An interactive R-based custom quantification program for semi-quantitative analysis of triacylglycerols in bovine milk.

The R programming language, RStudio, and open-source software solutions for analysis of liquid chromatography-mass spectrometry (LC-MS) data have been used with user-written R-based custom quantification programs (CQP) for semi-quantification of triacylglycerols (TAGs) in bovine milk lipid extracts. Using the peak-finding capabilities of the package "xcms" in RStudio, peaks were integrated, and retention times aligned, normalized, and then used for semi-quantitative analysis of a custom set of four extraction internal standards (EISs) and 29 TAG regioisomers using the choice of four analytical internal standards (AISs). Alternating stereospecific numbering (sn) 1,3 TAG regioisomers (standards 1, 3, and 5 of six calibration standards) and sn-1,2 TAG regioisomers (standards 2, 4, and 6 of six standards) were used to make a set of six calibration standards, which were used for quantification using a linear fit model, polynomial fit model, power fit model, level-bracketed linear fit, replicate-bracketed polynomial fit, replicate-bracketed power fit, and replicate- and level-bracketed linear fit and response factors. For example, the linear fit for EIS1 gave an unacceptable coefficient of determination (CoD), r2 = 0.9616, whereas the polynomial fit gave r2 = 0.9908 and the power fit gave r2 = 0.9928, while the double-bracketed linear fit gave CoDs of r2 = 0.9960, 0.9848, and 0.9781 for the three brackets, yet gave the least % difference to known calibration concentrations. For unparalleled transparency, the CQP produced webpages that allowed every step in the data processing and quantification sequence to be verified and reproduced, and contained interactive figures. The data are publicly available using a digital object identifier (DOI). The R code can be downloaded and used with the downloadable data to reproduce the results, to modify the code and further customize the results, or to copy and paste and adapt the code to other quantification applications.

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.

A Low-Starch and High-Fiber Diet Intervention Impacts the Microbial Community of Raw Bovine Milk.

Background: A more sustainable dairy cow diet was designed that minimizes use of feed components digestible by monogastric animals by increasing the quantity of forages. Objectives: This study determined if feeding lactating cows the more sustainable, low-starch and high-fiber (LSHF) diet was associated with changes in raw milk microbiota composition and somatic cell count (SCC). Methods: In a crossover design, 76 lactating Holstein cows were assigned to an LSHF diet or a high-starch and low-fiber (HSLF) diet, similar to common dairy cow diets in the United States, for 10 wk then placed on the opposite diet for 10 wk. The LSHF diet contained greater quantities of forages, beet pulp, and corn distillers' grain, but contained less canola meal and no high-moisture corn compared with the HSLF diet. Raw milk samples were collected from each cow 4-5 d before intervention and 5 wk into each diet treatment. Within 4 d, additional milk samples were collected for measurement of SCC using Fossmatic 7. The microbial community was determined by sequencing the 16S rRNA gene V4-V5 region and analyzing sequences with QIIME2. After quality filtering, 53 cows remained. Results: Raw milk microbial communities differed by diet and time. Taxa associated with fiber consumption, such as Lachnospiraceae, Lactobacillus, Bacteroides, and Methanobrevibacter, were enriched with the LSHF diet. Meanwhile, taxa associated with mastitis, such as Pseudomonas, Stenotrophomonas, and Enterobacteriaceae, were enriched with the HSLF diet. Relatedly, an interaction of diet and time was found to impact SCC. Conclusions: In raw milk, consumption of an LSHF diet compared with an HSLF diet was associated with changes in abundance of microbes previously associated with fiber consumption, udder health, and milk spoilage. Further research is needed to determine if an LSHF diet indeed leads to lower rates of mastitis and milk spoilage, which could benefit the dairy industry.

Dietary Fiber to Starch Ratio Affects Bovine Milk Oligosaccharide Profiles.

