population fluctuations in wild animals.

Effects of DNA Methylation of HPA-Axis Genes of F1 Juvenile Induced by Maternal Density Stress on Behavior and Immune Traits in Root Voles ()-A Field Experiment.

Extracellular vesicles (EVs) are important paracrine mediators derived from various cells and biological fluids, including plasma, that are capable of inducing regenerative effects by transferring bioactive molecules such as microRNAs (miRNAs). This study investigated the effect of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) isolated from umbilical cord blood and human umbilical cord plasma-derived extracellular vesicles (UCB-EVs) on wound healing and scar formation reduction. Spatial transcriptomics (ST) was used to study the effects of MSC-EVs and UCB-EVs on the heterogeneity of major cell types and wound healing pathways in mouse skin tissue. MSC-EVs and UCB-EVs were isolated using ultracentrifugation and identified using transmission electron microscopy, nanoparticle tracking analysis, and western blot. The effects of MSC-EVs and UCB-EVs on human dermal fibroblast-adult cell (HDF-a) migration and proliferation were evaluated using cell scratch assays, cell migration assays, and cell proliferation assays. In vivo, MSC-EVs and UCB-EVs were injected around full-cut wounds to evaluate their efficacy of wound healing by measuring wound closure rates and scar width and performing histological analysis. ST was performed on skin tissue samples from mice in each group after wound healing to analyze the heterogeneity of major cell types compared with the control group and investigate potential mechanisms affecting wound healing and scar formation. In vitro experiments demonstrated that MSC-EVs and UCB-EVs promoted the proliferation and migration of HDF-a cells. Local injection of MSC-EVs and UCB-EVs into the periphery of a mouse skin wound accelerated re-epithelialization, promoted wound healing, and reduced scar width. ST analysis of skin tissue from each group after wound healing revealed that MSC-EVs and UCB-EVs reduced the relative expression of marker genes in myofibroblasts, regulated wound healing, and decreased scar formation by reducing the expression of the TGF-β signaling pathway and increasing the expression of the Wnt signaling pathway. The results suggest that MSC-EVs and UCB-EVs play a significant role in the activity of cord blood plasma-derived mesenchymal stem cells and cord blood plasma. They can be considered promising new agents for promoting skin wound healing.

MSC-EVs and UCB-EVs promote skin wound healing and spatial transcriptome analysis.

Phosphatidylcholine cytidine transferase α (CCTα) is a key rate-limiting enzyme in the CDP-choline pathway, the primary pathway for phosphatidylcholine (PC) synthesis in mammals. This study investigated the role of CCTα in lipid droplet (LD) formation, phospholipid synthesis, LD fusion, and lipophagy in bovine mammary epithelial cells (BMECs) through CCTα gene knockout (CCT-KO) and overexpression (CCT-OE). CCTα mRNA expression was significantly increased in bovine mammary gland tissue after lactation. In BMECs, CCTα was transferred from the nucleus to the endoplasmic reticulum and localized on LD surfaces in the presence of linoleic acid. Compared with normal BMECs (NC), CCTα knockout (CCT-KO) cells had significantly greater LD diameters (1.53 μm vs. 1.68 μm,  < 0.05), lower proportions of small LDs (<1 µm; 11.39% vs. 5.42%), and higher proportions of large LDs (>3 µm; 0.67% vs. 2.88%). In contrast, CCTα overexpression (CCT-OE) decreased the diameter of LDs to 1.18 μm ( < 0.01), increased the proportion of small LDs to 35.48%, and decreased the proportion of large LDs to 0.24%. CCTα knockout significantly decreased the PC content and the ratio of PC to PE, whereas CCTα overexpression increased the PC content and the ratio of PC to phosphatidyl ethanolamine (PE) ( < 0.05). The lipidomics analysis indicated that PC synthesis was significantly influenced by CCTα gene expression. Live cell observations showed that CCTα knockout promoted the fusion of small LDs into large LDs. In cells with CCT α overexpression, the expression of the microtubule-associated protein 1 light chain 3 (LC3) protein and the number of lysosomes was elevated, and the lysosomal phagocytosis of LDs was observed through transmission electron microscopy, thus indicating that CCTα overexpression enhanced lipophagy. In conclusion, these results suggest that CCTα plays a role in regulating LD formation by influencing PC synthesis, LD fusion, and lipophagy in BMECs.

Phosphatidylcholine Cytidine Transferase α (CCTα) Affects LD Formation Through Fusion and Lipophagy in Bovine Mammary Epithelial Cells.

The bacteriophage vB_SaRS_FS19-1, isolated from urban sewage, produced numerous clear plaques, about 1 to 2 mm in diameter, on the S. aureus JP19 bacterial lawn. A broad host range of the bacteriophage vB_SaRS_FS19-1 was found, and the lysis rate of S. aureus was 84 % (42/50). Electron microscopic and genomic DNA analyses suggested that phage vB_SaRS_FS19-1 is a recently discovered absence of lysogeny genes phage, classified under the genus Kayvirus within the family Herelleviridae. Analyses of phylogeny and genomics indicated that there is a relatively close evolutionary relationship with Staphylococcus phage. Notably, phage vB_SaRS_FS19-1 demonstrated robust stability under diverse environmental conditions and showed stable lytic activity across a wide range of pH values (4-10) and temperatures (20-50 °C), even in a 3 M NaCl solution. Moreover, phage vB_SaRS_FS19-1 has the ability to inhibit and kill S. aureus JP19 in a dose-dependent manner in TSB. S. aureus JP19 in contaminated sausages and milk was successfully inhibited at phage MOIs of 100, 1 and 0.01 (P < 0.01). These results indicate that the lytic phage vB_SaRS_FS19-1 could be a promising candidate for managing S. aureus contamination in food products.

Characterization of vB_SaRS_FS19-1, a newly isolated broad-host-range and virulent bacteriophage infecting Staphylococcus aureus and its application in foods.

Public health problems caused by foodborne illnesses have become increasingly serious. Although it was usually regarded as an opportunistic pathogen causing urinary tract infections in humans, recent years have seen an increasing number of foodborne infections related to P. mirabilis. Therefore, it was crucial to develop effective strategies to control this bacterium. In this study, the bacteriophage T2 isolated from cattle dung formed numerous clear plaques with a diameter of approximately 3 mm on the bacterial lawn of P. mirabilis Y22 using a double-layer agar method. Electron microscopic and genomic DNA analyses suggested that phage T2 was a recently discovered virulent phage, classified under the genus Novosibovirus within the subfamily Slopekvirinae. Analyses of phylogeny and genomics indicated that there was a relatively close evolutionary relationship with Proteus phage. Notably, phage T2 exhibited excellent environmental adaptability and showed stable lytic activity across a wide range of pH values (4-10) and temperatures (20-50 °C), even in a 3 M NaCl solution. Phage T2 has the ability to inhibit and kill P. mirabilis Y22 in a dose-dependent manner in LB broth, and phage T2 was capable of significantly inhibiting biofilm formation and breaking down established biofilms. The growth of P. mirabilis Y22 in milk and sausage was successfully suppressed at an MOI of 100 (P < 0.01). Moreover, phage T2 can lyse and kill bacteria in G. mellonella, thereby increasing their survival rate. These results indicated that the lytic phage T2 could be a promising candidate for managing P. mirabilis contamination in food products.

Isolation and characterization of Proteus mirabilis bacteriophage T2 and its application.

The size of fat globules in milk, which is influenced mainly by the size of lipid droplets (LDs) in bovine mammary epithelial cells, affects the quality of the milk. The present study aimed to explore how lipophagy affected the catabolism of LDs in bovine mammary epithelial cells.

Lipophagy modifies the catabolism of intracellular triglycerides by preferentially targeting small-sized lipid droplets in bovine mammary epithelial cells.

Milk fat globules (MFGs) are derived from lipid droplets (LDs) in the mammary epithelial cells of ruminants and released into milk. This study aimed to detect the differences in LDs and MFGs to reveal the mechanism by which LDs transform into MFGs in the mammary cells of dairy goats. The mammary gland tissue and milk of lactating dairy goats were collected and analyzed using lipidomics. The results showed that a total of 1167 and 675 lipid molecules were detected in the LDs and MFGs, respectively. The top four lipids in terms of their contents in the LDs were triglycerides (TGs), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and diglycerol (DG), whereas the top four in the MFGs were TG, PE, PC, and phosphatidylinositol (PI). Furthermore, compared to the LDs, the contents of TG and DG in the MFGs were significantly increased ( < 0.05), while PC content was significantly decreased ( < 0.05). We also found that diglycerol (16:0:18), diglycerol (O-19:0:16), triglycerides (14:0:0_16:0), and phosphatidylethanolamine (P-15:0:20:3) were the key characteristic lipids in the transformation of LDs into MFGs. This study reveals the unique lipid profiles of LDs and MFGs and provides a theoretical basis for understanding the mechanism behind low-fat milk syndrome.

Lipidomics Reveals Differences in Lipid Composition Between Lipid Droplets and Milk Fat Globules in Dairy Goat Mammary Tissue.

Giardia duodenalis, Cryptosporidium spp., Blastocystis sp., and Enterocytozoon bieneusi are prevalent zoonotic intestinal parasitic protozoans that cause diseases in humans and various animals. In this study, a total of 319 fresh fecal samples from domestic and wild animals collected from the Qinghai-Tibetan Plateau Area (QTPA), China, were examined. The results showed that the overall prevalence of these four protozoa in this region was 6.6% (21/319). Specifically, the infection prevalence of G. duodenalis, Cryptosporidium spp., Blastocystis sp., and E. bieneusi was 3.4% (11/319), 1.3% (4/319), 1.9% (6/319), and 0% (0/319), respectively. Three G. duodenalis assemblages, A (n = 1), E (n = 4) and F (n = 1), and two zoonotic Cryptosporidium spp., C. ubiquitum and C. alticolis, were identified. Regarding Blastocystis sp., only one subtype (ST1) was identified in root vole (Microtus oeconomus), a potentially zoonotic subtype. PCR amplification at the internal transcribed spacer (ITS) locus did not detect E. bieneusi in any of the samples. This study provides fundamental data on these four pathogens in M. oeconomus and other domestic animals in the QTPA, China. Although the prevalence of the four parasites was relatively low, the zoonotic species C. ubiquitum and G. duodenalis assemblage A were identified, indicating that wild animals and domestic animals might act as potential reservoirs in the transmission between humans and animals. Further studies are needed to better understand the epidemiology of parasites in wild animals in the QTPA and their potential public health implications.

Prevalence of intestinal protozoan parasites (Giardia, Cryptosporidium, Blastocystis and Enterocytozoon) in animals from Qinghai-Tibetan Plateau area, China.

Article	Year
Is negative density-dependent reproduction regulated by density-induced stress in root voles? Two field experiments. Ecology and evolution\| PubMed ID: 35592070	2022
Proteomic analysis of milk fat globule membranes from small-sized milk fat globules and their function in promoting lipid droplet fusion in bovine mammary epithelial cells. Food & function\| PubMed ID: 36752527	2023
Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro. Journal of dairy science\| PubMed ID: 37678795	2023
Is milk fat globule size correlated with milk fat content in Ruminants? Food chemistry\| PubMed ID: 38043286	2024
Effects of DNA Methylation of HPA-Axis Genes of F1 Juvenile Induced by Maternal Density Stress on Behavior and Immune Traits in Root Voles ()-A Field Experiment. Animals : an open access journal from MDPI\| PubMed ID: 39272253	2024
MSC-EVs and UCB-EVs promote skin wound healing and spatial transcriptome analysis. Scientific reports\| PubMed ID: 39893214	2025
Phosphatidylcholine Cytidine Transferase α (CCTα) Affects LD Formation Through Fusion and Lipophagy in Bovine Mammary Epithelial Cells. International journal of molecular sciences\| PubMed ID: 40076755	2025
Characterization of vB_SaRS_FS19-1, a newly isolated broad-host-range and virulent bacteriophage infecting Staphylococcus aureus and its application in foods. Food chemistry\| PubMed ID: 40902380	2025
Isolation and characterization of Proteus mirabilis bacteriophage T2 and its application. Microbial pathogenesis\| PubMed ID: 40930438	2025
Lipophagy modifies the catabolism of intracellular triglycerides by preferentially targeting small-sized lipid droplets in bovine mammary epithelial cells. Journal of the science of food and agriculture\| PubMed ID: 40944479	2026
Lipidomics Reveals Differences in Lipid Composition Between Lipid Droplets and Milk Fat Globules in Dairy Goat Mammary Tissue. Animals : an open access journal from MDPI\| PubMed ID: 41302011	2025
Prevalence of intestinal protozoan parasites (Giardia, Cryptosporidium, Blastocystis and Enterocytozoon) in animals from Qinghai-Tibetan Plateau area, China. Acta tropica\| PubMed ID: 41812739	2026

Yanbin Yang

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