Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. II: Biohydrogenation and incorporation into bacterial lipids.

To maintain membrane homeostasis, ruminal bacteria synthesize branched-chain fatty acids (BCFA) or their derivatives (vinyl ethers) that are recovered during methylation procedures as branched-chain aldehydes (BCALD). Many strains of cellulolytic bacteria require 1 or more branched-chain volatile fatty acid (BCVFA). Therefore, the objective of this study was to investigate BCVFA incorporation into bacterial lipids under different dietary conditions. The study was an incomplete block design with 8 continuous culture fermenters used in 4 periods with treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were high (HF) or low forage (LF, 67 or 33% forage, 33:67 alfalfa:orchardgrass), without or with supplemental corn oil (CO; 3% dry matter, 1.5% linoleic fatty acid), and without or with 2.15 mmol/d (5 mg/d 13C each of isovalerate, isobutyrate, and 2-methylbutyrate). After methylation of bacterial pellets collected from each fermenter's effluent, fatty acids and fatty aldehydes were separated before analysis by gas chromatography and isotope ratio mass spectrometry. Supplementation of BCVFA did not influence biohydrogenation extent. Label was only recovered in branched-chain lipids. Lower forage inclusion decreased BCFA in bacterial fatty acid profile from 9.45% with HF to 7.06% with LF and decreased BCALD in bacterial aldehyde profile from 55.4% with HF to 51.4% with LF. Supplemental CO tended to decrease iso even-chain BCFA and decreased iso even-chain BCALD in their bacterial lipid profiles. The main 18:1 isomer was cis-9 18:1, which increased (P < 0.01) by 25% from CO (data not shown). Dose recovery in bacterial lipids was 43.3% lower with LF than HF. Supplemental CO decreased recovery in the HF diet but increased recovery with LF (diet × CO interaction). Recovery from anteiso odd-chain BCFA and BCALD was the greatest; therefore, 2-methylbutyrate was the BCVFA primer most used for branched-chain lipid synthesis. Recovery in iso odd-chain fatty acids (isovalerate as primer) was greater than label recovery in iso even-chain fatty acids (isobutyrate as primer). Fatty aldehydes were less than 6% of total bacterial lipids, but 26.0% of 13C recovered in lipids were recovered in BCALD because greater than 50% of aldehydes were branched-chain. Because BCFA and BCALD are important in the function and growth of bacteria, especially cellulolytics, BCVFA supplementation can support the rumen microbial consortium, increasing fiber degradation and efficiency of microbial protein synthesis.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. III: Protein metabolism and incorporation into bacterial protein.

Some cellulolytic bacteria cannot transport branched-chain AA (BCAA) and do not express complete synthesis pathways, thus depending on cross-feeding for branched-chain volatile fatty acid (BCVFA) precursors for membrane lipids or for reductive carboxylation to BCAA. Our objective was to assess BCVFA uptake for BCAA synthesis in continuous cultures administered high forage (HF) and low forage (LF) diets without or with corn oil (CO). We hypothesized that BCVFA would be used for BCAA synthesis more in the HF than in LF diets. To help overcome bacterial inhibition by polyunsaturated fatty acids in CO, BCVFA usage for bacterial BCAA synthesis was hypothesized to decrease when CO was added to HF diets. The study was an incomplete block design with 8 dual-flow fermenters used in 4 periods with 8 treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were: HF or LF (67 or 33% forage, 33:67 alfalfa:orchardgrass pellets), without or with supplemental CO (3% of dry matter), and without or with 2.15 mmol/d (5 mg/d 13C) each of isovalerate, isobutyrate, and 2-methylbutyrate for one combined BCVFA treatment. The flow of bacterial BCAA increased by 10.7% by supplementing BCVFA and 9.14% with LF versus HF; similarly, dosing BCVFA versus without BCVFA increased BCAA by 1.98% in total bacterial AA, whereas LF increased BCAA by 1.92% versus HF. Additionally, BCVFA supplementation increased bacterial AA flow by 16.6% when supplemented in HF - CO and 12.4% in LF + CO diets, but not in the HF + CO (-1.5%) or LF - CO (+6.7%) diets (Diet × CO × BCVFA interaction). The recovery of 13C in bacterial AA flow was 31% lower with LF than with HF. Of the total 13C recovered in bacteria, 13.8, 17.3, and 30.2% were recovered in Val, Ile, and Leu, respectively; negligible 13C was recovered in other AA. When fermenters were dosed with BCVFA, nonbacterial and total effluent flows of AA, particularly of alanine and proline, suggest decreased peptidolysis. Increased ruminal outflow of bacterial AA, especially BCAA, but also nonbacterial AA could potentially support postabsorptive responses from BCVFA supplementation to dairy cattle.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. I: Digestibility, microbial protein, and prokaryotic community structure.

Branched-chain amino acids are deaminated by amylolytic bacteria to branched-chain volatile fatty acids (BCVFA), which are growth factors for cellulolytic bacteria. Our objective was to determine the dietary conditions that would increase the uptake of BCVFA by rumen bacteria. We hypothesized that increased forage would increase cellulolytic bacterial abundance and incorporation of BCVFA into their structure. Supplemental polyunsaturated fatty acids, supplied via corn oil (CO), should inhibit cellulolytic bacteria growth, but we hypothesized that additional BCVFA would alleviate that inhibition. Further, supplemental BCVFA should increase neutral detergent fiber degradation and efficiency of bacterial protein synthesis more with the high forage and low polyunsaturated fatty acid dietary combination. The study was an incomplete block design with 8 dual-flow continuous cultures used in 4 periods with 8 treatments (n = 4 per treatment) arranged as a 2 × 2 × 2 factorial. The factors were: high forage (HF) or low forage (LF; 67 or 33%), without or with supplemental CO (3% dry matter), and without or with 2.15 mmol/d (which included 5 mg/d of 13C each of BCVFA isovalerate, isobutyrate, and 2-methylbutyrate). The isonitrogenous diets consisted of 33:67 alfalfa:orchardgrass pellet, and was replaced with a concentrate pellet that mainly consisted of ground corn, soybean meal, and soybean hulls for the LF diet. The main effect of supplementing BCVFA increased neutral detergent fiber (NDF) degradability by 7.6%, and CO increased NDF degradability only in LF diets. Supplemental BCVFA increased bacterial N by 1.5 g/kg organic matter truly degraded (6.6%) and 0.05 g/g truly degraded N (6.5%). The relative sequence abundance decreased with LF for Fibrobacter succinogenes, Ruminococcus flavefaciens, and genus Butyrivibrio compared with HF. Recovery of the total 13C dose in bacterial pellets decreased from 144 µg/ mg with HF to 98.9 µg/ mg with LF. Although isotope recovery in bacteria was greater with HF, BCVFA supplementation increased NDF degradability and efficiency of microbial protein synthesis under all dietary conditions. Therefore, supplemental BCVFA has potential to improve feed efficiency in dairy cows even with dietary conditions that might otherwise inhibit cellulolytic bacteria.

Assessing milk response to different combinations of branched-chain volatile fatty acids and valerate in Jersey cows.

Some cellulolytic bacteria require 1 or more branched-chain volatile fatty acids (BCVFA) for the synthesis of branched-chain AA and branched-chain long-chain fatty acids because they are not able to uptake branched-chain AA or lack 1 or more enzymes to synthesize branched-chain AA de novo. Supplemental BCVFA and valerate were included previously as a feed additive that was later removed from the market; these older studies and more current studies have noted improvements in neutral detergent fiber digestibility and milk efficiency. However, most studies provided a single BCVFA or else isobutyrate (IB), 2-methylbutyrate (MB), isovalerate, and valerate altogether without exploring optimal combinations. Our objective was to determine a combination of isoacids that is optimal for milk production. Sixty (28 primiparous and 32 multiparous) lactating Jersey cows (106 ± 54 days in milk) were blocked and assigned randomly to either a control (CON) treatment without any isoacids, MB [12.3 mmol/kg dry matter (DM)], MB + IB (7.7 and 12.6 mmol/kg DM of MB and IB, respectively), or all 4 isoacids (6.2, 7.3, 4.2, and 5.1 mmol/kg DM of MB, IB, isovalerate, and valerate, respectively). Cattle were fed the CON treatment for a 2-wk period, then were assigned randomly within a block to treatments for 8 wk (n = 15). There was a trend for an interaction of supplement and parity for milk components. There were no differences in components for primiparous cows, whereas MB + IB tended to increase protein concentration by 0.04 and 0.08 percentage units in multiparous cows compared with the CON and MB treatments, respectively. Feeding MB + IB increased fat concentration by 0.23 to 0.31 percentage units compared with all other treatments in multiparous cows. Milk yield and dry matter intake (DMI) did not change with treatment. Treatment interacted with week for milk net energy for lactation/DMI; MB + IB tended to increase milk net energy of lactation/DMI by 0.10 Mcal/kg compared with MB and approached a trend for CON, mainly during the early weeks of the treatment period, whereas differences decreased during the last 2 wk of the treatment period. Cows fed MB had the highest 15:0 anteiso fatty acids in the total milk fatty acid profile, which was greater than that for CON or MB + IB cows, but not cows supplemented with isoacids. Cows fed MB alone had the numerically lowest milk net energy for lactation/DMI. The combination of MB + IB appeared optimal for increasing feed efficiency in our study and was not at the expense of average daily gain. Further research is needed for evaluating how potential changes in supplemental isoacid dosage should vary under differing dietary conditions.

Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 1. Production, milk fatty acid profile, and nutrient digestibility.

The objectives of this experiment were to determine the effects of increased diet fermentability and polyunsaturated fatty acids (FA) with or without supplemental 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), isoacids (IA; isobutyrate, 2-methylbutyrate, isovalerate, and valerate) or the combination of these on milk fat depression (MFD). Ten Holstein cows (194 ± 58 DIM, 691 ± 69 kg BW, 28 ± 5 kg milk yield) were used in a replicated 5 × 5 Latin square design. Treatments included a high-forage control diet (HF-C), a low-forage control diet (LF-C) causing MFD by increasing starch and decreasing neutral detergent fiber (NDF), the LF-C diet supplemented with HMTBa at 0.11% (28 g/d), the LF-C diet supplemented with IA at 0.24% of dietary dry matter (60 g/d), and the LF-C diet supplemented with HMTBa and IA. Preplanned contrasts were used to compare HF-C versus LF-C and to examine the main effects of HMTBa or IA and their interactions within the LF diets. Dry matter intake was greater for LF-C versus HF-C, but milk yield remained unchanged. The LF-C diet decreased milk fat yield (0.87 vs. 0.98 kg/d) but increased protein yield compared with HF-C. As a result, energy-corrected milk was lower (28.5 vs. 29.6 kg/d) for LF-C versus HF-C. Although the concentration of total de novo synthesized FA in milk fat was not affected, some short- and medium-chain FA were lower for LF-C versus HF-C, but the concentrations of C18 trans-10 isomers were not different. Total-tract NDF apparent digestibility was numerically lower (42.4 vs. 45.6%) for LF-C versus HF-C. As the main effects, the decrease in milk fat yield observed in LF-C was alleviated by supplementation of HMTBa through increasing milk yield without altering milk fat content and by IA through increasing milk fat content without altering milk yield so that HMTBa or IA, as the main effects, increased milk fat yield within the LF diets. However, interactions for milk fat yield and ECM were observed between HMTBa and IA, suggesting no additive effect when used in combination. Minimal changes were found on milk FA profile when HMTBa was provided. However, de novo synthesized FA increased for IA supplementation. We detected no main effect of HMTBa, IA, and interaction between those on total-tract NDF digestibility. In conclusion, the addition of HMTBa and IA to a low-forage and high-starch diet alleviated moderate MFD. Although the mechanism by which MFD was alleviated was different between HMTBa and IA, no additive effects of the combination were observed on milk fat yield and ECM.

Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 2. Ruminal fermentation, fatty acid, and bacterial community structure.

The experiment was conducted to understand ruminal effects of diet modification during moderate milk fat depression (MFD) and ruminal effects of 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa) and isoacids on alleviating MFD. Five ruminally cannulated cows were used in a 5 × 5 Latin square design with the following 5 dietary treatments (dry matter basis): a high-forage and low-starch control diet with 1.5% safflower oil (HF-C); a low-forage and high-starch control diet with 1.5% safflower oil (LF-C); the LF-C diet supplemented with HMTBa (0.11%; 28 g/d; LF-HMTBa); the LF-C diet supplemented with isoacids [(IA) 0.24%; 60 g/d; LF-IA]; and the LF-C diet supplemented with HMTBa and IA (LF-COMB). The experiment consisted of 5 periods with 21 d per period (14-d diet adaptation and 7-d sampling). Ruminal samples were collected to determine fermentation characteristics (0, 1, 3, and 6 h after feeding), long-chain fatty acid (FA) profile (6 h after feeding), and bacterial community structure by analyzing 16S gene amplicon sequences (3 h after feeding). Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) in a Latin square design. Preplanned comparisons between HF-C and LF-C were conducted, and the main effects of HMTBa and IA and their interaction within the LF diets were examined. The LF-C diet decreased ruminal pH and the ratio of acetate to propionate, with no major changes detected in ruminal FA profile compared with HF-C. The α-diversity for LF-C was lower compared with HF-C, and ß-diversity also differed between LF-C and HF-C. The relative abundance of bacterial phyla and genera associated indirectly with fiber degradation was influenced by LF-C versus HF-C. As the main effect of HMTBa within the LF diets, HMTBa increased the ratio of acetate to propionate and butyrate molar proportion. Ruminal saturated FA were increased and unsaturated FA concentration were decreased by HMTBa, with minimal changes detected in ruminal bacterial diversity and community. As the main effect of IA, IA supplementation increased ruminal concentration of all branched-chain volatile FA and valerate and increased the percentage of trans-10 C18 isomers in total FA. In addition, α-diversity and the number of functional features were increased for IA. Changes in the abundances of bacterial phyla and genera were minimal for IA. Interactions between HMTBa and IA were observed for ruminal variables and some bacterial taxa abundances. In conclusion, increasing diet fermentability (LF-C vs. HF-C) influenced rumen fermentation and bacterial community structure without major changes in FA profile. Supplementation of HMTBa increased biohydrogenation capacity, and supplemental IA increased bacterial diversity, possibly alleviating MFD. The combination of HMTBa and IA had no associative effects in the rumen and need further studies to understand the interactive mechanism.

Conditions stimulating neutral detergent fiber degradation by dosing branched-chain volatile fatty acids. II: Relation with solid passage rate and pH on neutral detergent fiber degradation and microbial function in continuous culture.

To support improving genetic potential for increased milk production, intake of digestible carbohydrate must also increase to provide digestible energy and microbial protein synthesis. We hypothesized that the provision of exogenous branched-chain volatile fatty acids (BCVFA) would improve both neutral detergent fiber (NDF) degradability and efficiency of microbial protein synthesis. However, BCVFA should be more beneficial with increasing efficiency of bacterial protein synthesis associated with increasing passage rate (kp). We also hypothesized that decreasing pH would increase the need for isobutyrate over 2-methylbutyrate. To study these effects independent from other sources of variation in vivo, we evaluated continuous cultures without (control) versus with BCVFA (0 vs. 2 mmol/d each of isobutyrate, isovalerate, and 2-methylbutyrate), low versus high kp of the particulate phase (2.5 vs. 5.0%/h), and high versus low pH (ranging from 6.3 to 6.8 diurnally vs. 5.7 to 6.2) in a 2 × 2 × 2 factorial arrangement of treatments. Diets were 50% forage pellets and 50% grain pellets administered twice daily. Without an interaction, NDF degradability tended to increase from 29.7 to 35.0% for main effects of control compared with BCVFA treatments. Provision of BCVFA increased methanogenesis, presumably resulting from improved NDF degradability. Decreasing pH decreased methane production. Total volatile fatty acid (VFA) and acetate production were decreased with increasing kp, even though true organic matter degradability and bacterial nitrogen flow were not affected by treatments. Decreasing pH decreased acetate but increased propionate and valerate production, probably resulting from a shift in bacterial taxa and associated VFA stoichiometry. Decreasing pH decreased isobutyrate and isovalerate production while increasing 2-methylbutyrate production on a net basis (subtracting doses). Supplementing BCVFA improved NDF degradability in continuous cultures administered moderate (15.4%) crude protein diets (excluding urea in buffer) without major interactions with culture pH and kp.

Conditions stimulating neutral detergent fiber degradation by dosing branched-chain volatile fatty acids. I: Comparison with branched-chain amino acids and forage source in ruminal batch cultures.

Three experiments assessed branched-chain volatile fatty acid (BCVFA) stimulation of neutral detergent fiber (NDF) disappearance after 24 h of incubation in batch cultures derived from ruminal fluid inocula that were enriched with particulate-phase bacteria. In experiment 1, a control was compared with 3 treatments with isomolar doses of all 3 BCVFA (plus valerate), all 3 branched-chain AA (BCAA), or half of each BCVFA and BCAA mix with either alfalfa or grass hays (50%) and ground corn grain (50%). A portion of the BCAA and BCVFA doses were enriched with 13C, and valerate (also enriched with 13C) was added with BCVFA. Although BCAA yielded a similar production of BCVFA compared with dosing BCVFA, equimolar substitution of BCVFA for BCAA decreased the percentage of N in bacterial pellets when alfalfa hay was fed but increased N when grass hay was fed. Substituting BCVFA for BCAA increased total fatty acid (FA) concentration with alfalfa hay. Dosing of BCAA or BCVFA did not affect total branched-chain FA, iso-FA, or anteiso-FA percentages in bacterial total FA, whereas numerous individual FA isomers and their 13C enrichments were affected by these treatments. Increasing recovery of the 13C dose from respective labeled BCVFA primers indicated facilitated BCVFA uptake and incorporation into FA compared with BCAA, whereas increased recovery of 13C from labeled BCAA in the bacteria pellet but not in the FA fraction suggested direct assimilation into bacterial protein. The BCVFA and valerate were dosed in varying combinations that either summed to 4 mM (experiment 2) or had only 1 mM no matter what combination (experiment 3). In general, grass hay was more responsive to stimulation in NDF digestibility by BCVFA than was alfalfa hay, which was attributed to the higher degradable protein in the latter. The net production of the BCVFA (after subtracting dose) was affected by source and combination of BCVFA. Isovalerate dosing tended to increase its own net production; in contrast, isobutyrate seemed to be used more when it was added alone, but 2-methylbutyrate seemed to be preferred over isobutyrate when 2-methylbutyrate was added. Results supported potential interactions, including potential feedback in production from feed BCAA or increased concentration-dependent competition for dosed BCVFA into cellular products. Under our conditions, the BCVFA appear to be more readily available than BCAA, probably because of regulated BCAA transport and metabolism. Valerate consistently provided no benefit. Using nonparametric ranking, all 3 BCVFA or either isovalerate or isobutyrate (both yielding iso-FA) should be combined with 2-methylbutyrate (yielding anteiso-FA) as a potential opportunity to improve NDF digestibility when rumen-degraded BCAA are limited in diets to decrease environmental impact from N in waste.

Conditions stimulating neutral detergent fiber degradation by dosing branched-chain volatile fatty acids. III: Relation with solid passage rate and pH on prokaryotic fatty acid profile and community in continuous culture.

Our objectives were to evaluate potential interactions in culture conditions that influence how exogenously dosed branched-chain VFA (BCVFA) would be recovered as elongated fatty acids (FA) or would affect bacterial populations. A 2 × 2 × 2 factorial arrangement of treatments evaluated 3 factors: (1) without versus with BCVFA (0 vs. 2 mmol/d each of isobutyrate, isovalerate, and 2-methylbutyrate; each dose was partially substituted with 13C-enriched tracers before and during the collection period); (2) high versus low pH (ranging diurnally from 6.3 to 6.8 vs. 5.7 to 6.2); and (3) low versus high particulate-phase passage rate (kp; 2.5 vs. 5.0%/h) in continuous cultures administered a 50:50 forage:concentrate diet twice daily. Samples of effluent were collected and composited before harvesting bacteria from which FA and DNA were extracted. Profiles and enrichments of FA in bacteria were evaluated by gas chromatography and isotope-ratio mass spectrometry. The 13C enrichment in bacterial FA was calculated as percentage recovery of dosed 13C-labeled BCVFA. Dosing BCVFA increased the even-chain iso-FA, preventing the reduced concentration at higher kp and potentially as a physiological response to decreased pH. However, decreasing pH decreased recovery of 13C in these even-chain FA, suggesting greater reliance on isobutyrate produced from degradation of dietary valine. The iso-FA were decreased, whereas anteiso-FA and 16:0 increased with decreasing pH. Thus, 2-methylbutyrate still appeared to be important as a precursor for anteiso-FA to counter the increased rigidity of bacterial membranes that had more saturated straight-chain FA when pH decreased. Provision of BCVFA stimulated the relative sequence abundance of Fibrobacter and Treponema, both of which require isobutyrate and 2-methylbutyrate. Numerous bacterial community members were shifted by low pH, including increased Prevotella and genera within the phylum Proteobacteria, at the expense of members within phylum Firmicutes. Because of relatively few interactions with pH and kp, supplementation of BCVFA can stimulate neutral detergent fiber degradability via key fibrolytic bacteria across a range of conditions. Decreasing pH shifted bacterial populations and their FA composition, suggesting that further research is needed to distinguish pH from dietary changes.

Maternal supplementation with cobalt sources, folic acid, and rumen-protected methionine and its effects on molecular and functional correlates of the immune system in neonatal Holstein calves.

Calves born to multiparous Holstein cows fed during the last 30 d of pregnancy 2 different cobalt sources [cobalt glucoheptonate (CoPro) or cobalt pectin (CoPectin)], folic acid (FOA), and rumen-protected methionine (RPM) were used to study neonatal immune responses after ex vivo lipopolysaccharide (LPS) challenge. Groups were (n = 12 calves/group) CoPro, FOA+CoPro, FOA+CoPectin, and FOA+CoPectin+RPM. Calves were weighed at birth and blood collected at birth (before colostrum), 21 d of age, and 42 d of age (at weaning). Growth performance was recorded once a week during the first 6 wk of age. Energy metabolism, inflammation, and antioxidant status were assessed at birth through various plasma biomarkers. Whole blood was challenged with 3 µg/mL of LPS or used for phagocytosis and oxidative burst assays. Target genes evaluated by real-time quantitative PCR in whole blood samples were associated with immune response, antioxidant function, and 1-carbon metabolism. The response in mRNA abundance in LPS challenged versus nonchallenged samples was assessed via Δ = LPS challenged - LPS nonchallenged samples. Phagocytosis capacity and oxidative burst activity were measured in neutrophils and monocytes, with data reported as ratio (percentage) of CD14 to CH138A-positive cells. Data including all time points were subjected to ANOVA using PROC MIXED in SAS 9.4 (SAS Institute Inc.), with Treatment, Sex, Age, and Treatment × Age as fixed effects. A 1-way ANOVA was used to determine differences at birth, with Treatment and Sex as fixed effects. Calf birth body weight and other growth parameters did not differ between groups. At birth, plasma haptoglobin concentration was lower in FOA+CoPro compared with CoPro calves. We detected no effect for other plasma biomarkers or immune function due to maternal treatments at birth. Compared with CoPro, in response to LPS challenge, whole blood from FOA+CoPectin and FOA+CoPectin+RPM calves had greater mRNA abundance of intercellular adhesion molecule 1 (ICAM1). No effect for other genes was detectable. Regardless of maternal treatments, sex-specific responses were observed due to greater plasma concentrations of haptoglobin, paraoxonase, total reactive oxygen metabolites, nitrite, and ß-carotene in female versus male calves at birth. In contrast, whole blood from male calves had greater mRNA abundance of IRAK1, CADM1, and ITGAM in response to LPS challenge at birth. The longitudinal analysis of d 0, 21, and 42 data revealed greater bactericidal permeability-increasing protein (BPI) mRNA abundance in whole blood from FOA+CoPectin versus FOA+CoPro calves, coupled with greater abundance in FOA+CoPro compared with CoPro calves. Regardless of maternal treatments, most genes related to cytokines and cytokine receptors (IL1B, IL10, TNF, IRAK1, CXCR1), toll-like receptor pathway (TLR4, NFKB1), adhesion and migration (ICAM1, ITGAM), antimicrobial function (MPO), and antioxidant function (GPX1) were downregulated over time. Phagocytosis capacity and oxidative burst activity in both neutrophils and monocytes did not differ due to maternal treatment. Regardless of maternal treatments, we observed an increase in the percentage of neutrophils capable of phagocytosis and oxidative burst activity over time. Overall, these preliminary assessments suggested that maternal supplementation with FOA and Co combined with RPM had effects on a few plasma biomarkers of inflammation at birth and molecular responses associated with inflammatory mechanisms during the neonatal period.

Cross-sectional study of the effect of diet composition on plasma folate and vitamin B12 concentrations in Holstein cows in the United States and Canada.

The objective of this cross-sectional study was to assess the variability of plasma folate and vitamin B12 concentrations in lactating Holstein cows across the United States and Canada. We also evaluated the effect of diet composition and cow characteristics on folate and vitamin B12 plasma vitamin concentrations. A total of 22 and 24 US and Canadian dairy herds were enrolled, totaling 427 and 476 cows at 10 to 197 days in milk across all US and Canadian herds, respectively. Blood samples were taken to analyze plasma folate and vitamin B12 concentrations, and ingredients of the diet were collected to determine nutrient composition. To reduce the number of interdependent variables in the analysis of the association of diet composition with plasma vitamin concentrations, we conducted a principal component analysis. Plasma folate concentrations were lower for US cows [13.4 ng/mL, 95% confidence interval (CI): 12.7-14.2] than for Canadian cows (14.5 ng/mL, 95% CI: 13.7-15.2), and the opposite was observed for plasma vitamin B12 concentrations (US 206 pg/mL, 95% CI: 192-221; Canada 170 pg/mL, 95% CI: 159-181). The highest plasma concentrations of both vitamins were observed in the Northwest region of the United States (Oregon and Washington). Cows in California had the lowest plasma folate concentrations, and cows in Québec and New York State had the lowest plasma vitamin B12 concentrations. Plasma folate concentrations were higher for multiparous than for primiparous cows and plasma vitamin B12 concentrations progressively increased from parity 1 to 3 and higher. For both studied vitamins, plasma concentrations were lower at 0 to 55 than at 56 to 200 days in milk. Of 3 principal components, the one associated with dietary carbohydrates was significantly correlated with plasma folate and vitamin B12 concentrations. Indeed, plasma folate concentrations decreased with dietary fiber concentrations (i.e., neutral and acid detergent fibers and lignin) and increased with dietary nonfiber carbohydrate concentrations. We obtained the opposite results for plasma vitamin B12 concentrations. Both multivariable models explained 41% (pseudo-R2) of the variation in plasma folate and vitamin B12 concentrations. Information gathered in this study is the first step toward determining sources of variation in plasma folate and vitamin B12 concentrations, as well as the vitamin status of cows.

Supplementing Zn, Mn, and Cu from amino acid complexes and Co from cobalt glucoheptonate during the peripartal period benefits postpartal cow performance and blood neutrophil function.

The physiologic and metabolic stresses that dairy cows experience during the transition into early lactation can promote oxidative stress, inflammation, and immune dysfunction. Optimal supply of micronutrients such as trace minerals (e.g., Zn, Mn, Cu, and Co) via more bioavailable forms (e.g., AA complexes) might minimize these negative effects. Multiparous Holstein cows were enrolled at 60 d before dry-off (~110 d before calving) and remained on experiment until 30 d in milk (DIM). Cows were offered a common diet supplemented entirely with inorganic trace minerals (INO) from -110 to -30 d before calving. From -30 to calving cows received a common prepartal [1.5 Mcal/kg of dry matter (DM), 15% crude protein] diet, and from calving to 30 DIM a common postpartal (1.76 Mcal/kg of DM, 18% crude protein) diet. Both diets were partially supplemented with an INO mix of Zn, Mn, and Cu to supply 35, 45, and 6 mg/kg, respectively, of the total diet DM. Cows were assigned to treatments in a randomized complete block design to receive an oral bolus with a mix of INO (n=21) or organic AA complexes (AAC; n=16) of Zn, Mn, Cu, and Co to achieve supplemental levels of 75, 65, 11, and 1mg/kg, respectively, in the total diet DM. Inorganic trace minerals were provided in sulfate form and AAC were supplied via Availa Zn, Availa Mn, