Associations among Milk Microbiota, Milk Fatty Acids, Milk Glycans, and Inflammation from Lactating Holstein Cows.

Milk oligosaccharides (MOs) can be prebiotic and antiadhesive, while fatty acids (MFAs) can be antimicrobial. Both have been associated with milk microbes or mammary gland inflammation in humans. Relationships between these milk components and milk microbes or inflammation have not been determined for cows and could help elucidate a novel approach for the dairy industry to promote desired milk microbial composition for improvement of milk quality and reduction of milk waste. We aimed to determine relationships among milk microbiota, MFAs, MOs, lactose, and somatic cell counts (SCC) from Holstein cows, using our previously published data. Raw milk samples were collected at three time points, ranging from early to late lactation. Data were analyzed using linear mixed-effects modeling and repeated-measures correlation. Unsaturated MFA and short-chain MFA had mostly negative relationships with potentially pathogenic genera, including Corynebacterium, Pseudomonas, and an unknown Enterobacteriaceae genus but numerous positive relationships with symbionts Bifidobacterium and Bacteroides. Conversely, many MOs were positively correlated with potentially pathogenic genera (e.g., Corynebacterium, Enterococcus, and Pseudomonas), and numerous MOs were negatively correlated with the symbiont Bifidobacterium. The neutral, nonfucosylated MO composed of eight hexoses had a positive relationship with SCC, while lactose had a negative relationship with SCC. One interpretation of these trends might be that in milk, MFAs disrupt primarily pathogenic bacterial cells, causing a relative increase in abundance of beneficial microbial taxa, while MOs respond to and act on pathogenic taxa primarily through antiadhesive methods. Further research is needed to confirm the potential mechanisms driving these correlations. IMPORTANCE Bovine milk can harbor microbes that cause mastitis, milk spoilage, and foodborne illness. Fatty acids found in milk can be antimicrobial and milk oligosaccharides can have antiadhesive, prebiotic, and immune-modulatory effects. Relationships among milk microbes, fatty acids, oligosaccharides, and inflammation have been reported for humans. To our knowledge, associations among the milk microbial composition, fatty acids, oligosaccharides, and lactose have not been reported for healthy lactating cows. Identifying these potential relationships in bovine milk will inform future efforts to characterize direct and indirect interactions of the milk components with the milk microbiota. Since many milk components are associated with herd management practices, determining if these milk components impact milk microbes may provide valuable information for dairy cow management and breeding practices aimed at minimizing harmful and spoilage-causing microbes in raw milk.

Supranutrition of microalgal docosahexaenoic acid and calcidiol improved growth performance, tissue lipid profiles, and tibia characteristics of broiler chickens.

BACKGROUND: Docosahexaenoic acid (DHA) and calcidiol could be enriched in chicken for improving public nutrition and health. It remains unclear if supranutritional levels of DHA and calcidiol impair growth performance or metabolism of broiler chickens. This study was to determine singular and combined effects of high levels of supplemental DHA-rich microalgal biomass or oil and calcidiol on growth performance, concentrations of triglycerides, cholesterol, and nonesterfied fatty acids in plasma, liver, breast, and thigh, and biophysical properties of tibia. METHODS: In Exp. 1, 144 day-old Cornish chicks were divided into 4 groups (6 cages/treatment, 6 birds/cage), and were fed a corn-soybean meal basal diet (BD), BD + 10,000 IU calcidiol/kg (BD + Cal), BD + 1% DHA-rich Aurantiochytrium (1.2 g DHA/kg; BD + DHA), and BD + Cal + DHA for 6 weeks. In Exp. 2, 180 day-old chicks were divided into 5 groups, and were fed: BD, BD + DHA (0.33% to 0.66% oil, 1.5 to 3.0 g DHA/kg), BD + DHA + EPA (1.9% to 3.8% eicosapentaenoic acid-rich Nannochloropsis sp. CO18, 0.3 to 0.6 g EPA/kg), BD + DHA + calcidiol (6000 to 12,000 IU/kg diet), and BD + DHA + EPA + Cal for 6 weeks. RESULTS: Birds fed BD + Cal diet in Exp. 1 and BD + DHA + EPA diet in Exp. 2 had higher (P < 0.05) body weight gain (10%-11%) and gain:feed ratio (7%), and lower (P < 0.05) total cholesterol and triglyceride concentrations in plasma (18%-54%), liver (8%-26%), breast (19%-26%), and thigh (10%-19%), respectively, over the controls. The two diets also improved (P < 0.05) tibial breaking strength (8%-24%), total bone volume (2%-13%), and (or) bone mineral density (3%-19%) of chickens. CONCLUSION: Supranutrition of dietary calcidiol and DHA alone or together did not produce adverse effects, but led to moderate improvements of growth performance, lipid profiles of plasma and muscle, and bone properties of broiler chickens.

