Mining key circRNA-associated-ceRNA networks for milk fat metabolism in cows with varying milk fat percentages.

BACKGROUND: Cow milk fat is an essential indicator for evaluating and measuring milk quality and cow performance. Growing research has identified the molecular functions of circular RNAs (circRNAs) necessary for mammary gland development and lactation in mammals. METHOD: The present study analyzed circRNA expression profiling data in mammary epithelial cells (MECs) from cows with highly variable milk fat percentage (MFP) using differential expression analysis and weighted gene co-expression network analysis (WGCNA). RESULTS: A total of 309 differentially expressed circRNAs (DE-circRNAs) were identified in the high and low MFP groups. WGCNA analysis revealed that the pink module was significantly associated with MFP (r = - 0.85, P = 0.007). Parental genes of circRNAs in this module were enriched mainly in lipid metabolism-related signaling pathways, such as focal adhesion, ECM-receptor interaction, adherens junction and AMPK. Finally, six DE-circRNAs were screened from the pink module: circ_0010571, circ_0007797, circ_0002746, circ_0003052, circ_0004319, and circ_0012840. Among them, circ_0002746, circ_0003052, circ_0004319, and circ_0012840 had circular structures and were highly expressed in mammary tissues. Subcellular localization revealed that these four DE-circRNAs may play a regulatory role in the mammary glands of dairy cows, mainly as competitive endogenous RNAs (ceRNAs). Seven hub target genes (GNB1, GNG2, PLCB1, PLCG1, ATP6V0C, NDUFS4, and PIGH) were obtained by constructing the regulatory network of their ceRNAs and then analyzed by CytoHubba and MCODE plugins in Cytoscape. Functional enrichment analysis revealed that these genes are crucial and most probable ceRNA regulators in milk fat metabolism. CONCLUSIONS: Our study identified several vital circRNAs and ceRNAs affecting milk fat synthesis, providing new research ideas and a theoretical basis for cow lactation, milk quality, and breed improvement.

Comprehensive review of milk fat globule membrane proteins across mammals and lactation periods in health and disease.

Milk fat globule membrane (MFGM) is a three-layer membrane-like structure encasing natural milk fat globules (MFGs). MFGM holds promise as a nutritional supplement because of the numerous physiological functions of its constituent protein. This review summarizes and compares the differences in MFGM protein composition across various species, including bovines, goats, camels, mares, and donkeys, and different lactation periods, such as colostrum and mature milk, as assessed by techniques such as proteomics and mass spectrometry. We also discuss the health benefits of MFGM proteins throughout life. MFGM proteins promote intestinal development, neurodevelopment, and glucose and lipid metabolism by upregulating tight junction protein expression, brain function-related genes, and glucose and fatty acid biosynthesis processes. We focus on the mechanisms underlying these beneficial effects of MFGM proteins. MFGM proteins activate key substances in in signaling pathways, such as the phosphatidylinositol 3-kinase/protein kinase B, mitogen-activated protein kinase, and myosin light chain kinase signaling pathways. Overall, the consumption of MFGM proteins plays an essential role in conferring health benefits, some of which are important throughout the mammalian life cycle.

Types and amounts of MFGM proteins in mammals, as assessed by proteomic and mass spectrometry analysis, are summarized.Colostrum MFGM contains more acute phase proteins, whereas mature milk has higher levels of mucins (1 and 15), ADPH, XDH, and FABP.Health benefits of MFGM proteins, including intestinal development, neurodevelopment, and immune activity enhancement, are summarized.MFGM proteins have been shown to significantly activate the PI3K/Akt/mTOR signaling pathway, promoting cell proliferation and glycolipid metabolism.

Identification and anti-oxidative potential of milk fat globule membrane (MFGM)-derived bioactive peptides released through in vitro gastrointestinal digestion.

This study investigated the stability of milk fat globule membrane (MFGM) protein under simulated gastrointestinal conditions using an in vitro enzymatic digestion method. The optimal hydrolysis conditions were determined by monitoring the changes in particle size and zeta-potential of MFGM protein hydrolysates over time. Furthermore, the distribution of small molecular weight peptides with antioxidant activity was explored through DEAE-52 combined with in vitro cell experiments. Two novel antioxidant peptides (TGIIT and IITQ) were identified based on molecular docking technology and evaluated their potential scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS+) radicals. TGIIT and IITQ also demonstrated remarkable abilities in promoting mitochondrial biogenesis and activating Keap1/Nrf2 signaling pathway, which can effectively counteract skeletal muscle dysfunction induced by oxidative stress. Thus, MFGM-derived antioxidant peptides have the potential to be employed in food to regulate muscle protein metabolism and alleviate sarcopenia.

Human milk-specific fat components enhance the secretion of ghrelin by MGN3-1 cells.

