Tumor necrosis factor alpha and palmitate simulate bovine fatty liver disease in vitro when using abattoir-derived primary bovine hepatocytes isolated by a novel nonperfusion method.

Hepatic lipidosis (i.e., fatty liver) is a common periparturient disease in high-producing dairy cattle affecting nearly 50% of cows to some degree and costing an estimated 60 million dollars annually. Large animal studies are costly, labor intensive, and are not well suited to mechanistic studies. Traditionally, mechanistic studies employ in vitro methodologies, utilizing established cell lines or primary cell culture methods. However, with dairy cattle, established hepatic cell lines do not exist, and methods for primary cell culture studies typically involve complicated procedures that often utilize very young animals (typically bull calves). Several previously published papers have used abattoir-derived tissues as a source of primary cells; however, a simple method utilizing simple culture media has yet to be presented. In addition, we sought to develop a way to replicate the syndrome of fatty liver disease "in a dish" using adult cattle that should more closely represent the physiology of the periparturient dairy cow. Herein we present a non-perfusion-based method that results in robust growth and proliferation of abattoir-derived bovine hepatocytes that demonstrate lipid loading, elevated lactate dehydrogenase leakage, and cytotoxicity as demonstrated by elevated caspase 3/7 expression consistent with in vivo physiology of the periparturient dairy cow with fatty liver disease.

Non-coding RNA in raw and commercially processed milk and putative targets related to growth and immune-response.

BACKGROUND: Bovine milk contains extracellular vesicles (EVs) that play a role in cellular communication, acting in either an autocrine, paracrine, or an exocrine manner. The unique properties of the EVs protect the cargo against degradation. We profiled the ncRNAs (non-coding RNA) present in the EVs from seven dairy products - raw whole milk, heat-treated skim milk, homogenized heat-treated skim milk, pasteurized homogenized skim milk, pasteurized heavy whipping cream, sweet cream buttermilk and cultured buttermilk with four replicates each, obtained at different processing steps from a commercial dairy plant. EVs and their cargo were extracted by using a validated commercial kit that has been shown to be efficient and specific for EVs. Further, to find the annotation of ncRNAs, we probed bovine and other organism repositories(such as miRBase, miRTarBase, Ensemble) to find homolog ncRNA annotation in case the annotations of ncRNA are not available in Bos Taurus database. RESULTS: Specifically, 30 microRNAs (miRNAs), were isolated throughout all the seven milk samples, which later when annotated with their corresponding 1546 putative gene targets have functions associated with immune response and growth and development. This indicates the potential for these ncRNAs to beneficially support mammary health and growth for the cow as well as neonatal gut maturation. The most abundant miRNAs were bta-miR-125a and human homolog miR-718 based on the abundance values of read count obtained from the milk samples.bta-miR-125a is involved in host bacterial and viral immune response, and human homolog miR-718 is involved in the regulation of p53, VEGF, and IGF signaling pathways, respectively. Sixty-two miRNAs were up-regulated and 121 miRNAs were down-regulated throughout all the milk samples when compared to raw whole milk. In addition, our study explored the putative roles of other ncRNAs which included 88 piRNAs (piwi-interacting RNA), 64 antisense RNAs, and 105 lincRNAs (long-intergenic ncRNAs) contained in the bovine exosomes. CONCLUSION: Together, the results indicate that bovine milk contains significant numbers of ncRNAs with putative regulatory targets associated with immune- and developmental-functions important for neonatal bovine health, and that processing significantly affects the ncRNA expression values; but statistical testing of overall abundance(read counts) of all miRNA samples suggests abundance values aren't much affected. This can be attributed to the breakage of exosomal vesicles during the processing stages. It is worth noting, however, that these gene regulatory targets are putative, and further evidence could be generated through experimental validation.

Bacterial communities in the rumen and feces of lactating Holstein dairy cows are not affected when fed reduced-fat dried distillers' grains with solubles.