Background: Bovine milk oligosaccharides (BMOs) have several demonstrated and hypothesized benefits including roles in cognitive development and antipathogenic activities, making them promising ingredients for infant formulas and nutraceutical applications. BMO extraction from bovine milk is challenged by low concentrations relative to nonbioactive simple sugars like lactose. BMO abundances are known to vary with a cow's lactation stage, breed, and parity, but these characteristics are difficult to modify in existing dairy herds. In contrast, diet modification is an accessible target, and is already known to influence milk yield, lipid content, protein levels, and monosaccharide compositions. Objectives: To determine the impact of a low starch high fiber versus a high starch low fiber diet on overall BMO profiles and individual BMO abundances in Holstein dairy cattle. Methods: Milk samples were collected from 59 midlactation Holsteins in a crossover study featuring dietary modification with either a low starch high fiber or high starch low fiber feed. BMO profiles were evaluated by nano-LC quadrupole time-of-flight tandem MS, and differences in BMO abundances between diets were evaluated using linear mixed effects modeling. Results: A total of 19 BMOs were identified across the sample set, including 4 large fucosylated compounds. Seven BMOs were found to have significantly more positive percent changes in yield-adjusted abundance from the pre-experiment baseline period for milk samples collected during feeding with the low starch high fiber diet compared with the high starch low fiber diet. Conclusions: Consuming the low starch high fiber diet promoted greater overall BMO production than the high starch low fiber diet in a population of midlactation Holsteins. Additionally, this study afforded the opportunity to investigate the impact of other factors potentially influencing BMO abundances, furthering understanding of how dairy herd management practices can positively impact milk composition and support the potential use of BMOs as functional ingredients.

Identification of High and Low Branched-Chain Fatty Acid-Producing Phenotypes in Holstein Cows following High-Forage and Low-Forage Diets in a Crossover Designed Trial.

BACKGROUND: Branched-chain fatty acids (BCFAs) are rumen-derived fatty acids comprising ∼2% of bovine-milk fatty acids. BCFAs possess anti-inflammatory properties and enriching the BCFA content of bovine milk may provide human health benefits. OBJECTIVE: We determined whether forage content impacts the BCFA content of milk from Holstein cows and identified fatty acid phenotypes in high vs. low BCFA-containing milks. METHODS: Holstein cows (n = 62), fed for 67 d in a crossover design, consumed a diet with high forage and low concentrate (HF:C) and a diet with low forage and high concentrate (LF:C). Milk samples were collected at the end of each treatment period and fatty acid content determined. Paired t-tests, 1-factor ANOVA, sparse partial least-squares discriminant analysis (sPLSDA), and Pearson's correlation analysis were used to analyze the data. RESULTS: The total milk fatty acid concentration for cows fed the HF:C diet was greater than that of cows fed the LF:C diet (4.2 ± 0.7 g/100 mL vs. 3.9 ± 0.9 g/100 mL). sPLSDA demonstrated separation of the dietary treatments, with BCFAs and odd-chain fatty acids as primary determinants. Total BCFA content in milk fat was elevated by HF:C intake compared with LF:C intake (1.80 vs. 1.68%). Quintile separation of high vs. low BCFA milks resulted in 4 groups: HF:C /low BCFAs, HF:C /high BCFAs; LF:C /low BCFAs, and LF:C /high BCFAs. Milks from the high BCFA quintiles had lower palmitic acid content (29.6% vs. 34.4%) but higher oleic acid content than milks from the low BCFA quintiles (19.7% vs. 17.0%). Some cows were identified as high BCFA producers or low BCFA producers regardless of diet. CONCLUSIONS: BCFA content of milk is diet-sensitive but variation in responses exists. The potential to produce milk with high BCFA content and lower SFA content needs further study.

Evaluation of warm season annual forages for forage yield and quality in the north-central United States.

Grazing-based dairy operations require productive, high-quality forages capable of supporting the nutritional needs of mid-lactation dairy cows. Our objectives were to evaluate primary and regrowth harvests of two cultivars of sudangrass (SU), sorghum-sudangrass (S×SU), and pearl millet (PM) forages for growth and nutritive characteristics within the specific context of suitability for grazing by dairy cows. Three harvest cycles, including primary and regrowth cycles in 2016, and a single harvest cycle of primary growth in 2017, were evaluated at two locations (Prairie du Sac and Marshfield, WI). Within each cycle, sampling was initiated when canopy height was about 41 cm and continued thereafter on weekly intervals for 5 weeks, resulting in six equally spaced sampling dates per harvest cycle. Data were analyzed as a split-plot design with cultivars (6) as whole-plots arranged in randomized complete blocks and weekly harvest dates (6) as subplots. Yields of dry matter (DM) were less consistent at the more northern location (Marshfield), which is known for its heavier, poorly drained soils. Despite locational differences, the taller-growing cultivar within each forage type frequently exhibited yield advantages over dwarf or shorter-growing cultivars; this occurred for 7 of 9 intra-forage-type comparisons (P ≤ 0.021) across three harvest cycles at Prairie du Sac, and for 6 of 9 similar comparisons (P ≤ 0.032) at Marshfield. In 2016, shorter-growing cultivars had greater percentages of leaf in 4 of 6 intra-forage-type comparisons at both locations (P ≤ 0.004), which is especially relevant for grazing. Similarly, PM cultivars exhibited shorter canopy heights (P ≤ 0.002), but greater percentages of leaf (P < 0.001), than all other cultivars during all harvest cycles at both locations. However, the greater leaf percentages exhibited by PM cultivars did not translate into reduced percentages of structural plant fiber (asNDFom) on a whole-plant basis during any harvest cycle at either location; furthermore, asNDFom concentrations for PM cultivars were greater (P ≤ 0.047) than observed for other cultivars within 3 of 6 harvest cycles across both locations. Ruminal in-situ degradation of asNDFom for whole-plant forages based on a 48-h incubation was significantly greater (P ≤ 0.006) for PM compared with other cultivars in 4 of 6 harvest cycles. Pearl millet cultivars generally exhibited more suitable characteristics for grazing livestock than SU or S×SU cultivars.