Triacylglycerols (TAGs) are a major fat component in human milk. Since gastric lipase produces 1,2-diacylglycerol from TAGs, we focused on the bioactivity of human milk-derived diacylglycerols in stomach cells. Ghrelin is produced in the stomach and acts as an important regulator of growth hormone secretion and energy homeostasis. In this study, we showed that 1-oleoyl-2-palmitoylglycerol (OP) increased ghrelin secretion, whereas 1,3-dioleoyl-2-palmitoylglycerol (OPO), a major component of human milk TAGs, did not increase ghrelin secretion in the ghrelin-secreting cell line, MGN3-1. Therefore, diacylglycerol OP may directly contribute to the regulation of ghrelin secretion. We also found that 2-palmitoylglycerol and 1- and 2-oleoylglycerol increased ghrelin secretion. Finally, we demonstrated that intracellular cAMP levels and preproghrelin and ghrelin O-acyl transferase expression levels were enhanced by OP treatment in MGN3-1 cells. This may represent an example of a novel mother-infant interaction mediated by fat components derived from human breast milk.

Milk fat globule membranes for psychological and physical health: qualitative results from the Employing Milk Phospholipids to Observe Well-being and Emotional Resilience (EMPOWER) randomised trial.

BACKGROUND: Milk fat globule membranes (MFGM) present a nutritional intervention with the potential to improve psychological well-being and mitigate the negative effects of stress on health. The present study aimed to investigate participant's experience of different aspects of health during a trial of MFGM supplementation and determine the effect of MFGM on qualitative measures of psychological and physical well-being. METHODS: Seventy-three adults in New Zealand who were enrolled in a clinical trial to test MFGM supplementation for improvement of psychological well-being took part in a post-intervention interview. Participants and researchers remained blinded to intervention group allocation. Interviews were conducted over the video conferencing platform Zoom and transcribed. A mixed methods analytical approach included thematic analysis to identify emerging themes and χ2 regression models to examine frequency of improvements in different aspects of well-being between the MFGM and placebo groups. RESULTS: There were no significant demographic or psychological differences between interviewees and non-interviewed study participants. Four central themes emerged from the data for all participants: improved well-being, increased ability to cope with stress and improvements in mood, improvement in physical energy or activity, and improved sleep. The frequency of improved ability to cope with stress and improved sleep quality was significantly higher in participants who received MFGM supplementation compared to those receiving the placebo. CONCLUSIONS: Qualitative data may capture aspects of improved sleep or psychological well-being not measured by rating scales. The results suggest that MFGM supplementation may improve the ability to cope with stress and improve sleep quality in healthy adults.

Effects of nanoselenium supplementation on lactation performance, nutrient digestion and mammary gland development in dairy cows.

The objective of this experiment was to evaluate the influence of nanoselenium (NANO-Se) addition on milk production, milk fatty acid synthesis, the development and metabolism regulation of mammary gland in dairy cows. Forty-eight Holstein dairy cows averaging 720 ± 16.8 kg of body weight, 66.9 ± 3.84 d in milk (dry matter intake [DIM]) and 35.2 ± 1.66 kg/d of milk production were divided into four treatments blocked by DIM and milk yields. Treatments were control group, low-Se (LSe), medium-Se (MSe) and high-Se (HSe) with 0, 0.1, 0.2 and 0.3 mg Se, respectively, from NANO-Se per kg dietary dry matter (DM). Production of energy- and fat-corrected milk (FCM) and milk fat quadratically increased (p < 0.05), while milk lactose yields linearly increased (p < 0.05) with increasing NANO-Se addition. The proportion of saturated fatty acids (SFAs) linearly decreased (p < 0.05), while proportions of monounsaturated fatty acids (MUFAs) linearly increased and polyunsaturated fatty acids (PUFAs) quadratically increased. The digestibility of dietary DM, organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) quadratically increased (p < 0.05). Ruminal pH quadratically decreased (p < 0.01), while total VFA linearly increased (p < 0.05) with increasing NANO-Se addition. The acetic to propionic ratio decreased (p < 0.05) linearly due to the unaltered acetic molar percentage and a quadratical increase in propionic molar percentage. The activity of CMCase, xylanase, cellobiase and pectinase increased linearly (p < 0.05) following NANO-Se addition. The activity of α-amylase increased linearly (p < 0.01) with an increase in NANO-Se dosage. Blood glucose, total protein, estradiol, prolactin, IGF-1 and Se linearly increased (p < 0.05), while urea nitrogen concentration quadratically decreased (p = 0.04). Moreover, the addition of Se at 0.3 mg/kg from NANO-Se promoted (p < 0.05) mRNA and protein expression of PPARγ, SREBP1, ACACA, FASN, SCD, CCNA2, CCND1, PCNA, Bcl-2 and the ratios of p-ACACA/ACACA and BCL2/BAX4, but decreased (p < 0.05) mRNA and protein expressions of Bax, Caspase-3 and Caspase-9. The results suggest that milk production and milk fat synthesis increased by NANO-Se addition by stimulating rumen fermentation, nutrients digestion, gene and protein expressions concerned with milk fat synthesis and mammary gland development.