Reduced-fat dried distillers' grains with solubles (RF-DDGSs) are co-products of ethanol production and contain less fat than traditional distillers' grains. The fat in corn is ~91% unsaturated, and it is toxic to rumen microorganisms so it could influence the composition of the rumen microbiome. It has been demonstrated that RF-DDGS is a suitable ration ingredient to support the high-producing dairy cow, and this feedstuff is a promising alternative protein source for lactating dairy cows. The current study aims to better understand the effect of RF-DDGS on the rumen and fecal bacterial composition in lactating dairy cows. Thirty-six multiparous (two or three), mid-lactation Holstein cows (BW = 680 ± 11 kg; 106 ± 27 DIM) were randomly assigned to two groups which were fed a control diet made up of corn, corn silage, and alfalfa hay supplemented with expeller soybean meal or with added RF-DDGS (20% of the DM) containing approximately 6.0% fat. Whole rumen contents (rumen fluid and digesta; esophageal tubing method) and feces (free-catch method) were collected on day 35 of the experimental period, after the 14-d acclimation period. Rumen contents and feces from each cow were used for DNA extraction. The bacterial community composition in rumen and fecal samples was assessed via the 16S rRNA gene by using the Illumina MiSeq sequencing platform. Bacteroidetes, Actinobacteria, and Firmicutes were the most abundant phyla in rumen contents. The fecal microbiota was dominated by the phyla Firmicutes and Bacteroidetes, as well as Actinobacteria and Chloroflexi. RF-DGGS increased bacterial richness, evenness, and Shannon diversity in both rumen and fecal samples and was associated with several taxa that had different abundance in treatment versus control comparisons. The RF-DGGS, however, did not significantly alter the bacterial community in the rumen or feces. In general, these findings demonstrated that dietary inclusion of RF-DDGS did not impose any serious short-term (within 30 days) health or production consequences, as would be expected. With this study, we present further evidence that inclusion of 20% (DM basis) RF-DDGS in the diet of lactating dairy cows can be done without consequence on the microbiome of the rumen.

Feeding reduced-fat dried distillers grains with solubles to lactating Holstein dairy cows does not alter milk composition or cause late blowing in cheese.

Feeding dried distillers grains with solubles (DDGS) to lactating dairy cows has been implicated as a cause of late blowing defects in the production of Swiss-style cheeses. Our objectives were (1) to test the effect of feeding reduced-fat DDGS (RF-DDGS; ∼6% fat) to lactating dairy cows on the composition of milk and on the suitability of the milk for production of baby Swiss cheese and (2) to evaluate the effect of diet on cow lactation performance. Lactating Holstein dairy cows were fed both dietary treatments in a 2 × 2 crossover design. Cows were housed in a 48-cow freestall pen equipped with individual feeding gates to record feed intake. The control diet was a corn, corn silage, and alfalfa hay diet supplemented with mechanically expelled soybean meal. The experimental diet was the same base ration, but 20% (dry matter basis) RF-DDGS were included in place of the expelled soybean meal. The RF-DDGS diet was additionally supplemented with rumen-protected lysine; diets were formulated to be isoenergetic and isonitrogenous. Cows were allowed ad libitum access to feed and water, fed twice daily, and milked 3 times daily. For cheese production, milk was collected and pooled 6 times for each dietary treatment. There was no treatment effect on milk yield (35.66 and 35.39 kg/d), milk fat production (1.27 and 1.25 kg/d), milk fat percentage (3.65 and 3.61%), milk protein production (1.05 and 1.08 kg/d), lactose percentage (4.62 and 4.64%), milk total solids (12.19 and 12.28%), and somatic cell count (232.57 and 287.22 × 103 cells/mL) for control and RF-DDGS, respectively. However, dry matter intake was increased by treatment, which implied a reduction in feed efficiency. Milk protein percentage also increased (3.01 and 3.11%), whereas milk urea nitrogen decreased (14.18 and 12.99 mg/dL), indicating that protein utilization may be more efficient when cows are fed RF-DDGS. No differences in cheese were observed by a trained panel except cheese appearance; control cheese eyes were significantly, but not practically, larger than the RF-DDGS cheese. These results indicate that RF-DDGS can be effectively used in the rations of lactating Holstein cows with no deleterious effects on milk production and composition and metrics of the physiology of the cow (i.e., blood glucose and nonesterified fatty acids); however, feeding RF-DDGS increased dry matter intake, which decreased feed efficiency. Finally, feeding RF-DDGS did not negatively influence quality and suitability of milk for production of baby Swiss cheese.

Short communication: No antimicrobial effects from one source of commercial dried distillers grains with solubles.

Because residual antibiotics in dried distillers grains with solubles (DDGS) could lead to inadvertent feeding of antibiotics to animals, the objective of our study was to determine if a commercial DDGS contained antibiotics. The DDGS used in a feeding study, and milk from cows fed the DDGS, were below the detection limits for at least 17 antibiotics. Additionally, we evaluated if DDGS had any antimicrobial effect against Salmonella Typhimurium, Listeria innocua, Escherichia coli ATCC 25922, Staphylococcus aureus, Pediococcus acidilactici, Lactobacillus casei, Lactobacillus acidophilus, Bacillus licheniformis, Paenibacillus odorifer, Pseudomonas fluorescens, and Paenibacillus amylolyticus using the disk diffusion seeded agar overlay method. Neither the buffered nor nonbuffered water-soluble fractions of DDGS yielded clear zones around disks, indicating that the water-soluble DDGS fraction had no antimicrobial properties against any of the microorganisms tested. The absence of antibiotic residues in DDGS and milk samples in this study confirmed that this source of DDGS can be used as livestock feed without fear of inadvertent feeding of antibiotics.

Dried distillers grains with solubles do not always cause late blowing in baby Swiss cheese.

Late blowing in Swiss cheese, a result of unwanted gas production, is unacceptable to consumers and causes economic loss to manufacturers. Cheese processors have raised concerns that feeding dried distillers grains with solubles (DDGS) to cows leads to this defect, in part because of clostridial spores. We hypothesized that spores in DDGS would affect the quality of milk and baby Swiss cheese by promoting late-blowing defects. Thirty healthy multiparous and mid-lactation Holstein cows were fed total mixed ration containing DDGS (0, 10, and 20%; 10 cows per treatment group) by dietary dry matter in a 3×3 Latin square design. One complete milking from all cows within a treatment was collected and pooled for baby Swiss cheese, twice within each month of the 3-mo study. Additionally, individual milk samples from the 3 milkings of one day were collected weekly for proximate analysis. Incubation in reinforced clostridial medium-lactate medium tubes inoculated with milk, cheese, total mixed ration, or manure showed gas formation. Conversely, the DDGS used in our study did not contain gas-producing, spore-forming bacteria. Feeding 20% DDGS decreased milk fat percent and increased the solids nonfat, protein, and lactose percent of milk. After 60 d of ripening, baby Swiss cheese had typical propionic acid Swiss cheese aroma. Regardless of dietary treatment, pinholes, slits, splits, cracks, or a combination of these, were seen throughout most cheeses. Feeding of DDGS increased the amount of long-chain unsaturated fatty acids and decreased short-chain and most medium-chain fatty acids in the baby Swiss cheese. Although feeding cows diets with DDGS modified milk composition, and subsequently cheese composition, DDGS was not a source for gas-producing, spore-forming bacteria or for quality defects in Swiss cheese. Rather, the gas-producing, spore-forming bacteria likely originated from the environment or the cows themselves.

Feeding dried distillers grains with solubles affects composition but not oxidative stability of milk.

Feeding lactating dairy cows dried distillers grains with solubles (DDGS) increases the concentration of unsaturated fatty acids in the milk from those cows, potentially leading to increased susceptibility to development of off-flavors. Feeding DDGS has been loosely implicated to be a cause of development of spontaneous oxidative off-flavor in milk. We hypothesized that increased feeding of DDGS would accelerate development of off-flavors and that fortification with vitamin E (0.06% wt/wt) or C (0.06% wt/wt) would prevent spontaneous oxidative off-flavors. The objective of this research was to determine the effects of feeding DDGS to lactating dairy cows on several parameters of milk quality as determined by both chemical and sensory evaluations. Twenty-four healthy mid-lactation Holstein dairy cows were fed total mixed rations containing DDGS (0, 10, or 25% dry matter). Cows were blocked by parity and randomly assigned to 1 of 2 groups (12 cows each). Each group received all 3 treatments in a 3-period Youden square design so that each cow served as her own control. Samples of milk from individual cows for proximate analysis and pooled milk for pasteurization and sensory analysis were collected on d 14, 21, and 28 of each experimental period. Pooled milk was assayed for peroxides and free fatty acids and evaluated by a trained sensory panel for the presence of 7 off-flavors common to milk on d 1, 3, and 7. Feeding 25% DDGS caused a significant decrease in daily milk yield. Increased dietary inclusion of DDGS also caused a concomitant decrease in percentage of milk fat and an increase in percentages of both solids nonfat and protein. Milk peroxides and free fatty acids were almost all below the detection limit, and the few exceptions were not found in replicated analyses. Sensory analysis revealed off-flavors only in milk from cows fed 0% DDGS when that milk was stored for 7d and when milk from cows fed 25% DDGS was fortified with 0.06% (wt/wt) vitamin C. Those few detected off-flavor scores were less than 1.5cm on a 15-cm line scale, indicating that the differences are not practically significant. Peroxide values support the findings by the sensory panel that both feeding DDGS at 10 and 25% and vitamin E and C fortification did not practically change the oxidative stability of milk. These results, taken together, indicate that feeding DDGS under our experimental conditions modified milk composition, but did not contribute to the development of off-flavors in milk.