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The gastrointestinal microbial consortium in dairy cattle is critical to determining the energetic status of the dairy cow from birth through her final lactation. The ruminant's microbial community can degrade a wide variety of feedstuffs, which can affect growth, as well as production rate and efficiency on the farm, but can also affect food safety, animal health, and environmental impacts of dairy production. Gut microbial diversity and density are powerful tools that can be harnessed to benefit both producers and consumers. The incentives in the United States to develop Alternatives to Antibiotics for use in food-animal production have been largely driven by the Veterinary Feed Directive and have led to an increased use of probiotic approaches to alter the gastrointestinal microbial community composition, resulting in improved heifer growth, milk production and efficiency, and animal health. However, the efficacy of direct-fed microbials or probiotics in dairy cattle has been highly variable due to specific microbial ecological factors within the host gut and its native microflora. Interactions (both synergistic and antagonistic) between the microbial ecosystem and the host animal physiology (including epithelial cells, immune system, hormones, enzyme activities, and epigenetics) are critical to understanding why some probiotics work but others do not. Increasing availability of next-generation sequencing approaches provides novel insights into how probiotic approaches change the microbial community composition in the gut that can potentially affect animal health (e.g., diarrhea or scours, gut integrity, foodborne pathogens), as well as animal performance (e.g., growth, reproduction, productivity) and fermentation parameters (e.g., pH, short-chain fatty acids, methane production, and microbial profiles) of cattle. However, it remains clear that all direct-fed microbials are not created equal and their efficacy remains highly variable and dependent on stage of production and farm environment. Collectively, data have demonstrated that probiotic effects are not limited to the simple mechanisms that have been traditionally hypothesized, but instead are part of a complex cascade of microbial ecological and host animal physiological effects that ultimately impact dairy production and profitability., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Calf diarrhea, a common disease mainly induced by Escherichia coli infection, is one of the main reasons for nonpredator losses. Hence, an effective nonantibacterial approach to prevent calf diarrhea has become an emerging requirement. This study evaluated the microalgae Schizochytrium sp. (SZ) and lactoferrin (LF) as a nutrient intervention approach against E. coli O101:K99-induced preweaning calve diarrhea. Fifty 1-d-old male Holstein calves were randomly divided into 5 groups (n = 10): (1) control, (2) blank (no supplement or challenge), (3) 1 g/d LF, (4) 20 g/d SZ, or (5) 1 g/d LF plus 20 g/d SZ (LFSZ). The experimental period lasted 14 d. On the morning of d 7, calves were challenged with 1 × 10 11 cfu of E. coli O101:K99, and rectum feces were collected on 3, 12, 24, and 168 h postchallenge for the control, LF, SZ, and LFSZ groups. The rectal feces of the blank group were collected on d 14. Data were analyzed using the mixed procedure of SAS (version 9.4; SAS Institute Inc.). The E. coli K99 challenge decreased the average daily gain (ADG) and increased feed-to-gain ratio (F:G) and diarrhea frequency (control vs. blank). Compared with the control group, the LFSZ group had a higher ADG and lower F:G, and the LFSZ and SZ groups had lower diarrhea frequency compared with the control group. In addition, the LFSZ and SZ groups have no differences in diarrhea frequency compared with the blank group. Compared with the control group, the blank group had lower serum nitric oxide (NO), endothelin-1, d-lactic acid (D-LA), and lipopolysaccharide (LPS) concentrations, as well as serum IgG, IL-1β, IL-6, IL-10, and TNF-α levels on d 7 and 14. On d 7, compared with the control group, all treatment groups had lower serum NO level, the SZ group had a lower serum D-LA concentration, and the LF and LFSZ groups had lower serum LPS concentration. On d 14, compared with the control group, the fecal microbiota of the blank group had lower Shannon, Simpson, Chao1, and ACE indexes, the LFSZ group had lower Shannon and Simpson indexes, the SZ and LFSZ groups had a higher Chao1 index, and all treatment groups had a higher ACE index. In fecal microbiota, Bifidobacterium and Actinobacteria were negatively associated with IL-10 and d-lactate, while Akkermansia was negatively associated with endothelin-1 and positively correlated with