The impact of genetic variants related to the fatty acid metabolic process pathway on milk production traits in Jersey cows.

The synthesis of fatty acids plays a critical role in shaping milk production characteristics in dairy cattle. Thus, identifying effective haplotypes within the fatty acid metabolism pathway will provide novel and robust insights into the genetics of dairy cattle. This study aimed to comprehensively examine the individual and combined impacts of fundamental genes within the fatty acid metabolic process pathway in Jersey cows. A comprehensive phenotypic dataset was compiled, considering milk production traits, to summarize a cow's productivity across three lactations. Genotyping was conducted through PCR-RFLP and Sanger sequencing, while the association between genotype and phenotype was quantified using linear mixed models. Moderate biodiversity and abundant variation suitable for haplotype analysis were observed across all examined markers. The individual effects of the FABP3, LTF and ANXA9 genes significantly influenced both milk yield and milk fat production. Additionally, this study reveals novel two-way interactions between genes in the fatty acid metabolism pathway that directly affect milk fat properties. Notably, we identified that the GGAAGG haplotype in FABP3×LTF×ANXA9 interaction may be a robust genetic marker concerning both milk fat yield and percentage. Consequently, the genotype combinations highlighted in this study serve as novel and efficient markers for assessing the fat content in cow's milk.

Effect of monensin on milk production efficiency and milk composition in lactating dairy cows fed modern diets.

Since the US Food and Drug Administration's approval of monensin in 2004, significant nutritional advances have been made to increase feed efficiency and milk fat production. Recent evidence suggests monensin's adverse effect on milk fat percentage may be absent when diets are formulated to address known diet-induced milk fat depression risk factors. Thus, study objectives were to evaluate effects of monensin level on dry matter intake (DMI), milk production and composition, and efficiency of high-producing cows fed diets formulated to optimize milk fat. Ninety-six lactating Holstein cows (36 primiparous, 60 multiparous; 106 ± 17 d in milk [DIM]) were balanced by parity, DIM, and milk production and were randomly assigned to 1 of 12 pens with 8 cows per pen. All cows received 11 g/t monensin for 5 wk after which pens received 1 of 4 dietary treatments (n = 3) formulated to provide 0 (CON), 11 (R11), 14.5 (R14.5), or 18 (R18) g/t monensin for 9 wk. The basal diet was 54% forage, 27% NDF, 29% starch, and 2.3% rumen unsaturated fatty acid load. Pen was the experimental unit and data were analyzed using the Fit Model Procedure of JMP. Effects of treatment, time, and treatment × time interaction were included as fixed effects and pen as a random effect. Least squares means were determined and linear and quadratic contrasts were tested. Dry matter intake tended to decrease linearly with increasing monensin dose. Milk yield, fat percentage, and protein percentage and yield were unaffected by treatment while fat yield was quadratically increased. Milk de novo and mixed fatty acid (FA) yields (g/d) increased quadratically with monensin whereas preformed FA linearly decreased during the experimental period. Energy-corrected milk (ECM) was quadratically increased by monensin. Milk urea nitrogen concentrations increased linearly with increasing monensin dose. Monensin linearly increased feed efficiency (ECM/DMI, 3.5% fat-corrected milk/DMI, and solids-corrected milk/DMI). Body weight gain did not differ between treatments. Estimated dietary energy tended to increase linearly with increasing monensin level. These data suggest monensin improves component-corrected milk production efficiency, estimated dietary energy, and does not negatively affect milk fat percentage or FA profile.

Effects of abomasal infusion of soybean or sunflower phospholipids on nutrient digestibility and milk production in lactating dairy cows.

The fatty acid (FA) and phospholipid composition of dietary lecithin may influence FA digestibility and milk production in cattle. Eight multiparous Holstein cows (99.4 ± 9.2 d in milk [DIM]; 48.9 ± 3.8 kg milk/d) were enrolled in a 3 × 3 incomplete Latin square design with 3 treatments provided as continuous abomasal infusates spanning 14-d experimental periods: water (CON), soybean phospholipids (SOY; 74.5 g of deoiled soy lecithin), or sunflower phospholipids (SUN; 133.5 g of hydrolyzed sunflower lecithin). Cows were fed the same diet, which contained (% dry matter) 27.0% neutral detergent fiber (NDF), 15.6% crude protein (CP), 26.2% starch, and 5.87% FA. Treatments did not modify body weight, milk fat, protein, or lactose contents, or the efficiency of producing energy-corrected milk. Cows infused with SUN had greater milk yields than those receiving SOY or CON treatments. Cows infused with SUN had higher total solids, protein, and lactose yields than cows receiving the SOY or CON treatments. Sunflower phospholipids enhanced feed efficiency (milk yield/dry matter intake) relative to SOY or CON. Treatment did not affect intakes or apparent total-tract digestibilities for NDF, CP, starch, or 16-carbon (16C) FA. Cows receiving SUN had greater total FA and 18-carbon (18C) FA intakes than SOY or CON, but treatments did not impact their digestibility. Milk FA composition was modified by treatment. Cows receiving SUN had a greater concentration of polyunsaturated FA and lower concentrations of saturated FA and monounsaturated FA in milk relative to SOY or CON. In conclusion, the abomasal infusion of SUN improved milk production and milk FA composition, indicating potential benefits for dairy cow nutrition and milk quality.