Validating the Use of Bovine Buccal Sampling as a Proxy for the Rumen Microbiota by Using a Time Course and Random Forest Classification Approach.

Analysis of the cow microbiome, as well as host genetic influences on the establishment and colonization of the rumen microbiota, is critical for development of strategies to manipulate ruminal function toward more efficient and environmentally friendly milk production. To this end, the development and validation of noninvasive methods to sample the rumen microbiota at a large scale are required. In this study, we further optimized the analysis of buccal swab samples as a proxy for direct bacterial samples of the rumen of dairy cows. To identify an optimal time for sampling, we collected buccal swab and rumen samples at six different time points relative to animal feeding. We then evaluated several biases in these samples using a machine learning classifier (random forest) to select taxa that discriminate between buccal swab and rumen samples. Differences in the inverse Simpson's diversity, Shannon's evenness, and Bray-Curtis dissimilarities between methods were significantly less apparent when sampling was performed prior to morning feeding (P < 0.05), suggesting that this time point was optimal for representative sampling. In addition, the random forest classifier was able to accurately identify nonrumen taxa, including 10 oral and putative feed-associated taxa. Two highly prevalent (>60%) taxa in buccal and rumen samples had significant variance in relative abundances between sampling methods but could be qualitatively assessed via regular buccal swab sampling. This work not only provides new insights into the oral community of ruminants but also further validates and refines buccal swabbing as a method to assess the rumen bacterial in large herds.IMPORTANCE The gastrointestinal tracts of ruminants harbor a diverse microbial community that coevolved symbiotically with the host, influencing its nutrition, health, and performance. While the influence of environmental factors on rumen microbes is well documented, the process by which host genetics influences the establishment and colonization of the rumen microbiota still needs to be elucidated. This knowledge gap is due largely to our inability to easily sample the rumen microbiota. There are three common methods for rumen sampling but all of them present at least one disadvantage, including animal welfare, sample quality, labor, and scalability. The development and validation of noninvasive methods, such as buccal swabbing, for large-scale rumen sampling is needed to support studies that require large sample sizes to generate reliable results. The validation of buccal swabbing will also support the development of molecular tools for the early diagnosis of metabolic disorders associated with microbial changes in large herds.

Key Considerations for the Use of Seaweed to Reduce Enteric Methane Emissions From Cattle.

Enteric methane emissions are the single largest source of direct greenhouse gas emissions (GHG) in beef and dairy value chains and a substantial contributor to anthropogenic methane emissions globally. In late 2019, the World Wildlife Fund (WWF), the Advanced Research Projects Agency-Energy (ARPA-E) and the Foundation for Food and Agriculture Research (FFAR) convened approximately 50 stakeholders representing research and production of seaweeds, animal feeds, dairy cattle, and beef and dairy foods to discuss challenges and opportunities associated with the use of seaweed-based ingredients to reduce enteric methane emissions. This Perspective article describes the considerations identified by the workshop participants and suggests next steps for the further development and evaluation of seaweed-based feed ingredients as enteric methane mitigants. Although numerous compounds derived from sources other than seaweed have been identified as having enteric methane mitigation potential, these mitigants are outside the scope of this article.

Ruminal fermentation, kinetics, and total-tract digestibility of lactating dairy cows fed distillers dried grains with solubles in low- and high-forage diets.

The objective of this study was to investigate the effects of concentrations of forages and corn distillers dried grains with solubles (DDGS) on ruminal fermentation, ruminal kinetics, and total-tract digestibility of nutrients in lactating dairy cows. Four lactating Holstein cows with ruminal cannulas were assigned to a 4 × 4 Latin square in a 2 × 2 factorial arrangement of treatments. Diets were formulated to contain low forage [LF; 17% forage neutral detergent fiber (NDF)] or high forage (HF; 24.5% forage NDF) and DDGS at 0 or 18% (0DG or 18DG) of diet dry matter (DM). Intake of DM was not affected by the diets. Daily mean ruminal pH was affected by forage NDF × DDGS interactions, as the lowest ruminal pH was observed among cows fed LF18DG (6.02). Apparent total-tract digestibility for DM, organic matter, crude protein, NDF, acid detergent fiber, and starch was not affected by diets. Cows fed LF diets had a greater total volatile fatty acid concentration compared with cows fed HF (122 vs. 116 mM). Molar proportions of acetate were greater for HF compared with that of LF diets (62.6 vs. 57.5 mmol/100 mmol) and greater for 0DG diet compared with that of 18DG diets (61.3 vs. 58.7 mmol/100 mmol) diets. The molar proportion of propionate was affected by forage × DDGS interaction as the greatest propionate molar proportion was observed with cows fed LF18DG diet (27.7 mmol/100 mmol). Also, molar proportion of butyrate was affected by forage × DDGS interaction, as the greatest butyrate molar proportion was observed in cows fed HF18DG diet (13.5 mmol/100 mmol). Average fractional dilution rate for all diets was 11.9%/h and was not affected by diets. Fractional passage rate of the solid phase was greater for HF than for LF (4.40 vs. 3.76%/h). The ruminal retention time of solid phase was greater for LF compared with HF diets (27.3 vs. 23.3 h). Fractional passage rate of DDGS was affected by forage × DG interaction, as the highest fractional passage rate of DDGS was observed with cows fed HF18DG diet (7.72%/h). Our results demonstrated that concentrations of forage, DDGS, and their interaction influence ruminal degradation and kinetics of diets fed. Diets formulated at 17% forage NDF at 17% (DM basis) can decrease milk fat concentration compared with diets formulated at 25% forage NDF. Additionally, feeding DDGS at 18% DM basis to lactating dairy cows did not affect milk fat concentration or yield.

Production of dairy cows fed distillers dried grains with solubles in low- and high-forage diets.

The objective of the study was to investigate the effects of dietary forage and distillers dried grains with solubles (DDGS) concentration on the performance of lactating dairy cows. Twelve Holstein cows were blocked by parity and milk production and assigned to replicated 4 × 4 Latin squares with a 2 × 2 factorial arrangement of treatments. Diets were formulated to contain low forage [LF; 17% forage neutral detergent fiber (NDF)] or high forage (HF; 24.5% forage NDF) and DDGS at 0 or 18% of diet dry matter. The forage portion of the diet consisted of 80% corn silage and 20% alfalfa hay (dry matter basis). A portion of the ground corn and all of the expeller soybean meal and extruded soybeans from 0% DDGS diets were replaced with DDGS to formulate 18% DDGS diets. Overall, we found no interactions of forage × DDGS concentrations for any of the production measures. We observed no effect of diet on dry matter intake. Milk yield was greater when cows were fed LF diets compared with HF diets (43.3 vs. 41.5 kg/d). Milk fat concentration (3.03 vs. 3.38%) was lower for cows fed LF diets compared with HF diets, whereas protein concentration (3.11 vs. 2.98%) and yield (1.34 vs. 1.24 kg/d) were greater for cows fed LF diets compared with HF diets. Yields of fat, total solids, energy-corrected milk, and feed efficiency were not affected by diets. Cows partitioned equally for milk, maintenance, and body reserves. Replacing starch from ground corn and protein from soybean feeds with DDGS at either 17 or 24.5% of forage NDF concentration in the diet was cost-effective and did not affect the production performance of lactating dairy cows.

Effects of feeding alfalfa stemlage or wheat straw for dietary energy dilution on nutrient intake and digestibility, growth performance, and feeding behavior of Holstein dairy heifers.

Feeding high-quality forage diets may lead to excessive weight gains and over-conditioning for dairy heifers. Restriction of energy density and dry matter intake by using low-energy forages, such as straw, is a good approach for controlling this problem. Alfalfa stems contain high fiber and moderate protein content and have the potential to be used to replace straw to reduce dietary energy. The objective of this study was to compare nutrient intakes, digestibilities, growth performance, and feeding behaviors of dairy heifers offered an alfalfa silage/corn silage high-energy diet (HE; 13.1% crude protein, 65.4% total digestible nutrients, 39.7% neutral detergent fiber) with 2 energy-diluted diets that replaced various proportions of the corn or alfalfa silages with either alfalfa stemlage (STM; 12.6% crude protein, 59.1% total digestible nutrients, 46.4% neutral detergent fiber) or chopped wheat straw (WS; 12.6% crude protein, 61.9% total digestible nutrients, 43.7% neutral detergent fiber). Seventy-two pregnant Holstein heifers (16.8 ± 1.3 mo) were stratified into 3 blocks (24 heifers/block) by initial body weight (light, 440 ± 18.0 kg; medium, 486 ± 18.6 kg; heavy, 534 ± 25.1 kg), with each block composed of 3 pens (8 heifers/pen), with diets assigned randomly to 1 pen within the block. Diets were offered in a 56-d feeding trial. Both dry matter intake and energy intake were decreased with the addition of low-energy forages to the diets, but no differences in dry matter intake were observed across diluted diets. Digestibility of dry matter, organic matter, neutral detergent fiber, and apparent N were greater for HE compared with diluted diets, and for WS compared with STM. Total body weight gain (74 vs. 56 kg) and average daily gain (1.32 vs. 1.00 kg/d) were greater for heifers offered HE compared with diluted diets. Feed efficiency tended to be less for heifers offered the diluted diets compared with HE (10.7 vs. 8.6 kg of feed/kg of gain). Heifers did not sort for or against particles when offered HE. However, increased sorting behavior was observed for diluted diets. Compared with ad libitum feeding dairy heifers a diet with high nutrient content forages (corn silage and alfalfa silage), use of diet diluted with alfalfa stemlage or wheat straw is an effective feeding management strategy to control total daily dry matter and energy intake by increasing gut fill, and maintain desirable body condition and growth rates, even though the diluted diets had greater sortability.

Fermentation characteristics and aerobic stability of wet corn distillers grains with solubles ensiled in combination with whole plant corn.

BACKGROUND: Wet corn distillers grains with solubles (WDG) are prone to aerobic spoilage when stored for an extended period of time. The objective of this study was to evaluate the fermentation characteristics of ensiling WDG with whole plant corn (WPC) using the following combinations: (1) 100% WPC; (2) 75% WPC + 25% WDG; (3) 50% WPC + 50% WDG; and (4) 100% WDG. RESULTS: The initial pH was greatest for 100% WPC and lowest for 100% WDG (5.7, 4.6, 4.0, and 3.1, respectively). Concentrations of ammonia nitrogen (12.0, 26.8, 40.7, and 50.8 g kg(-1) dry matter (DM)) and Crude protein (CP) (98.7, 155.8, 206.8, and 307.9 g kg(-1) of DM) increased with increasing concentrations of WDG. Lactic acid concentration prior to ensiling was greatest for 100% WDG (9.0 g kg(-1) DM) and decreased with WPC in the silage. Acetic, propionic, and butyric acids were not present prior to ensiling. The pH of the ensiled feeds dropped below 4.0 by day 3, with no further decrease over time. Acetic acid increased from undetected amounts at day 0 to 38.8, 43.9, 43.2, and 2.2 g kg(-1) of DM at day 129 as concentration of WDG increased. Aerobic stability was enhanced with increasing WDG concentration in the silage. CONCLUSION: Fermentation, nutrient profile, and aerobic stability can be improved when ensiling wet distillers grains with whole plant corn.

Duodenal and milk trans octadecenoic acid and conjugated linoleic acid (CLA) isomers indicate that postabsorptive synthesis is the predominant source of cis-9-containing CLA in lactating dairy cows.

Duodenal and milk samples obtained from lactating cows in a previous study were analyzed to compare the content and isomer distribution of conjugated linoleic acids (CLA) and trans-18:1 fatty acids (tFA). Four diets containing either low [25 g/100 g dry matter (DM)] or high (60 g/100 g DM) forage were fed with or without 2% added buffer to four multiparous Holstein dairy cows in a 2 x 2 factorial, 4 x 4 Latin square design with 3-wk experimental periods. Duodenal flows of CLA were low (1.02-1.84 g/d), compared with that of tFA (57-120 g/d), regardless of diet. The greatest amounts of CLA and tFA, as well as the greatest proportions of trans-10-18:1 (P < 0.02), and cis-9, trans-11 (P < 0.01) and trans-10, cis-12 CLA (P < 0.01) were in the duodenal flow of cows fed the low forage unbuffered diet. In milk fat, tFA were increased by the low forage unbuffered diet and the trans-10-18:1 (P < 0.02) replaced trans-11-18:1 as the major 18:1 isomer. Milk CLA secretion (7.2-9.1 g/d) was greater (P < 0.001) than that in the duodenal flow with each diet. This was due to the increase in cis-9, trans-11-18:2 and trans-7, cis-9 CLA, resulting most likely from endogenous synthesis via Delta9-desaturation of ruminally derived tFA. For other CLA isomers, duodenal flow was always greater than milk secretion, suggesting that they essentially were produced in the rumen.