LPS, fecal scores, and d-lactate levels. Our results indicated that LF and SZ supplements could alleviate E. coli O101:K99-induced calf diarrhea individually or in combination. Supplementing 1 g/d LF and 20 g/d SZ could be a potential nutrient intervention approach to prevent bacterial diarrhea in calves., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Cashew nut shell extract (CNSE) is a byproduct of the cashew nut industry, containing bioactive compounds that alter rumen fermentation patterns. Therefore, study objectives were to evaluate the effects of CNSE (59% anacardic acid and 18% cardol) on production, rumen fermentation variables, metabolism, and inflammation in transition dairy cows. A total of 51 multiparous Holstein cows were used in a randomized design and assigned to treatment based on their previous 305-d mature equivalent milk and parity. Cows were assigned to 1 of 2 treatments 21 d before expected calving: (1) CON (control diet; n = 17) or (2) CNSE-5.0 (control diet and 5.0 g/d CNSE granule [containing 50% CNSE]; n = 34). Following parturition, 17 cows (preselected at initial treatment assignment) from the CNSE-5.0 treatment were reallocated into a third treatment group: CNSE-2.5 (control diet and 2.5 g/d CNSE granule; n = 17), resulting in 3 total treatments postpartum: (1) CON, (2) CNSE-2.5, and (3) CNSE-5.0. Prepartum rumen pH was unaltered by treatment; however, postpartum rumen pH was increased (0.31 units) in CNSE cows relative to CON. Prepartum rumen ammonia N concentration tended to be decreased (34%) in CNSE-5.0 cows compared with CON, and there tended to be a quadratic effect on postpartum ammonia N, as it was decreased in CNSE-2.5 compared with CON and CNSE-5.0. Prepartum dry matter intake (DMI) was unaffected by treatment; however, postpartum DMI was increased (8%) in CNSE cows relative to CON. No treatment differences were observed in pre- or postpartum digestibility measurements. Milk and protein yields from cows fed CNSE tended to be increased (6% and 7%, respectively) relative to CON. No treatment differences were detected for energy-corrected milk, feed efficiency, body weight, body condition score, energy balance, milk composition, milk urea nitrogen, or somatic cell count. Prepartum fecal pH decreased (0.12 units) in CNSE-5.0 cows relative to CON cows but was similar between treatments postpartum. Supplementing CNSE did not affect prepartum glucose, nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHB), or insulin. However, prepartum circulating blood urea nitrogen tended to be decreased and glucagon was decreased in CNSE-5.0 cows compared with CON (9 and 20%, respectively). Additionally, CNSE supplementation decreased glucose and insulin concentrations postpartum relative to CON cows (6% and 20%, respectively). Quadratic effects were detected for postpartum circulating NEFA and BHB such that their levels were increased in CNSE-2.5 cows relative to CON and CNSE-5.0. Pre- and postpartum circulating serum amyloid A, lipopolysaccharide-binding protein, and haptoglobin were unaffected by treatment. Overall, CNSE influenced some key rumen fermentation variables, altered postabsorptive metabolism, and increased production parameters in transition dairy cows., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Subacute rumen acidosis may cause postruminal intestinal barrier dysfunction, but this does not appear to be due to increased hindgut fermentation. Alternatively, intestinal hyperpermeability may be explained by the plethora of potentially harmful substances (e.g., ethanol, endotoxin, and amines) produced in the rumen during subacute rumen acidosis, which are difficult to isolate in traditional in vivo experiments. Therefore, objectives were to evaluate whether abomasal infusion of acidotic rumen fluid collected from donor (Donor) cows elicits systemic inflammation or alters metabolism or production in healthy recipients. Ten rumen-cannulated lactating dairy cows [249 ± 63 d in milk; 753 ± 32 kg of body weight (BW)] were randomly assigned to 1 of 2 abomasal infusion treatments: (1) healthy rumen fluid (HF; 5 L/h; n = 5) or (2) acidotic rumen fluid (AF; 5 L/h; n = 5) infused. Eight rumen-cannulated cows [4 dry, 4 lactating (lactating = 391 ± 220 d in milk); 760 ± 70 kg of BW] were used as Donor cows. All 18 cows were acclimated to a high-fiber diet (46% neutral detergent fiber; 14% starch) during an 11-d prefeeding period during which rumen fluid was collected for the eventual infusion into HF cows. During period (P) 1 (5 d), baseline data were obtained and on d 5 Donor were corn-challenged (2.75% BW ground corn after 16 h of 75% feed restriction). Cows were fasted until 36 h relative to rumen acidosis induction (RAI), and data were collected through 96 h RAI. At 