Effect of increasing dietary inclusion of whole cottonseed on nutrient digestibility and milk production of high-producing dairy cows.

We determined the effects of increasing the dietary inclusion of whole cottonseed (WCS) on nutrient digestibility and the milk production responses of high-producing dairy cows. Twenty-four multiparous Holstein cows (mean ± SD; 52.7 ± 2.63 kg/d milk; 104 ± 23 DIM) were randomly assigned to treatment sequences in a replicated 4 × 4 Latin square design with 21-d periods. Treatments were increasing doses of WCS at 0%, 8%, 16%, and 24% DM, with WCS replacing soybean meal and hulls to maintain similar diet nutrient composition (% DM) of NDF (32%), forage NDF (21%), starch (27%), and CP (17%). Total fatty acid (FA) content of each treatment was 1.70%, 2.96%, 4.20%, and 5.40% DM, respectively. Three preplanned contrasts were used to test the linear, quadratic, and cubic effects of increasing dietary WCS. Increasing dietary WCS from 0% to 24% DM quadratically influenced the intake of DM and NDF, with the highest value being the inclusion of 8% WCS, and intakes of 16- and 18-carbon, and total FA, with maximum values obtained up to 24% WCS. Increasing dietary WCS affected digestibility of DM (cubic) and NDF (quadratic), with the lowest values being the inclusion of 8% WCS. Increasing WCS increased 16-carbon digestibility (quadratic) but decreased digestibility of 18-carbon and total FA (both quadratic), with highest and lowest values for the inclusion of 24% WCS, respectively. Increasing dietary WCS quadratically increased absorbed 16- and 18-carbon, and total FA, with maximum values obtained for 24% WCS. Increasing dietary WCS quadratically increased yields of milk, milk fat, milk protein, milk lactose, 3.5% FCM, and ECM, and linearly increased BW gain. The source of milk FA was affected by dietary WCS, with a quadratic decrease in the yield of de novo and mixed milk FA and a quadratic increase in preformed milk FA. Increasing dietary WCS linearly increased trans-10 C18:1 milk FA content. As dietary WCS increased, plasma insulin linearly decreased, and plasma gossypol levels linearly increased. Despite the decrease in total FA digestibility, increasing dietary WCS from 0% to 24% DM increased FA absorption. Increasing the dietary inclusion of WCS up to 16% DM increased milk production responses and DM intake. Under the current dietary conditions, high-producing dairy cows benefited best from a diet containing an inclusion of 8% to 16% WCS in DM.

Mammary gland responses to altering the supply of de novo fatty acid substrates and preformed fatty acids on the yields of milk components and milk fatty acids.

The objective of our study was to evaluate the effect of altering the dietary supply of acetate, palmitic acid (PA), and cottonseed on the yields of milk components and milk fatty acids (FA) in lactating dairy cows. Thirty-two multiparous Holstein cows (133 ± 57 d in milk, 50.5 ± 7.2 kg/d milk) were used in a 4 × 4 Latin square split plot design with a 2 × 2 factorial arrangement of subplot treatments. Cows were blocked by energy-corrected milk (ECM) yield and allocated to a main plot receiving a basal diet (n = 16) with no supplemental PA (Low PA) or a basal diet (n = 16) with 1.5% inclusion of a FA supplement containing ~85% PA (High PA). In each main plot, the following subplots of treatment diets were fed in a Latin square arrangement consisting of 14-d periods: 1) a control diet (CON), 2) the control diet supplemented with 3% sodium acetate (AC), 3) the control diet supplemented with 12% whole cottonseed (CS), and 4) the control diet supplemented with 3% sodium acetate and 12% whole cottonseed (CS+AC). The PA supplement and sodium acetate replaced soyhulls, and whole cottonseed replaced cottonseed hulls and meal. All diets were balanced for 30% neutral detergent fiber (NDF), 23% forage NDF, 28% starch, and 17% crude protein (CP). Sources of FA were classified as de novo (<16 carbons), mixed (16-carbon), and preformed (>16 carbons). The statistical model included the random effect of cow nested within basal diet and fixed effect of period, basal diet, acetate, cottonseed, and their interactions. Three-way interactions among basal diet, acetate, and cottonseed were observed for the yields of milk fat, 3.5% fat-corrected milk (FCM), and the molar yields of de novo FA, mixed FA, and preformed FA. In the Low PA diets, AC and CS+AC increased the yields of milk fat and FCM compared with CON and CS, whereas, in the High PA diets, CS+AC increased the yields of milk fat and FCM compared with the other treatments and AC increased milk fat yield compared with CON and CS. Compared with Low PA, High PA increased milk fat content, mixed FA yield, and tended to increase C4:0 yield. Diets containing acetate increased DMI and the yields of milk fat, ECM, FCM, de novo FA, mixed FA, and preformed FA compared with diets without acetate. Diets containing cottonseed increased the yields of milk and preformed FA, tended to increase the yields of FCM and protein, and decreased DMI and the yields of de novo FA and mixed FA compared with diets without cottonseed. In summary, in high PA diets, the inclusion of acetate plus cottonseed increased milk fat yield compared with the other treatments. The CON diet in High PA increased milk fat yield to the same extent as AC and CS+AC in Low PA suggesting PA is important for initiating milk TG synthesis. Balancing the supply of de novo FA substrates and preformed FA is important for increasing the synthesis of milk fat triglycerides and milk fat production.

A study of the effect of hypothyroidism during pregnancy on human milk quality based on rheological properties.

Hypothyroidism has been found to have an effect on the nutritional composition of human milk during pregnancy. This study aims to explore the combined influence of rheological properties, macronutrient content, particle size, and the zeta potential of milk fat globules, as well as the composition of milk fat globule membrane (MFGM) proteins on the quality of human milk in gestational hypothyroidism. The study revealed that human milk from the group with hypothyroidism during pregnancy (AHM) was less viscoelastic and stable when compared with normal pregnancy group human milk (NHM). Furthermore, the particle size and macronutrient content of NHM were found to be larger than that of AHM. In contrast, the zeta potential of AHM was greater than that of NHM. The sodium dodecyl sulfate-PAGE results disclosed that the composition of MFGM proteins in these 2 groups were generally the same, but the content of AHM was lower than that of NHM. In conclusion, this study confirms that hypothyroidism during pregnancy can have a significant effect on the quality of human milk.

Increasing palmitic acid and reducing stearic acid content of supplemental fatty acid blends improves production performance of mid-lactation dairy cows.

We determined the effects of altering the ratio of palmitic (C16:0) and stearic (C18:0) acids in supplemental fatty acid (FA) blends on production responses of mid-lactation dairy cows. Twenty-four multiparous Holstein cows (mean ± standard deviation; 47.1 ± 5.8 kg of milk yield, 109 ± 23 DIM) were randomly assigned to treatment sequences in a replicated 4 × 4 Latin square design with 21-d periods. Treatments were a control diet not supplemented with FA (CON), and 3 diets incorporating 1.5% of dry matter (DM) FA supplement blends containing 30% C16:0 + 50% C18:0, 50% C16:0 + 30% C18:0, and 80% C16:0 + 10% C18:0. Additionally, the FA blends were balanced to contain 10% of oleic acid (cis-9 C18:1). The FA blends replaced soyhulls in the CON diet. Diets were formulated to contain (% of DM) 31.0% neutral detergent fiber, 27.0% starch, and 16.9% crude protein. The statistical model included the random effect of cow within square and the fixed effects of period, treatment, and their interaction. Preplanned contrasts included CON versus overall effect of FA supplementation and the linear and quadratic effects of increasing C16:0 in FA blends. Overall FA treatment had no effect on dry matter intake (DMI), but increasing C16:0 linearly increased DMI. Compared with CON, overall FA treatment increased yields of milk, 3.5% of fat-corrected milk, energy-corrected milk, and milk fat but did not affect milk protein yield. Increasing C16:0 linearly increased milk fat yield and tended to linearly increase the yields of 3.5% of fat-corrected milk and energy-corrected milk. Fatty acid supplementation decreased the yield of de novo milk FA but increased yields of mixed and preformed milk FA compared with CON. Increasing C16:0 in FA treatments did not affect the yield of de novo milk FA, linearly increased the yield of mixed, and decreased the yield of preformed milk FA. In summary, feeding FA supplements containing C16:0 and C18:0 increased milk production responses with no effect on DMI compared with a control diet. Mid-lactation cows producing ∼40 to 50 kg/d milk yield responded best to increasing supplemental C16:0 in FA supplements, demonstrating that FA supplements higher in C16:0 and limited in C18:0 improves production responses.

Effects of direct-fed microbial supplement on ruminal and plasma metabolome of early-lactation dairy cows: Untargeted metabolomics approach.

We examined the effects of 2 multispecies direct-fed microbial (DFM) supplements on ruminal and plasma metabolome of early-lactation dairy cows using a high-coverage untargeted metabolomics approach. A total of 45 multiparous Holstein cows (41 ± 7 DIM) were enrolled for the 14-d pre-experimental and 91-d experimental period and were a subset from a lactation performance study, which used 114 cows. Cows were blocked using pre-experimental energy-corrected milk yield and randomly assigned within each block to 1 of 3 treatments: (1) corn silage-based diet with no DFM supplement (control; CON), (2) basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/d (PRO-A), or (3) basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 109 cfu/d (PRO-B). The basal diet was fed ad libitum daily as a TMR at 0600 and 1200 h for a duration of 91 d. Rumen fluid and blood samples were taken on d -3, 28, 49, 70, and 91 and immediately stored at -80°C. Before analysis, ruminal and plasma samples from d 28, 49, 70, and 91 were composited. An in-depth, untargeted metabolome profile of the composite rumen and plasma samples and the d -3 samples was developed by using a chemical isotope labeling/liquid chromatography-mass spectrometry (LC-MS)-based technique. Differentially abundant metabolites (taking into account fold change [FC] values and false discovery rates [FDR]) were identified with a volcano plot. In the rumen, compared with the CON diet, supplemental PRO-A increased (FC ≥1.2; FDR ≤0.05) the relative concentrations of 9 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, quinolinic acid, and shikimic acid, and PRO-B increased relative concentrations of 16 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, 16-hydroxypalmitic acid, and 2 propionate precursors (succinic and methylsuccinic acids). Relative to PRO-A, supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative rumen concentrations of 3 metabolites, 16-hydroxypalmitic acid, indole-3-carboxylic acid, and 5-aminopentanoic acid, but reduced relative rumen concentrations of 13 metabolites, including carnitine, threonic acid, and shikimic acid. Compared with the CON diet, relative concentrations of 13 plasma metabolites, including myxochelin A and glyceraldehyde, were increased (FC ≥1.2; FDR ≤0.05) by PRO-A supplementation, whereas those of 9 plasma metabolites, including 4-(2-aminophenyl)-2,4-dioxobutanoic acid, N-acetylornithine, and S-norlaudanosolin, were reduced (FC ≤0.83; FDR ≤0.05). Supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative concentrations of 9 plasma metabolites, including trans-o-hydroxybenzylidenepyruvic acid and 3-methylsalicylaldehyde, and reduced relative concentrations of 4 plasma metabolites, including ß-ethynylserine and kynurenine. Pathway analysis of the differentially abundant metabolites in both rumen and plasma revealed that these metabolites are involved in AA and fatty acid metabolism and have antimicrobial and immune-stimulating properties. The results of this study demonstrated that dietary supplementation with either PRO-A or PRO-B altered the plasma and ruminal metabolome. Notably, ruminal and plasma metabolites involved in the metabolism of AA and fatty acids and those with immunomodulatory properties were altered by either or both of the 2 microbial additives.

Changes in the rumen microbial community composition of dairy cows subjected to an acidogenic diet.

In modern breeding systems, cows are subjected to many stress factors. Animals fed a high-grain diet may have a decreased rumen pH, which would lead to subacute ruminal acidosis syndrome. The aim of this study was to investigate the evolution of microbial community composition in cows undergoing a dietary stress challenge. Twelve cows were subjected to a challenge period that consisted of a rapid change of ration, from a normal (45.4:54.6 forage:concentrate) to a high-grain content diet (24.8:75.2 forage:concentrate) to induce subacute ruminal acidosis. Individual rumen fluid content samples were collected before (T0) and during the challenge (T3, T14, T28). The DNA from rumen contents was extracted, purified, and sequenced to evaluate bacterial populations, and sequencing was performed on Illumina MiSeq. The effect of animal conditions on rumen microbial community was quantified through a linear mixed model. The acidogenic diet created 2 main clusters: ruminal hypomotility (RH) and milk fat depression (MFD). The microbial composition did not differ in T0 between the 2 groups, whereas during the challenge Ruminococcus spp., Treponema spp., Methanobrevibacter spp., and Methanosphaera spp. concentrations increased in RH cows; and Succinivibrio spp. and Butyrivibrio spp. concentrations increased in MFD cows. Prevotella spp. and Ruminococcus spp. were negatively correlated, whereas the Christenellaceae family was positively correlated with both Methanobrevibacter spp. and Methanosphaera spp. Moreover, the same diet affected cows' microbiota composition differently, underlying the impact of the host effect. Other studies are necessary to deepen the relationship between microbiota composition and host.

The affinity of milk fat globule membrane fragments and buttermilk proteins to hydroxyapatite.

Buttermilk differs from skim milk by the presence of milk fat globule membrane (MFGM) fragments that are released during cream churning. Milk fat globule membrane is rich in health-promoting components, such as phospholipids and membrane proteins, but these compounds have a negative impact on buttermilk techno-functional properties in dairy applications. The isolation of MFGM from buttermilk improved its functionality while also recovering the MFGM bioactive components. Hydroxyapatite (HA) can be used to extract MFGM by adsorption via charged site interactions. However, the affinity of HA to MFGM or the main buttermilk proteins (casein micelles [CM], ß-LG, and α-LA) is not known. The influence of important physicochemical parameters such as pH and temperature on these interactions is also unclear. For each buttermilk component, a quartz crystal microbalance diffusion analysis was performed to determine the maximum adsorption time and the attached mass density on HA-coated gold sensors. The influence of pH, ionic strength (IS), and temperature (T) on the affinity of each buttermilk component for HA particles was assessed using a 3-levels and 3-factors Box-Behnken design. The absorption rate was highest for the CM, followed by ß-LG and α-LA, and then by the MFGM. Nevertheless, the final maximal attached mass densities to the HA were similar for the MFGM and CM, and 2.5 times higher than for ß-LG and α-LA. This difference can be explained by the higher number of binding sites found in CM and their heavier mass. The model obtained by the Box-Behnken design plan showed that the adsorption of the CM changed with T, pH, and IS. These results suggest that the techno-functional properties of buttermilk may be restored by specifically extracting MFGM with HA. Experiments are ongoing to determine conditions for fractionating MFGM directly from buttermilk.

Effects of feeding whole-cracked rapeseeds, nitrate, and 3-nitrooxypropanol on composition and functional properties of the milk fat fraction from Danish Holstein cows.

The aim of this study was to determine the individual and combined effects of supplementing fat with whole-cracked rapeseed (FAT), nitrate (NITRATE), and 3-nitrooxypropanol (3-NOP) on compositional and functional properties of milk fat. An 8 × 8 incomplete Latin square design was conducted with 48 lactating Danish Holstein cows over 6 periods of 21 d each. Eight diets were 2 × 2 × 2 factorially arranged: FAT (30 or 63 g crude fat/kg DM), NITRATE (0 or 10 g nitrate/kg DM), and 3-NOP (0 or 80 mg 3-NOP/kg DM), and cows were fed ad libitum. Milk samples were analyzed for general composition, fatty acids (FA) and thermal properties of milk fat. Milk fat content was decreased by supplementing fat but increased by 3-NOP. The changes in FA composition were mainly driven by the FAT × 3-NOP interaction. Fat supplementation shifted milk FA composition toward a lower content of SFA and greater contents of MUFA and PUFA, whereas these effects became smaller in combination with 3-NOP. However, 3-NOP had no effects on SFA, MUFA, or PUFA in low-fat diets. Fat supplementation lowered solid-fat content in milk fat because of the decreased SFA content. The onset crystallization temperature of milk fat was decreased by 3-NOP when supplemented in low-fat diets. According to the FAT × 3-NOP interaction, supplementation of fat without 3-NOP shifted the peak temperature of the low-melting fraction of milk fat toward low temperature as a result of a decreased proportion of C16:0 and increased proportions of C18:1 cis-9, C18:1 trans-11, C18:2 cis-9, and CLA cis-9,trans-11. In conclusion, no additive effects were observed among FAT, NITRATE, and 3-NOP on chemical and thermal properties of milk fat, and fat supplementation largely changed milk FA composition, which in turn affected the thermal properties of milk fat.

Regulation of gene expression in milk secretory pathways during secretory activation in Holstein cows.

Increased transcription of genes involved in lactose synthesis is a key feature of secretory activation of the mammary gland. We determined which genes are transcriptionally regulated during the transition from colostrum to milk in cows and whether more frequent milking of one udder half would increase milk yields and alter gene expression. We enrolled 12 Holstein cows immediately after calving, harvested colostrum, then randomly assigned one udder half to 12 h (2X) or 6 h (4X) milking intervals for 48 h after first milking. After 48 h, all quarters were milked twice daily until d7, when final quarter milk yields were collected. Yields and composition of colostrum and milk were recorded for each 12 h interval. After each milking, a strip sample of hind milk was collected for isolation of RNA from milk fat and quantification of selected transcripts via qPCR. Milk, milk fat, total protein, and lactose yields increased significantly over the initial 48 h and at d7 after calving. Quarters on 4X treatment produced more milk than the contralateral 2X quarters. Genes upregulated concomitantly with milk yields encoded α-lactalbumin (LALBA), ß-1,4-galactosyltransferase (B4GALT1), fatty acid synthase (FASN), ß casein (CSN2), and folate receptor α (FOLR1). Downregulated genes encoded monosaccharide transporters (SLC2A3, SLC2A8, SLC35A2) and enzymes involved in galactose synthesis (HK1, PGM1, GALE). Three genes were initially downregulated but later upregulated at d7 (LPIN1, SLC2A1, UGP2). Notably, milking frequency had no effect on gene expression. Sequential upregulation first of genes encoding the former enzymes, then of UGP2, may be necessary for copious milk production. However, the local effects of milking frequency on milk production during lactogenesis do not appear to be controlled by transcription of these genes related to lactose synthesis.

Effect of feeding increasing levels of whole cottonseed on milk and milk components, milk fatty acid profile, and total-tract digestibility in lactating dairy cows.

Dietary fat is fed to increase energy intake and provide fatty acids (FA) to support milk fat production. Oilseeds contain unsaturated FA that increase the risk for biohydrogenation-induced milk fat depression, but FA in whole cottonseed (WCS) are expected to be slowly released in the rumen and thus have a lower risk for biohydrogenation-induced milk fat depression. Our hypothesis was that increasing dietary WCS would increase milk fat yield by providing additional dietary FA without induction of milk fat depression. Four primiparous and 8 multiparous lactating Holstein cows, 136 ± 35 and 127 ± 4 DIM, respectively, were arranged in a replicated 4 × 4 Latin square design with 21-d periods. Treatments were WCS provided at 0%, 3.4%, 6.8%, and 9.9% of dietary dry matter, and WCS was substituted for cottonseed hulls and soybean meal to maintain dietary fiber and protein. Treatment did not change milk yield. There was a treatment-by-parity interaction for milk fat percent and yield with a quadratic decreased in primiparous cows but no effect of WCS in multiparous cows. Cottonseed linearly increased milk fat trans-10 18:1 in primiparous cows but not in multiparous cows. Increasing WCS increased milk preformed (18C) FA yield and partially overcame the trans-10 18:1 inhibition of de novo FA synthesis in the primiparous cows. Apparent transfer of 18C FA from feed to milk decreased in all cows as WCS increased, but the magnitude of the change was greater in primiparous cows. Increasing WCS decreased total-tract apparent dry matter, organic matter, and neutral detergent fiber digestibility. There was no change in total FA digestibility. However, 18C FA digestibility tended to be decreased in both parities and 16C FA digestibility was quadratically increased in multiparous cows but not changed in primiparous cows. Total fecal flow of intact WCS increased as WCS level increased, but fecal flow of intact seeds as a percentage consumed was similar across treatments. Fecal flow of intact seeds was greater in multiparous cows (4.3% vs. 1.1% of consumed). Plasma concentrations of glucose, nonesterified FA, triglycerides, and insulin were not changed. However, plasma urea-N increased with increasing WCS. Plasma gossypol increased with WCS (0.08-1.15 µg/mL) but was well below expected toxic levels. In conclusion, WCS maintained milk and milk component yield when fed at up to 9.9% of the diet to multiparous cows without concerns of gossypol toxicity, but primiparous cows were more susceptible to biohydrogenation-induced milk fat depression in the current trial. This highlights the interactions of parity with diet composition when feeding rumen-available unsaturated fat to dairy cows.

Invited review: Camel milk and gut health-Understanding digestibility and the effect on gut microbiota.

Camel milk (CM), known for its immune-regulatory, anti-inflammatory, antiapoptotic, and antidiabetic properties, is a natural healthy food. It is easily digestible due to the high levels of ß-casein and diverse secreted antibodies, exhibiting superior antibacterial and antiviral activities compared with bovine milk. ß-casein is less allergic and more digestible because it is more susceptible to digestive hydrolysis in the gut; therefore, higher levels of ß-casein make CM advantageous for human health. Furthermore, antibodies help the digestive system by destroying the antigens, which are then overwhelmed and digested by macrophages. The connection between the gut microbiota and human health has gained substantial research attention, as it offers potential benefits and supports disease treatment. The gut microbiota has a vital role in regulating the host's health because it helps in several biological functions, such as protection against pathogens, immune function regulation, energy harvesting from digested foods, and reinforcement of digestive tract biochemical barriers. These functions could be affected by the changes in the gut microbiota profile, and gut microbiota differences are associated with several diseases, such as inflammatory bowel disease, colon cancer, irritable bowel disorder, mental illness, allergy, and obesity. This review focuses on the digestibility of CM components, particularly protein and fat, and their influence on gut microbiota modulation. Notably, the hypoallergenic properties and small fat globules of CM contribute to its enhanced digestibility. Considering the rapid digestion of its proteins under conditions simulating infant gastrointestinal digestion, CM exhibits promise as a potential alternative for infant formula preparation due to the high ß-/αs-casein ratio and protective proteins, in addition to the absence of ß-lactoglobulin.