Microwave Dielectric Response of Bovine Milk as Pregnancy Detection Tool in Dairy Cows.

The most reliable methods for pregnancy diagnosis in dairy herds include rectal palpation, ultrasound examination, and evaluation of plasma progesterone concentrations. However, these methods are expensive, labor-intensive, and invasive. Thus, there is a need to develop a practical, non-invasive, cost-effective method that can be implemented on the farm to detect pregnancy. This study suggests employing microwave dielectric spectroscopy (MDS, 0.5-40 GHz) as a method to evaluate reproduction events in dairy cows. The approach involves the integration of MDS data with information on milk solids to detect pregnancy and identify early embryonic loss in dairy cows. To test the ability to predict pregnancy according to these measurements, milk samples were collected from (i) pregnant and non-pregnant randomly selected cows, (ii) weekly from selected cows (n = 12) before insemination until a positive pregnancy test, and (iii) daily from selected cows (n = 10) prior to insemination until a positive pregnancy test. The results indicated that the dielectric strength of Δε and the relaxation time, τ, exhibited reduced variability in the case of a positive pregnancy diagnosis. Using principal component analysis (PCA), a clear distinction between pregnancy and nonpregnancy status was observed, with improved differentiation upon a higher sampling frequency. Additionally, a neural network machine learning technique was employed to develop a prediction algorithm with an accuracy of 73%. These findings demonstrate that MDS can be used to detect changes in milk upon pregnancy. The developed machine learning provides a broad classification that could be further enhanced with additional data.

Meat crust as a novel food ingredient to regulate lipid peroxidation and oxidative stress.

Pan Fry (PF) is a common heating treatment however, there is limited data on meat oxidation after PF using direct contact with an uncoated iron pan. After PF, a crust is formed, and in this study, we aim to evaluate the potential anti-oxidation and anti-lipid peroxidation capacity of such crust. Ground beef and turkey meat were heat treated using PF or microwave. Lipid peroxidation was evaluated using malondialdehyde accumulation. PF meat generated lower lipid peroxidation levels versus microwave-heated meat. Iron PF has decreased lipid peroxidation versus Teflon pan heating. The crust significantly lowered lipid peroxidation and possessed millard reaction products (MRPs), strong reducing abilities, iodine removal capacity, and some iron chelation capacity. We demonstrated that the crust substantially decreases lipid peroxidation levels in various systems and can be used as a novel seminatural antioxidant ingredient, which may lead to extended shelf life and protects various food products.

Milk fat globule size: Unraveling the intricate relationship between metabolism, homeostasis, and stress signaling.

Fat is an important component of milk which delivers energy, nutrients, and bioactive molecules from the lactating mother to the suckling neonate. Milk fat consists of a complex mixture of different types of lipids; hundreds of fatty acids, triglycerides, phospholipids, sphingolipids, cholesterol and cholesteryl ester, and glycoconjugates, secreted by the mammary gland epithelial cells (MEC) in the form of a lipid-protein assembly termed the milk fat globule (MFG). The mammary gland in general, and specifically that of modern dairy cows, faces metabolic stress once lactation commences, which changes the lipogenic capacity of MECs directly by reducing available energy and reducing factors required for both lipid synthesis and secretion or indirectly by activating a proinflammatory response. Both processes have the capacity to change the morphometric features (e.g., number and size) of the secreted MFG and its precursor-the intracellular lipid droplet (LD). The MFG size is tightly associated with its lipidome and proteome and also affects the bioavailability of milk fat and protein. Thus, MFG size has the potential to regulate the bioactivity of milk and dairy products. MFG size also plays a central role in the functional properties of milk and dairy products such as texture and stability. To understand how stress affects the structure-function of the MFG, we cover: (i) The mechanism of production and secretion of the MFG and the implications of MFG size, (ii) How the response mechanisms to stress can change the morphometric features of MFGs, and (iii) The possible consequences of such modifications.

The role of milk fat globule size in modulating the composition of postbiotics produced by Bacillus subtilis and their effect on mammary epithelial cells.