12 h RAI, an additional 0.50% BW of ground corn was added, and acidotic fluid collections began (7 L/Donor every 2 h; 6 M HCl was added to collected fluid until pH was between 5.0 and 5.2). On d 1 of P2 (4 d), HF/AF cows were abomasally infused with their respective treatments for 16 h, and data were collected for 96 h relative to the first infusion. Data were analyzed in SAS (SAS Institute Inc.) using PROC MIXED. Following the corn challenge in the Donor cows, rumen pH only mildly decreased at nadir (pH = 5.64 at 8 h RAI) and remained above the desired threshold for both acute (5.2) and subacute (5.6) acidosis. In contrast, fecal and blood pH markedly decreased to acidotic levels (nadir = 4.65 and 7.28 at 36 and 30 h RAI, respectively), and fecal pH remained below 5 from 22 to 36 h RAI. In Donor cows, dry matter intake remained decreased through d 4 (36% relative to baseline) and serum amyloid A and lipopolysaccharide-binding protein markedly increased by 48 h RAI in Donor cows (30- and 3-fold, respectively). In cows that received the abomasal infusions, fecal pH decreased in AF from 6 to 12 h relative to the first infusion (7.07 vs. 6.33) compared with HF; however, milk yield, dry matter intake, energy-corrected milk, rectal temperature, serum amyloid A, and lipopolysaccharide-binding protein were unaffected. Overall, the corn challenge did not cause subacute rumen acidosis but markedly decreased fecal and blood pH and stimulated a delayed inflammatory response in the Donor cows. Abomasal infusion of rumen fluid from corn-challenged Donor cows decreased fecal pH but did not cause inflammation, nor did it create an immune-activated phenotype in recipient cows., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to investigate the effects of dietary energy levels and rumen-protected lysine supplementation on serum free fatty acid levels, β-hydroxybutyrate levels, dry matter (DM) intake, and milk production and composition. Treatments were arranged in a 2 × 2 factorial design with 2 dietary energy levels [high net energy for lactation (NE L ) = 1.53 Mcal/kg of DM vs. low NE L = 1.37 Mcal/kg of DM; HE vs. LE) fed either with rumen-protected lysine (bypass lysine; 40 g/cow per day) or without rumen-protected lysine (control). Sixty-eight third-lactation Holstein dairy cows entering their fourth lactation were randomly allocated to 4 treatments groups: HE with bypass lysine, HE without bypass lysine, LE with bypass lysine, and LE without bypass lysine. Groups were balanced based upon their expected calving date, previous milk yields, and body condition score. All cows were fed the same diet (NE L = 1.34 Mcal/kg of DM) during the dry period prior to the trial. Rumen-protected lysine was top-dressed on a total mixed ration to deliver 9.68 g/d of metabolizable lysine to pre- and postpartum cows. After calving, all cows received the same TMR (1.69 Mcal/kg of DM). Blood samples were collected at -21, -14, -7, 0, 3, 7, 14, and 21 d relative to calving, and free fatty acids and β-hydroxybutyrate concentrations were measured. Amount of feed offered and orts were collected and measured for individual cows 4 d/wk. Milk samples were collected once per week following calving, and milk composition was analyzed. Feeding high NE L to close-up cows decreased the concentrations of free fatty acid and β-hydroxybutyrate in prepartum cows but not in postpartum cows. Addition of rumen-protected lysine increased postpartum DM intake, and decreased serum free fatty acid and β-hydroxybutyrate concentrations. Neither energy nor rumen-protected lysine supplementation nor their interaction affected milk yield or fat or lactose yields. However, cows in the group receiving HE with bypass lysine tended to produce more milk compared with other groups and had a lower blood β-hydroxybutyrate concentration postpartum. These results indicate that feeding a high-energy diet together with rumen-protected lysine improved DM intake and lowered serum free fatty acid and β-hydroxybutyrate concentrations in transition cows., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of the current study was to examine the effect of pasteurization of waste milk, used to feed dairy calves, on the bacterial diversity of their lower gut. Using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing, fecal samples from dairy calves, ages 1 wk to 6 mo old and fed either pasteurized or nonpasteurized waste milk, were analyzed for bacterial diversity. Calves were maintained on 2 separate farms and, aside from how the waste milk was treated, were housed and cared for similarly. Fifteen calves were sampled from each age group (1, 2, and 4 wk, and 2, 4, and 6 mo of age; n=90 samples per milk treatment, 180 total samples) on each farm via rectal palpation and the