Changes in the Fecal Metabolome Accompany an Increase in Aberrant Crypt Foci in the Colon of C57BL/6 Mice Fed with a High-Fat Diet.

High-fat diet (HFD)-induced obesity is a risk factor for colon cancer. Our previous data show that compared to an AIN-93 diet (AIN), a HFD promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation and microbial dysbiosis in C57BL/6 mice. To explore the underlying metabolic basis, we hypothesize that AOM treatment triggers a different fecal metabolomic profile in C57BL/6 mice fed the HFD or the AIN. We found that 65 of 196 identified metabolites were significantly different among the four groups of mice (AIN, AIN + AOM, HFD, and HFD + AOM). A sparse partial least squares discriminant analysis (sPLSDA) showed that concentrations of nine fecal lipid metabolites were increased in the HFD + AOM compared to the HFD, which played a key role in overall metabolome group separation. These nine fecal lipid metabolite concentrations were positively associated with the number of colonic ACF, the cell proliferation of Ki67 proteins, and the abundance of dysbiotic bacteria. These data suggest that the process of AOM-induced ACF formation may increase selective fecal lipid concentrations in mice fed with a HFD but not an AIN. Collectively, the accumulation of these critical fecal lipid species may alter the overall metabolome during tumorigenesis in the colon.

Quantifying Sphingomyelin in Dairy through Infusion-Based Shotgun Mass Spectrometry with Lithium-Ion-Induced Fragmentation.

Quantifying sphingomyelin (SM) species by infusion-based mass spectrometry (MS) is complicated by the presence of isobaric phosphatidylcholine (PC) species, which generate a common m/z 184 product ion in the presence of ammonium ions as a result of the phosphocholine headgroup. Lithium ion adducts of SM undergo a selective dehydration [Li + H2O + (CH3)3NC2H4PO4] with a corresponding neutral loss of -207 Da. This neutral loss was employed to create a SM-selective method for identifying target species, which were quantitated using multiple reaction monitoring (MRM). SM-selective fragments in MS3 were used to characterize the sphingosine base and acyl chain. These methods were used to identify 50 individual SM species in bovine milk ranging from SM 28:1 to SM 44:2, with d16:1, d17:1, d18:1, d19:1, and d20:1 bases, and acyl fatty acids ranging from 10 to 25 carbons and 0-1 desaturations. Spiked SM standards into milk had a recovery of 99.7%, and endogenous milk SM had <10% coefficient of variation for both intra- and interday variability, with limits of detection of 1.4-5.55 nM and limits of quantitation of 11.8-178.1 nM. This MS-MRM method was employed to accurately and precisely quantify SM species in dairy products, including bovine-derived whole milk, half and half, whipping cream, and goat milk.

Replacing fish oil and astaxanthin by microalgal sources produced different metabolic responses in juvenile rainbow trout fed 2 types of practical diets.

Dietary fish oil supplementation provides n-3 long-chained polyunsaturated fatty acids for supporting fish growth and metabolism and enriching fillet with eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; c22:6n-3). Two experiments were performed as a 3 × 2 factorial arrangement of dietary treatments for 16 wk to determine effects and mechanisms of replacing 0%, 50%, and 100% fish oil with DHA-rich microalgae in combination with synthetic vs. microalgal source of astaxanthin in plant protein meal (PM)- or fishmeal (FM)- based diets for juvenile rainbow trout (Oncorhynchus mykiss). Fish (22 ± 0.26 g) were stocked at 17/tank and 3 tanks/diet. The 100% fish oil replacement impaired (P < 0.0001) growth performance, dietary protein and energy utilization, body indices, and tissue accumulation of DHA and EPA in both diet series. The impairments were associated (P < 0.05) with upregulation of hepatic gene expression related to growth (ghr1and igf1) and biosynthesis of DHA and EPA (fads6 and evol5) that was more dramatic in the FM than PM diet-fed fish, and more pronounced on tissue EPA than DHA concentrations. The source of astaxanthin exerted interaction effects with the fish oil replacement on several measures including muscle total cholesterol concentrations. In conclusion, replacing fish oil by the DHA-rich microalgae produced more negative metabolic responses than the substitution of synthetic astaxanthin by the microalgal source in juvenile rainbow trout fed 2 types of practical diets.

Dietary supplemental microalgal astaxanthin modulates molecular profiles of stress, inflammation, and lipid metabolism in broiler chickens and laying hens under high ambient temperatures.

This research was to determine effects of supplemental dietary microalgal astaxanthin (AST) on hepatic gene expression and protein production of redox enzymes, heat shock proteins (HSPs), cytokines, and lipid metabolism in broilers (BR) and laying hens (LH) under high ambient temperatures. A total of 240 (day old) Cornish male BR and 50 (19 wk old) White Leghorn Shavers LH were allotted in 5 dietary treatments with 6 and 10 cages/treatment (8 BR or 1 LH/cage), respectively. The birds were fed corn-soybean meal basal diets supplemented with microalgal (Haematococcus pluvialis) AST at 0, 10, 20, 40, and 80 mg/kg diet for 6 wk. Supplemental AST to the BR diet linearly decreased (P < 0.10, R2 = 0.18-0.36) hepatic mRNA levels of several redox status-controlling genes, heat shock protein 70 (HSP70), heat shock transcription factor 1 (HSTF1), c-Jun N-terminal kinase 1 (JNK1), tumor necrosis factor-α, and sterol regulatory element-binding protein 1 (SREBP1). The supplementation linearly elevated (P = 0.04, R2 = 0.20) diacylglycerol acyltransferase 2 (DGAT2) mRNA level and produced quadratic changes (P < 0.10, R2 = 0.15-0.47) in mRNA levels of glutathione S-transferase (GST), serine/threonine kinase (AKT1), P38 mitogen-activated protein kinase (P38MAKP), lipid metabolism-controlling genes, and the protein production of HSP90 and P38MAPK in the liver. Supplementing AST to the LH diets linearly decreased (P < 0.10, R2 = 0.18-0.56) mRNA levels of GST, HSF1, JNK1, and interleukin 10; lipogenesis genes; and JNK1 protein production. However, supplemental dietary AST produced quadratic changes (P < 0.10, R2 = 0.26-0.72) in mRNA levels of most antioxidant-, stress-responsive, and lipid metabolism-related genes in the liver of LH. In conclusion, supplemental dietary AST affected the hepatic gene expression and protein production related to redox status, heat stress and inflammation, and lipid metabolism in both BR and LH. The impacts varied with the chicken type and demonstrated linear and quadratic regressions with the inclusion levels of AST.

Supplemental methionine and stocking density affect antioxidant status, fatty acid profiles, and growth performance of broiler chickens.

Broilers stocked in high densities may be prone to oxidative and inflammatory insults, resulting in impaired health status, growth performance, and meat quality. This study was to determine if 30% extra supplemental dl-methionine alleviated or prevented those adverse effects of a higher stocking density in broiler chickens. A total of 560 male Cornish Cross cockerels (day old) were divided into four groups: two stocking densities (9 and 12 birds/m2) and two supplementations of methionine (grower: 2.90 or 3.77 g/kg and finisher: 2.60 or 3.38 g/kg). Growth performance was recorded weekly. Blood and tissues were sampled at the end of each period. High stocking density decreased (P < 0.05) body weight and growth performance of growers and (or) finishers. Those differences were partially attenuated by the extra methionine supplementation. The high methionine elevated (P < 0.05) glutathione (GSH) concentration in the thigh at both ages (> 24%). The high stocking density elevated (>28%, P < 0.05) glutathione concentration in the plasma, breast, and thigh of growers, but decreased (P < 0.05) it in the liver of growers and thigh of finishers. Interaction effects (P < 0.05) between dietary methionine and stocking density were found on activities of the antioxidant enzyme glutathione S-transferase in the liver of growers and breast, thigh, and adipose tissue of finishers. The interaction effect was also found on activities of glutathione peroxidase and superoxide dismutase in the thigh of growers. The extra methionine decreased (P < 0.05) hepatic gene expression of heat shock protein 90 (18%) and thigh and breast malondialdehyde concentrations of the finishers (35%). In conclusion, the 30% extra dl-methionine supplementation was able to partially mitigate adverse effects caused by the higher stocking density and to improve the redox status of the broilers.

Supplemental methionine exerted chemical form-dependent effects on antioxidant status, inflammation-related gene expression, and fatty acid profiles of broiler chicks raised at high ambient temperature1.

This study was to explore metabolic effects of two forms and concentrations of supplemental methionine in grower and finisher diets for broiler chickens raised at high temperature. Male Cornish cockerel chicks (total = 360, day-old) were divided into four groups (10 pens/treatment, 9 chicks/pen) and fed with 100% or 130% required methionine in the diets as DL-methionine (DL-MET) or 2-hydroxy-4-(methylthio)butanoate (HMTBA). The room was maintained at 4 to 13 °C above the suggested thermoneutral temperature. The higher concentration of both DL-MET and HMTBA enhanced (P < 0.05) hepatic GSH concentrations of the growers and plasma ferric reducing ability of the finishers. The DL-MET-fed growers had greater (P < 0.05%) muscle GSH and hepatic unsaturated fatty acid concentrations than those fed HMTBA. Expression of inflammation-related genes in the liver of finishers was affected (P < 0.05) by interaction effects of the methionine form and concentration. In conclusion, effects of the extra methionine supplementation on the high ambient temperature-related metabolic responses of broilers varied with their age and(or) tissue and the methionine form.

Supplemental Docosahexaenoic-Acid-Enriched Microalgae Affected Fatty Acid and Metabolic Profiles and Related Gene Expression in Several Tissues of Broiler Chicks.

This experiment was to enrich docosahexaenoic acid (DHA) in broiler tissues through feeding a DHA-rich microalgal biomass and to explore the underlying metabolic and molecular mechanisms. Hatchling Cornish male broilers (total = 192) were fed a corn-soybean meal basal diet containing a full-fatted microalgae ( Aurantiochytrium) at 0%, 1%, 2%, and 4% for 6 weeks ( n = 6 cages/treatment, 8 birds/cage). The inclusion of microalgae led to dose-dependent ( P < 0.01) enrichments of DHA and decreases ( P < 0.01) of n-6/n-3 fatty acids (FAs) in plasma, liver, muscle, and adipose tissue. The microalgae supplementation also lowered ( P < 0.05-0.1) nonesterified FAs concentrations in the plasma, liver and adipose tissue. The mRNA abundances of most assayed genes involved in lipid metabolism were decreased ( P < 0.05) in the liver but elevated ( P < 0.05) in the adipose in response to the biomass supplementation. In conclusion, the biomass-resultant DHA enrichments in the broiler tissues were associated with a distinctive difference in the expression of lipid metabolism-controlling genes between the liver and adipose tissue.

Inclusion of Dietary Defatted Microalgae Dose-Dependently Enriches ω-3 Fatty Acids in Egg Yolk and Tissues of Laying Hens.

BACKGROUND: The potential for dietary microalgae to enrich eggs of laying hens with ω-3 (n-3) fatty acids, and the mechanisms involved, are unclear. OBJECTIVES: The aim of this study was to determine the effects and molecular regulation of a defatted Nannochloropsis oceanica microalgae (DNOM) biomass on the enrichment of the eggs and tissues of laying hens with ω-3 fatty acids. METHODS: Fifty Shaver-White Leghorn hens (46 wk of age, body weight: 1.70 ± 0.27 kg) were individually caged (n = 10) and fed a corn-soy-based diet supplemented with DNOM at 0% (control), 2.86%, 5.75%, 11.5%, and 23% for 6 wk. Fatty acid profiles, health status, and related gene expression in eggs, blood, and tissues were performed at weeks 0, 2, 4, and 6. Data were analyzed by a combination of 1-factor ANOVA and correlation between DNOM doses and measures. RESULTS: The DNOM produced linear (P < 0.01) enrichments of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and total ω-3 fatty acids in the egg yolk (R2 > 0.9) and of DHA in the liver, breast, and thigh (R2 = 0.66-0.82). Concentrations of EPA + DHA in the egg yolk and these 3 tissues of hens fed 11.5% and 23% DNOM were 1.4-2.1, 0.6-1, 3.3-5.3, and 6-7 times greater (P < 0.001) than those in the controls, respectively. The DNOM caused dose-dependent elevations (P < 0.01) of malic enzyme and elongases 3, 4, and 5 mRNA levels (R2 = 0.97, 0.78, 0.97, and 0.86, respectively), along with increased (P < 0.01) Δ5- and Δ6-desaturases and decreased (P < 0.01) Δ9-desaturase and acyl-coenzyme A thioesterase 4 mRNA levels in the liver. CONCLUSIONS: Feeding DNOM to laying hens produced dose-dependent enrichments of DHA in their eggs, liver, and muscles by regulating key genes involved in the elongation and desaturation of polyunsaturated fatty acids. Our findings will help produce DHA-enriched eggs.

Defatted Microalgae-Mediated Enrichment of n-3 Polyunsaturated Fatty Acids in Chicken Muscle Is Not Affected by Dietary Selenium, Vitamin E, or Corn Oil.

Background: We previously showed enrichments of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in broiler chicks fed defatted microalgae. Objectives: The aims of this study were to determine 1) if the enrichments affected meat texture and were enhanced by manipulating dietary corn oil, selenium, and vitamin E concentrations and 2) how the enrichments corroborated with hepatic gene expression involved in biosynthesis and oxidation of EPA and DHA. Methods: Day-old hatching Cornish Giant cockerels (n = 216) were divided into 6 groups (6 cages/group and 6 chicks/cage). Chicks were fed 1 of the 6 diets: a control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg (4CO), 4CO + 10% microalgae (defatted Nannochloropsis oceanica; 4CO+ MA), 4CO+ MA - 2% corn oil (2CO+MA), 2CO+MA + 75 IU vitamin E/kg (2CO+MA+E), 2CO+MA + 0.3 mg Se/kg (2CO+MA+Se), and 2CO+MA+E + 0.3 mg Se/kg (2CO+MA+E+Se). After 6 wk, fatty acid profiles, DHA and EPA biosynthesis and oxidation, gene expression, lipid peroxidation, antioxidant status, and meat texture were measured in liver, muscles, or both. Results: Compared with the control diet, defatted microalgae (4CO+MA) enriched (P < 0.05) DHA and EPA by ≤116 and 24 mg/100 g tissue in the liver and muscles, respectively, and downregulated (41-76%, P < 0.01) hepatic mRNA abundance of 4 cytochrome P450 (CYP) enzymes (CYP2C23b, CYP2D6, CYP3A5, CYP4V2). Supplemental microalgae decreased (50-82%, P < 0.05) lipid peroxidation and improved (16-28%, P < 0.05) antioxidant status in the liver, muscles, or both. However, the microalgae-mediated enrichments in the muscles were not elevated by altering dietary corn oil, vitamin E, or selenium and did not affect meat texture. Conclusion: The microalgae-mediated enrichments of DHA and EPA in the chicken muscles were associated with decreased hepatic gene expression of their oxidation, but were not further enhanced by altering dietary corn oil, vitamin E, or selenium.

Dose-Dependent Enrichments and Improved Redox Status in Tissues of Broiler Chicks under Heat Stress by Dietary Supplemental Microalgal Astaxanthin.

Astaxanthin (AST) is a well-known carotenoid with a high antioxidant capacity. This study was designed to evaluate the nutritional and metabolic effects of microalgal AST added to the diets of broiler chicks under heat stress. A total of 240 Cornish male chicks (1 day old) were divided into six cages per treatment (eight chicks per cage) and fed a corn-soybean meal diet supplemented with AST from Haematococcus pluvialis at 0, 10, 20, 40, and 80 mg/kg for 6 weeks. Heat stress was employed during weeks 4-6. The supplementation led to dose-dependent enrichments ( P < 0.05) of AST and total carotenoids in the plasma, the liver, and the breast and thigh muscles. There were similar enhancements ( P < 0.05) of oxygen-radical-absorbance capacities, but there were decreases or mixed responses ( P < 0.05) of glutathione concentrations and glutathione peroxidase activities in the tissues. In conclusion, supplemental dietary microalgal AST was bioavailable to the chicks and enriched in their tissues independent of heat stress, leading to coordinated changes in their endogenous antioxidant defense and meat quality.