Turning weeds into feed: Ensiling Calotropis gigantea (Giant milkweed) reduces its toxicity and enhances its palatability for dairy cows.

Calotropis gigantea (Giant milkweed, GM) has the potential to be utilized as a new feed additive for ruminants, however, the presence of unpalatable or toxic compounds decreases animal feed intake. This study aimed to valorize GM as a potential new feed resource through the chemical and microbial biotransformation of toxic compounds that will henceforth, make the plant palatable for cows. After GM's ensiling using fermentative bacteria, the plant was sampled for UHPLC-MS/MS to analyse the metabolomic changes. Illumina Miseq of the 16 S rRNA fragment genes and ITS1 were used to describe the microbial composition and structure colonizing GM silage and contributing to the biodegradation of toxic compounds. Microbial functions were predicted from metataxonomic data and KEGG pathways analysis. Eight Holstein dairy cows assigned in a cross-over design were supplemented with GM and GM silage to evaluate palatability and effects on milk yield and milk protein. Cows were fed their typical diet prior to the experiment (positive control). After ensiling, 23 flavonoids, 47 amino acids and derivatives increased, while the other 14 flavonoids, 9 amino acids and derivatives decreased, indicating active metabolism during the GM ensiling process. Lactobacillus buchneri, Bacteroides ovatus, and Megasphaera elsdenii were specific to ensiled GM and correlated to functional plant metabolites, while Sphingomonas paucimobilis and Staphylococcus saprophyticus were specific to non-ensiled GM and correlated to the toxic metabolite 5-hydroxymethylfurfural."Xenobiotics biodegradation and metabolism", "cancer overview" and "neurodegenerative disease" were the highly expressed microbial KEGG pathways in non-ensiled GM. Non-ensiled GM is unpalatable for cows and drastically reduces the animal's feed intake, whereas ensiled GM does not reduce feed intake, milk yield and milk protein. This study provides essential information for sustainable animal production by valorizing GM as a new feed additive.

Could soil microplastic pollution exacerbate climate change? A meta-analysis of greenhouse gas emissions and global warming potential.

Microplastics pollution and climate change are primarily investigated in isolation, despite their joint threat to the environment. Greenhouse gases (GHGs) are emitted during: the production of plastic and rubber, the use and degradation of plastic, and after contamination of environment. This is the first meta-analysis to assess underlying causal relationships and the influence of likely mediators. We included 60 peer-reviewed empirical studies; estimating GHGs emissions effect size and global warming potential (GWP), according to key microplastics properties and soil conditions. We investigated interrelationships with microbe functional gene expression. Overall, microplastics contamination was associated with increased GHGs emissions, with the strongest effect (60%) on CH4 emissions. Polylactic-acid caused 32% higher CO2 emissions, but only 1% of total GWP. Phenol-formaldehyde had the greatest (175%) GWP via 182% increased N2O emissions. Only polystyrene resulted in reduced GWP by 50%, due to N2O mitigation. Polyethylene caused the maximum (60%) CH4 emissions. Shapes of microplastics differed in GWP: fiber had the greatest GWP (66%) whereas beads reduced GWP by 53%. Films substantially increased emissions of all GHGs: 14% CO2, 10% N2O and 60% CH4. Larger-sized microplastics had higher GWP (125%) due to their 9% CO2 and 63% N2O emissions. GWP rose sharply if soil microplastics content exceeded 0.5%. Higher CO2 emissions, ranging from 4% to 20%, arose from soil which was either fine, saturated or had high-carbon content. Higher N2O emissions, ranging from 10% to 95%, arose from soils that had either medium texture, saturated water content or low-carbon content. Both CO2 and N2O emissions were 43%-56% higher from soils with neutral pH. We conclude that microplastics contamination can cause raised GHGs emissions, posing a risk of exacerbating climate-change. We show clear links between GHGs emissions, microplastics properties, soil characteristics and soil microbe functional gene expression. Further research is needed regarding underlying mechanisms and processes.

Multi-omics dataset of bovine mammary epithelial cells stimulated by ten different essential amino acids.

Application of high-throughput sequencing and screening help to detect the transcriptional and metabolic discrepancies in organs provided with various levels of nutrients. The influences of individual essential amino acid (EAA) administration on transcriptomic and metabolomic profilings of bovine mammary epithelial cells (BMECs) were systematically investigated. A RNA sequencing and liquid chromatography-tandem mass spectrometry generated a comprehensive comparison of transcriptomics, non-targeted metabolomics and targeted amino acids profilings of BMECs with individual EAA stimulation by turn. The sequencing data and raw LC-MS/MS data of samples were presented in the databases of Gene Expression Omnibus, MetaboLights and Figshare for efficient reuse, including exploring the divergences in metabolisms between different EAAs and screening valuable genes and metabolites regulating casein synthesis.

Enhancing docosahexaenoic acid production of Schizochytrium sp. by optimizing fermentation using central composite design.

Docosahexaenoic acid (DHA) can improve human and animal health, particularly including anti-inflammatory, antioxidant, anticancer, neurological, and visual functions. Schizochytrium sp. is a marine heterotrophic protist producing oil with high DHA content, which is widely used in animal and food production. However, different fermentation conditions have intensive impacts on the growth and DHA content of Schizochytrium sp. Thus, this study aimed to enhance the DHA yield and concentration of Schizochytrium sp. I-F-9 by optimizing the fermentation medium. First, a single-factor design was conducted to select a target carbon and nitrogen source from several generic sources (glucose, sucrose, glycerol, maltose, corn syrup, yeast extract, urea, peptone, and ammonium sulfate). The Plackett-Burman design and the central composite design (CCD) were utilized to optimize the fermentation mediums. Schizochytrium sp. in 50-mL fermentation broth was cultured in a 250 mL shake flask at 28 °C and 200 rpm for 120 h before collecting the cell pellet. Subsequently, the cell walls were destroyed with hydrochloric acid to extract the fatty acid using n-hexane. The DHA content was detected by gas chromatography. The single-factor test indicated that glucose and peptone, respectively, significantly improved the DHA content of Schizochytrium sp. compared to the other carbon and nitrogen sources. Glucose, sodium glutamate, and sea crystal were the key factors affecting DHA production in the Plackett-Burman test (P = 0.0247). The CCD result showed that DHA production was elevated by 34.73% compared with the initial yield (from 6.18 ± 0.063 to 8.33 ± 0.052 g/L). Therefore, the results of this study demonstrated an efficient strategy to increase the yield and content of DHA of Schizochytrium sp.

Fresh Phyllanthus emblica (Amla) Fruit Supplementation Enhances Milk Fatty Acid Profiles and the Antioxidant Capacities of Milk and Blood in Dairy Cows.

The objective of this study was to investigate the effect of a diet supplemented with fresh amla fruit as a natural feed additive on blood metabolic parameters, milk antioxidant capacity, and milk fatty acid (FA) proportions in lactating dairy cows. Eight ruminally cannulated mid-lactation dairy cows were used in a repeated crossover design. The first group of four cows received total mixed ration (TMR) feed without fresh amla fruit (control group). The remaining four cows sequentially supplemented fresh amla fruit (FAF) at three levels (200, 400, then 600 g/d) (treatment group) at 14-day intervals. In second period, control and treatment groups were exchanged. The first ten days were adjusted to diet adaptation for each sub-period, and the last four days for sampling milk and blood. A total of 514 metabolites were detected from FAF using UPLC-ESI-MS/MS. The five main metabolites in FAF were phenolic acids (22%), flavonoids (20%), lipids (20%), amino acids and derivatives (9%), and tannins (7%). Amla fruit supplementation reduced total saturated fatty acid and the omega-6/omega-3 ratio at 200 or 400 g/d FAF dose compared to controls. In addition, amla fruit increased unsaturated FA, such as C20:5 (Eicosapentaenoic acid, EPA) and C22:6 (docosahexaenoic acid, DHA), and branched-chain FA in a dose-dependent manner at 200 or 400 g/d compared to controls. In addition, amla fruit increased the antioxidant capacity biomarkers in the blood, such as superoxide dismutase (SOD) and albumin; this confirms that amla fruit is an excellent antioxidant, inhibiting reactive oxygen species' (ROS) metabolism, and can thereby protect cells from oxidative stress. Moreover, the most remarkable improvement of ferric reducing-antioxidant power (FRAP) and total antioxidant capacity (TAC) in milk was recorded at 400 g/d FAF doses compared to controls. Therefore, fresh amla fruit doses for lactating cows at 400 g/d on an as-fed basis can be used as an alternative additive feed in dairy cow diets to improve antioxidant capacity, protein efficiency, butter quality, and to produce more desirable milk fatty acid profiles for human consumption.

Changes in Metabolites from Bovine Milk with ß-Casein Variants Revealed by Metabolomics.

ß-casein is a primary protein in milk, and its variants have been associated with changes in the protein content of bovine milk. However, there has been little research focused on the effects of ß-casein variants on milk metabolites. In the present study, dairy cows producing milk with ß-casein variant A1/A1 (A1), A2/A2 (A2), and their heterozygote A1/A2 (A12) were screened by a high-resolution melting method. Individual milk samples were then collected from each of the cows, and the milk metabolites were separated and analyzed using nuclear magnetic resonance spectroscopy- and liquid-chromatography mass spectrometry-based metabolomics techniques. Differences in metabolites among the variant groups were evaluated by multivariate statistical analysis. The relative abundances of methionine, proline, and α-lactose were the highest in ß-casein variant A2 milk, whereas choline, glycine, citric acid, and cyclic adenosine monophosphate (cAMP) showed the highest abundances in variant A1 milk. Metabolic pathways analysis indicated that the differential metabolites between variants A1 and A2 were involved in pantothenate and coenzyme A biosynthesis, butanoate metabolism, and valine, leucine, and isoleucine biosynthesis. Our results reveal the differences in milk metabolites among the ß-casein variants A1, A2, and the heterozygote. These findings, thus, provide novel insights into the effects of ß-casein variants on milk metabolites, facilitating further research into the mechanism of the biosynthesis of milk components in the mammary gland and the potential physiological function of milk associated with ß-casein variants.

Rubber seed oil and flaxseed oil supplementation on serum fatty acid profile, oxidation stability of serum and milk, and immune function of dairy cows

Objective: This study was designed to investigate the effect of diet supplementation with rubber seed oil and flaxseed oil on serum fatty acids profile, oxidation stability of serum and milk, and immune function of dairy cows. Methods: Forty-eight mid-lactation Holstein dairy cows were randomly assigned to one of four treatments for 8wk, including basal diet (CON) or the basal diet supplemented with 4% rubber seed oil (RO), 4% flaxseed oil (FO) or 2% rubber seed oil plus 2% flaxseed oil (RFO) on a DM basis. Results: Compared with CON, all the oil groups increased the levels of trans-11 C18:1 (vaccenic acid, VA), cis-9, trans-11 C18:2 (conjugated linoleic acid, CLA) and C18:3 (α-linolenic acid, ALA) in serum. Both the activity of glutathione peroxidase and catalase in serum and milk in oil groups were decreased, which were negatively correlated with the levels of cis-9, trans-11 CLA and ALA. The concentrations of proinflammatory factors (TNF-α and INF-γ) in serum of oil groups were lower than that from the CON cows. Conclusion: These results indicate that diet supplementation with rubber seed oil or flaxseed oil could alter serum fatty acid profile and enhance the immune function of dairy cows. However, the negative effect on milk oxidation stability should be considered when feeding these n-3 PUFA-enriched oils in dairy production.

Effects of Arginine concentration on the in vitro expression of Casein and mTOR pathway related genes in mammary epithelial cells from dairy cattle.

Arginine (Arg) is a conditionally-essential amino acid that is taken up by bovine mammary gland in excess of its output in milk. In this study we evaluated the effects of Arg concentration on the expression of casein and signaling pathway-related genes in mammary epithelial cells. The treatments (applied for 24 h) were designed to be devoid of Arg 0X (control; 0.00 mg/L), resemble the profile of Arg in casein (Arg 1X; 278.00 mg/L), be deficient [Arg 0.25X (69.50 mg/L) and Arg 0.5X (139.00 mg/L)], or be in excess of the amount in casein [Arg 2X (556.00 mg/L), Arg 4X (1,112 mg/L), and Arg 8X (2,224 mg/L)]. The expression of CSN1S, CSN3 and mTOR in the experimental groups was higher than those of the control group (P<0.05). Except for Arg 0.25X and Arg 8X (P>0.05), the expression of CSN1S2, CSN2 and JAK2 in other experimental groups was higher (P<0.05) than those in the control group. Except for Arg 8X (P>0.05), the expression of STAT5 in the other experimental groups was higher than those of the control (P<0.05). It also was observed that except for Arg 0.5X, the S6K expression was higher in other experimental groups than the control (P<0.05). In contrast, except for Arg 0.25X the other experimental groups resulted in lower 4EBP1 expression than the control (P<0.05). Among groups, the expression of CSN1S1, CSN1S2, CSN2, CSN3, JAK2, STAT5, mTOR and S6K gene was highest with Arg 2X (P<0.05); the reverse was true for 4EBP1 gene, with the lowest expression in this group (P<0.05). Taken together, Arg appears to play an important role in the transcriptional regulation of casein genes and mTOR-related genes in bovine mammary epithelial cells.

Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.

In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow.

BACKGROUND: The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. METHODOLOGY: Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. PRINCIPAL FINDINGS: The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n=60. Furthermore, they were capable of synthesizing beta-casein (CSN2), acetyl-CoA carboxylase-alpha (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. CONCLUSIONS: The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).