samples shipped and frozen before analysis. In general, bacterial diversity, as represented by the total number of different species, was greater for the calves fed pasteurized waste milk at all ages (except 1 wk of age) and increased with increasing age in both treatments. Proteobacteria, Bacteroidetes, and Firmicutes were the predominant phyla. Differences in phyla and class were observed among treatments and age of calf but with no consistent trends. Salmonella were detected in 9 out of 14 (64%) of the 1-wk-old calves fed nonpasteurized milk. Treponema, an important beneficial bacterium in cattle rumen, was more prevalent in the pasteurized waste milk-fed animals and became higher in the older animals from this group. Escherichia-Shigella were detected among treatments at all ages, and highest at 1 wk of age, averaging approximately 21 and 20% of all bacteria for calves fed pasteurized and nonpasteurized waste milk, respectively, and decreasing as calves aged (2.6 and 1.3%). The consistent detection of Salmonella in the younger animals fed nonpasteurized milk and its absence in all other groups is an important finding related to this feeding practice., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to evaluate the fermentation dynamics of 2 commonly fed corn (co)products in their intact and defatted forms, using the in vitro gas production (IVGP) technique, and to investigate the shifts of the predominant rumen bacterial populations using the 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) technique. The bTEFAP technique was used to determine the bacterial profile of each fermentation time at 24 and 48 h. Bacterial populations were identified at the species level. Species were grouped by substrate affinities (guilds) for cellulose, hemicellulose, pectin, starch, sugars, protein, lipids, and lactate. The 2 (co)products were a dried distillers grain (DDG) plus solubles produced from a low-heat drying process (BPX) and a high-protein DDG without solubles (HP). Chemical analysis revealed that BPX contained about 11.4% ether extract, whereas HP contained only 3.88%. Previous studies have indicated that processing methods, as well as fat content, of corn (co)products directly affect fermentation rate and substrate availability, but little information is available regarding changes in rumen bacterial populations. Fermentation profiles of intact and defatted BPX and HP were compared with alfalfa hay as a standard profile. Defatting before incubation had no effect on total gas production in BPX or HP, but reduced lag time and the fractional rate of fermentation of BPX by at least half, whereas there was no effect for HP. The HP feed supported a greater percentage of fibrolytic and proteolytic bacteria than did BPX. Defatting both DDG increased the fibrolytic (26.8 to 38.7%) and proteolytic (26.1 to 37.2%) bacterial guild populations and decreased the lactate-utilizing bacterial guild (3.06 to 1.44%). Information regarding the fermentation kinetics and bacterial population shifts when feeding corn (co)products may lead to more innovative processing methods that improve feed quality (e.g., deoiling) and consequently allow greater inclusion rates in dairy cow rations., (Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Salmonella is one of the most serious foodborne pathogenic bacteria in the United States, causing an estimated 1.3 million human illnesses each year. Dairy cows can be reservoirs of foodborne pathogenic bacteria, including Salmonella spp.; it is estimated that from 27 to 31% of dairy herds across the United States are colonized by Salmonella. The present study was designed to examine the occurrence of Salmonella spp. on dairies and to examine the serotypic diversity of Salmonella isolates on sampled dairies from across the United States. Fecal samples (n = 60 per dairy) were collected from 4 dairies in each of 4 states for a total of 960 fecal samples representing a total population of 13,200 dairy cattle. In the present study, 93 of 960 samples (9.96%) collected were culture-positive for Salmonella enterica. At least one Salmonella fecal-shedding cow was found in 9 of the 16 herds (56%) and the within-herd prevalence varied in our study from 0% in 7 herds to a maximum of 37% in 2 herds, with a mean prevalence among Salmonella-positive herds of 17%. Seventeen different serotypes were isolated, representing 7 different Salmonella serogroups. There were 2 or more different serogroups and serotypes present on 7 of the 9 Salmonella-positive farms. Serotypes Montevideo and Muenster were the most frequent and widespread. From our data, it appears that subclinical colonization with Salmonella enterica is relatively common on dairy farms and is represented by diverse serotypes on US dairy farms.