Milk lipids are secreted into the milk collecting ducts as milk fat globule (MFG) where they are exposed to microflora of the udder. We hypothesized that MFG size modulates the metabolic fingerprint of B. subtilis. Accordingly, small and large (2.3 and 7.0 µm, respectively) MFG were isolated from cow milk and used as a substrate for B. subtilis. Small MFG enhanced growth, whereas large MFG enhanced biofilm formation. Bacteria incubated with small MFG had increased concentration of metabolites related to energy production whereas metabolome of the bacteria incubated with large MFG had reduced concentrations of metabolites important for biofilm formation. Postbiotics from bacteria grown on large MFG exacerbated the proinflammatory response of MEC to LPS, and changed the expression of key enzymes involved in lipid and protein synthesis. Our results suggest that MFG size modulate growth trajectories and metabolome of B. subtilis, and consequently the stress response of host cells.

Effect of milk fat globules on growth and metabolism in rats fed an unbalanced diet.

We assessed the effects of supplementing milk fat globules (MFG) on the growth and development of the skeleton in rats fed a Western unbalanced diet (UBD). The UBD is high in sugar and fat, low in protein, fiber, and micronutrients, and negatively impacts health. The MFG-a complex lipid-protein assembly secreted into milk-has a unique structure and composition, which differs significantly from isolated and processed dietary ingredients. Rats consuming the UBD exhibited growth retardation and disrupted bone structural and mechanical parameters; these were improved by supplementation with small MFG. The addition of small MFG increased the efficiency of protein utilization for growth, and improved trabecular and cortical bone parameters. Furthermore, consumption of UBD led to a decreased concentration of saturated fatty acids and increased levels of polyunsaturated fatty acids (PUFA), particularly omega-6 PUFA, in the serum, liver, and adipose tissue. The addition of small MFG restored PUFA concentration and the ratio of omega-6 to omega-3 PUFA in bone marrow and adipose tissue. Finally, large but not small MFG supplementation affected the cecal microbiome in rats. Overall, our results suggest that natural structure MFG supplementation can improve metabolism and bone development in rats fed an UBD, with the effects depending on MFG size. Moreover, the benefits of small MFG to bone development and metabolism were not mediated by the microbiome, as the detrimental effects of an UBD on the microbiome were not mitigated by MFG supplementation.

The milk fat globule size governs a physiological switch for biofilm formation by Bacillus subtilis.

Milk lipids are organized in the form of milk fat globules (MFG), ranging in size from 0. 1 to 15 µm. The MFG size is closely associated with the composition of fatty acids, polar lipids, sphingolipids, cholesterol and the content of the MFG membrane (MFGM). Also, the MFGM integral proteins and glycoconjugates differ in composition and structure between different MFG size groups. These compositional differences may modulate the functionality of the MFG and its interaction with microbial cells. We report that small (2.3 µm) MFG facilitates the growth of the Gram-positive bacterium Bacillus subtilis whereas induction of biofilm formation was found in the presence of large (7.0 µm) MFG. Attempting to distinguish between the role played by the size from that played by the composition of the MFG, we compared phospholipid composition between treatments. We found that adjusting the phosphatidylethanolamine (PE) level to the concentration found in the small MFG, increased growth but suppressed biofilm formation in the presence of large MFG. The same normalization protocol for phosphatidylinositol (PI) or sphingomyeline (SM) did not exert a similar effect, suggesting a specific role for PE in regulating bacteria proliferation. We suggest that the content of MFGM, affected by MFG size, governs the ability of B. subtilis to utilize lipids from milk fat. This process might affect the bacterial decision-making toward biofilm formation or growth.

Association between Fatty Acid Composition, Cryotolerance and Fertility Competence of Progressively Motile Bovine Spermatozoa.