Although Escherichia coli are commensal organisms that reside within the host gut, some pathogenic strains of E. coli can cause hemorrhagic colitis in humans. The most notable enterohemorrhagic E. coli (EHEC) strain is O157:H7. Cattle are asymptomatic natural reservoirs of E. coli O157:H7, and it has been reported that as many as 30% of all cattle are carriers of this pathogen, and in some circumstances this can be as high as 80%. Feedlot and high-producing dairy cattle are fed large grain rations in order to increase feed efficiency. When cattle are fed large grain rations, some starch escapes ruminal microbial degradation and passes to the hind-gut where it is fermented. EHEC are capable of fermenting sugars released from starch breakdown in the colon, and populations of E. coli have been shown to be higher in grain fed cattle, and this has been correlated with E. coli O157:H7 shedding in barley fed cattle. When cattle were abruptly switched from a high grain (corn) diet to a forage diet, generic E. coli populations declined 1000-fold within 5 d, and the ability of the fecal generic E. coli population to survive an acid shock similar to the human gastric stomach decreased. Other researchers have shown that a switch from grain to hay caused a smaller decrease in E. coli populations, but did not observe the same effect on gastric shock survivability. In a study that used cattle naturally infected with E. coli O157:H7, fewer cattle shed E. coli O157:H7 when switched from a feedlot ration to a forage-based diet compared with cattle continuously fed a feedlot ration. Results indicate that switching cattle from grain to forage could potentially reduce EHEC populations in cattle prior to slaughter; however the economic impact of this needs to be examined.

The objective of this study was to determine the concentration of malate in forage varieties at different stages of maturity. Five alfalfa varieties (Alfagraze, Apollo Supreme Cimarron, Crockett, and Magnum III) and three bermudagrass varieties (Coastal, Tifton-78, and Tifton-85) were collected at different stages of maturity. Samples were collected from replicate plots (n = 3) of each alfalfa variety at 9, 18, 28, 35, and 42 d of maturity; bermudagrass hay samples were composited from six bales of each variety from two cuttings staged to be harvested at 27 and 41 d of maturity. Malate was extracted from the samples and quantitated by high performance liquid chromatography using an organic acid column. As maturity increased, the concentration of malate declined in both plant species. Concentrations of malate were numerically higher in two alfalfa varieties (Crockett and Magnum III) at 35 and 42 d of maturity than in all other alfalfa varieties. Concentrations of malate in bermudagrass at 41 d of maturity were lower than concentrations of malate in all alfalfa varieties at 42 d of maturity. Malate declined as maturity increased in the Coastal and Tifton-78 varieties. Because malate stimulates the utilization of lactate by the predominant ruminal bacterium Selenomonas ruminantium, some of the benefits associated with alfalfa in the diets of dairy cattle may be due to the malate in this forage.

The objective of this study was to determine the effects of cellobiose and monensin on the in vitro fermentation of organic acids (L-aspartate, fumarate, and DL-malate) by mixed ruminal bacteria. Ruminal fluid was collected from a steer fed 36.7 kg of forage and 4.5 kg of concentrate supplement once per day. Ruminal fluid was centrifuged to sediment feed particles and protozoa, and the resulting supernatant, which contained bacteria, was added (33%, vol/vol) to anaerobic media (500 ml). Incubations (n = 2) were performed in batch culture at 39 degrees C and sampled at 0, 2, 4, 6, 8, 12, and 24 h. Organic acids were added to achieve a final concentration of 7.5 mM. Cellobiose was added to obtain a final concentration of 5 mM, and monensin dissolved in ethanol was included at concentrations of 0 or 5 ppm. Addition of cellobiose to organic acid fermentations increased the rate of organic acid utilization by the mixed bacterial population. Total concentrations of volatile fatty acids were increased by the addition of cellobiose to all fermentations. A lag period (< or = 8 h) occurred in fermentations that were treated with monensin before organic acids were utilized. Total concentrations of volatile fatty acids were increased, and the acetate to propionate ratio was decreased, by monensin treatment. When cellobiose and monensin were added together, propionate production and organic acid utilization were increased. Both cellobiose and monensin affected the in vitro fermentation of organic acids by mixed ruminal bacteria by providing a carbon and energy source and by influencing electron disposal.