An association between progressive motility (PM) and spermatozoa fertility competence has been suggested. However, the mechanism that underlies PM is not clear enough. We examined physiological characteristics and fatty acid composition of fresh spermatozoa with high and low PM. Additional analysis of fatty acid composition and structural characteristics was performed on spermatozoa samples with high and low progressively motile spermatozoa's survival (PMSS), i.e., the ratio between the proportion of progressively motile spermatozoa after and before cryopreservation. Finally, a fertility field trial was conducted to examine the association between the number of PM spermatozoa within the insemination straw post thawing and conception rate. Analysis of fresh spermatozoa revealed a higher omega-6 to omega-3 ratio in ejaculates with low PM relative to those with high PM (p < 0.01). The proportion of polyunsaturated fatty acids was higher in low-PMSS fresh samples (p < 0.05) relative to their high-PMSS counterparts. Fresh samples with high-PMSS expressed a higher mitochondrial membrane potential (p < 0.05) and a higher proportion of viable cells that expressed reactive oxygen species (ROS; p < 0.05). Post-thawing evaluation revealed a reduced proportion of progressively motile sperm, with a prominent effect in samples with high PM relative to low PM, defined before freezing (p < 0.01). No differences in spermatozoa mitochondrial membrane potential or ROS level were found post-thawing. A fertility study revealed a positive correlation between the number of progressively motile spermatozoa within a standard insemination straw and conception rate (p < 0.05). Considering these, the bull PMSS is suggested to be taken into account at the time of straw preparation.

Direct effects of phenolic compounds on the mammary gland: In vivo and ex vivo evidence.

We assessed the potential of Pistacia lentiscus (lentisk) phenolic compounds to enhance production of milk composition in lactating goats and caprine primary mammary epithelial cells (MEC). Damascus goats were given a lentisk infusion (LI) or fresh water (FW) to drink, in a crossover design. Milk from LI vs. FW goats was 0.43% richer in fat and 30% in omega 3 fatty acids. Lentisk infusion enhanced antioxidant capacity of plasma and milk by 37% and 30% respectively, and induced transcriptional activation of antioxidant genes. To assess the direct effect of polyphenols on milk quality in terms of composition and antioxidant capacity, we used plasma collected from goats fed hay (HP) or browsed on phenolic compounds-rich pasture (primarily lentisk; PP) as a conditioning medium for primary culture of MEC. PP increased 2-fold cellular triglyceride content and 2.4-fold intracellular casein, and increased ATP production and non-mitochondrial oxygen consumption. Taken together, the results imply that lentisk phenolic compounds affect blood, MEC and milk oxidative status, which increase fat production by the mammary gland.

Lipidome changes, with a focus on phospholipids, due to feeding systems and processing in goat milk.

We evaluated the effects of processing - pasteurization and yoghurt manufacturing - on some health-promoting lipidome components in milk from two feeding treatments - brushland grazing or hay-feeding in confinement - in dairy goats. The contents of fat and protein were higher, and of urea, lower, in grazing goats. Fatty acid composition - at the exception of saturated fatty acids - was affected by dietary management and milk processing. Phospholipid contents was lower in confined goats, with little effect for processing. The phospholipid-to-triglyceride ratio was decreased by pasteurization. Sensitivity to pasteurization of phospholipid composition differed between feeding treatments. The percentage of sphingomyelin increased following pasteurization, with no response for fermentation to yoghurt. These results can be exploited to modulate health-promoting fats in dairy products.

The Role of Omega-3 Polyunsaturated Fatty Acids from Different Sources in Bone Development.

N-3 polyunsaturated fatty acids (PUFAs) are essential nutrients that must be obtained from the diet. We have previously showed that endogenous n-3 PUFAs contribute to skeletal development and bone quality in fat-1 mice. Unlike other mammals, these transgenic mice, carry the n-3 desaturase gene and thus can convert n-6 to n-3 PUFAs endogenously. Since this model does not mimic dietary exposure to n-3 PUFAs, diets rich in fish and flaxseed oils were used to further elucidate the role of n-3 PUFAs in bone development. Our investigation reveals that dietary n-3 PUFAs decrease fat accumulation in the liver, lower serum fat levels, and alter fatty acid (FA) content in liver and serum. Bone analyses show that n-3 PUFAs improve mechanical properties, which were measured using a three-point bending test, but exert complex effects on bone structure that vary according to its source. In a micro-CT analysis, we found that the flaxseed oil diet improves trabecular bone micro-architecture, whereas the fish oil diet promotes higher bone mineral density (BMD) with no effect on trabecular bone. The transcriptome characterization of bone by RNA-seq identified regulatory mechanisms of n-3 PUFAs via modulation of the cell cycle and peripheral circadian rhythm genes. These results extend our knowledge and provide insights into the molecular mechanisms of bone remodeling regulation induced by different sources of dietary n-3 PUFAs.

Progesterone Regulation of Milk Fat Globule Size Is VLDL Dependent.

Progesterone plays a pivotal role during mammogenesis and serves as an inhibitor of the secretory activation of mammary cells in the last days of gestation. However, its role during lactogenesis, in particular its involvement in lipid metabolism, and milk fat content and composition, is unknown. Here, we provide new evidence of progesterone's involvement in the regulation of milk fat globule (MFG) synthesis and secretion. Findings from both in vivo and in vitro studies indicated that the concentration and the direction (increase vs. decrease) of progesterone concentration to which the mammary epithelial cells (MECs) are exposed affect MFG size. This was found to be very-low-density lipoprotein (VLDL) dependent: in the presence of VLDL, the proportion of MEC with small lipid droplets (<1 µm) increased 2.4-fold, and the proportion of large lipid droplets (>1 µm) increased 4-fold; in the absence of VLDL, no differences were found. The findings add to our understanding of the mechanism underlying the regulation of MFG size and provide new evidence for progesterone's role in lipid metabolism in the mammary gland during lactogenesis. The fact that the size, synthesis, and composition of MFG are affected by the cyclic pattern of progesterone concentration in the circulation might have physiologically relevant consequences, in particular on milk as a nutritional source.

Dietary pomegranate peel improves milk quality of lactating ewes: Emphasis on milk fat globule membrane properties and antioxidative traits.

Concentrated pomegranate peel extract (CPE) was supplemented to ewes, and milk yield and fat content-fatty acid (FA) and phospholipid (PL) composition-were monitored. CPE-fed ewes had higher milk yield, and fat, protein and lactose contents than controls. Milk PL content-20% higher in the CPE-supplemented group-was regulated by treatment and not by total fat content; milk phosphatidylethanolamine and phosphatidylcholine increased by 22 and 26%, respectively, in CPE-supplemented vs. control ewes. Milk saturated FA concentration was higher, and total polyunsaturated and monounsaturated FA content lower in the CPE vs. control group, regardless of milk total fat content. CPE supplementation increased milk antioxidant capacity, suggesting antioxidant transfer from dietary source to milk, increasing stability and nutritive value. Our study provides first evidence for milk quality improvement in terms of antioxidants and PL enrichment without compromising total milk fat, suggesting strategies to improve dairy animals' milk composition without compromising total production.

Atrazine-induced toxicity in goat spermatozoa is alleviated to some extent by polyphenol-enriched feed.

Atrazine (ATZ) is one of the most extensively used herbicides to control growth of broadleaf and grassy weeds in crops. ATZ and its metabolites have deleterious effect on sperm quality. ATZ is also known for its ability to induce oxidative stress. Pistacia lentiscus (PL) is an evergreen shrub, with a high content of polyphenols in leaf extracts, with a known anti-inflammatory and antioxidant properties. The protective effect of PL or its extracts against ATZ-induced damage have not been yet evaluated. We examined the harmful effects of atrazine (ATZ) exposure on male reproductive system, using goat (Capra hircus) model spermatozoa and the protective effects of PL and PL ethanolic extract (PLE). In in-vivo experiments, male goats were fed a standard ration or one supplemented with 15 mg ATZ/kg body weight daily, for 6 months. Exposure to ATZ impaired the spermatozoa's morphology, viability, mitochondrial membrane potential and cell lipid composition. These alterations may in turn lead to reduced fertilization competence of the exposed spermatozoa. In an ex-vivo experiment, spermatozoa from male goats fed a standard ration or one supplemented with PL or PLE for 90 days and then were exposed to 1 µM ATZ or 10 µM of its major metabolite diaminochlorotriazine (DACT) through in-vitro capacitation. Prefeeding with PL or PLE partially attenuated the harmful effects of ATZ and DACT. Dietary supplementation with polyphenol-enriched feed can protect, to a certain extent, spermatozoa in males exposed to environmental toxicants.

Milk Lipids: A Complex Nutrient Delivery System.

The evolution of lactation and the composition, structures, and functions of milk's biopolymers illustrates that the Darwinian pressure on lactation selected for gene products with considerable structural complexity and diverse functions within the digestive system. For example, complex sugar polymers - oligosaccharides - possess unique properties in guiding the growth of intestinal bacteria that are not possible by feeding their simple sugars. The proteins of milk are diverse with some exhibiting enzymatic activities towards other milk components rendering those components both more digestible but also releasing biologically active products. Thus, research into milk's biopolymers has been most enlightening when milk was investigated for the formation and disassembly of its structures and for the functions within the infant. To date however, the most complex structure in mammalian milk, the fat globule, has not been effectively examined beyond its simple composition. The globules of milk are heterogeneous in size, composition, and function. With new research tools, scientists are beginning to understand the mechanisms that control the assembly of globules in the mammary gland and the disassembly within the infant.

Symposium review: Milk fat globule size: Practical implications and metabolic regulation.

Milk fat globule (MFG) size ranges over 3 orders of magnitude, from less than 200 nm to over 15 µm. The significance of MFG size derives from its tight association with its lipidome and proteome. More specifically, small MFG have relatively higher content of membrane compared with large globules, and this membrane exerts diverse positive health effects, as reported in human and animal studies. In addition, MFG size has industrial significance, as it affects the physicochemical and sensory characteristics of dairy products. Studies on the size regulation of MFG are scarce, mainly because various confounders indirectly affect MFG size. Because MFG size is determined before and during its secretion from mammary epithelial cells, studies on the size regulation of its precursors, the intracellular lipid droplets (LD), have been used as a proxy for understanding the mechanisms controlling MFG size. In this review, we provide evidence for 2 distinct mechanisms regulating LD size in mammary epithelial cells: co-regulation of fat content and triglyceride-synthesis capacity of the cells, and fusion between LD. The latter is controlled by the membrane's polar lipid composition and involves mitochondrial enzymes. Accordingly, this review also discusses MFG size regulation in the in vivo metabolic context, as MFG morphometric features are often modulated under conditions that involve animals' altered energy status.

Changes in lipid droplets morphometric features in mammary epithelial cells upon exposure to non-esterified free fatty acids compared with VLDL.

The effects of the macrostructure of long chain fatty acids on the lipid metabolism and biosynthesis of lipid droplets (LD) was studied in mammary epithelial cells (MEC). MEC were exposed to similar compositions and concentrations of fatty acids in the form of either triglycerides (Tg), as part of the very-low-density lipids (VLDL) isolated from lactating cow plasma, or as non-esterified- free fatty acids (FFA). Exposing MEC to FFA resulted in two distinct processes; each independently could increase LD size: an elevation in Tg production and alterations in phospholipid (PL) composition. In particular, the lower PC/PE ratio in the FFA treatment indicated membrane destabilization, which was concomitant with the biosynthesis of larger LD. In addition, 6 fold increase in the cellular concentration of the exogenously added linoleic acid (C18:2) was found in MEC treated with FFA, implying that long chain fatty acids administrated as FFA have higher availability to MEC, enabling greater PL synthesis, more material for the LD envelope, thereby enhancing LD formation. Availability of long chain fatty acids administrated as VLDL-Tg, is dependent on LPL which its activity can be inhibited by the hydrolysis products. Therefore, we used increasing concentrations of albumin, to reduce the allosteric inhibition on LPL by the hydrolysis products. Indeed, a combined treatment of VLDL and albumin, increased LD size and number, similar to the phenotype found in the FFA treatment. These results reveal the role played by the macrostructure of long chain fatty acids in the regulation of LD size in MEC which determine the size of the secreted MFG.

Plasma Lipoprotein Particle Subclasses in Preterm Infants.

OBJECTIVE: A pilot study to determine lipoprotein classes and subclasses in premature infants and examine associations with nutritional intake, gestational age (GA), and morbidity. STUDY DESIGN: Plasma lipoprotein particle concentrations were analyzed in a cohort of 15 premature infants in the first 5 days of life and again at 2 weeks. Breast milk samples were analyzed for fatty acid content. Associations between lipoprotein particle subclasses and GA, breast milk intake, milk fatty acid intake, and chronic lung disease (CLD) were determined. RESULTS: At 2 weeks of age, more premature infants had higher concentrations of total very low-density lipoprotein and lower concentrations of total high-density lipoprotein (HDL) and large HDL particles (similar to profiles seen in adults and children with infectious disease, cardiometabolic disease, and diabetes). Lower total HDL, large HDL, and medium HDL and a higher small HDL:total HDL ratio at 2 weeks were each associated with CLD with GA a likely confounder. Intake of human milk C18 and C20 fatty acids was inversely correlated with plasma total LDL concentration at 2 weeks of age. CONCLUSION: Dyslipidemia was common in extremely premature infants and was associated with CLD and with lower intake of specific long chain fatty acids.

Lipid Droplet Fusion in Mammary Epithelial Cells is Regulated by Phosphatidylethanolamine Metabolism.

Mammary epithelial cells (MEC) secrete fat in the form of milk fat globules (MFG) which are found in milk in diverse sizes. MFG originate from intracellular lipid droplets, and the mechanism underlying their size regulation is still elusive. Two main mechanisms have been suggested to control lipid droplet size. The first is a well-documented pathway, which involves regulation of cellular triglyceride content. The second is the fusion pathway, which is less-documented, especially in mammalian cells, and its importance in the regulation of droplet size is still unclear. Using biochemical and molecular inhibitors, we provide evidence that in MEC, lipid droplet size is determined by fusion, independent of cellular triglyceride content. The extent of fusion is determined by the cell membrane's phospholipid composition. In particular, increasing phosphatidylethanolamine (PE) content enhances fusion between lipid droplets and hence increases lipid droplet size. We further identified the underlying biochemical mechanism that controls this content as the mitochondrial enzyme phosphatidylserine decarboxylase; siRNA knockdown of this enzyme reduced the number of large lipid droplets threefold. Further, inhibition of phosphatidylserine transfer to the mitochondria, where its conversion to PE occurs, diminished the large lipid droplet phenotype in these cells. These results reveal, for the first time to our knowledge in mammalian cells and specifically in mammary epithelium, the missing biochemical link between the metabolism of cellular complex lipids and lipid-droplet fusion, which ultimately defines lipid droplet size.

Influence of gestational diabetes mellitus on fatty acid concentrations in human colostrum.

OBJECTIVE: The aim of this study was to examine differences in fatty acid concentrations in colostrum of women with and without gestational diabetes mellitus (GDM). The effect of GDM on fatty acid composition of colostrum is not fully understood, although rates of GDM are increasing globally. METHODS: A prospective case-control study was conducted of postpartum women with and without GDM. Gas chromatographic analysis was conducted to examine differences in colostral fatty acids of the colostrum samples of 29 women with and 34 without GDM. RESULTS: Analyses of the fatty acid composition revealed significantly higher concentrations of four essential ω-6 polyunsaturated fatty acids-γ-linolenic, eicosatrienoic, arachidonic, and docosatetraenoic-in the colostrum of GDM women compared with non-GDM women. Timing of collection influenced saturated medium chain fatty acid and monounsaturated fatty acid levels. CONCLUSIONS: Differences in concentrations of ω-6 fatty acids but not in dietary linoleic fatty acid or ω-3 fatty acids suggest that altered concentrations are attributed to changes in specific endogenous metabolic pathways. Implications of higher concentrations of ω-6 fatty acids in the colostrum of women with GDM have yet to be determined. Timing of colostrum collection is critical in determining colostral fatty acid and metabolite concentrations.

Human milk fatty acids composition is affected by maternal age.

Human colostrums and transition milk were collected from women under the age of 37 years and women aged 37 years and older. Transition milk of the younger group had lower fat content and 10-fold higher concentrations of omega 6 FA, eicosadecanoic, and arachdonic acids. Gestational age affected the colostrum concentration of total fat and omega 3 and omega 6 FA composition only in the older group. We concluded that age may be a factor in the FA composition of human milk. This should be taken into account when planning diets for pregnant women of different ages.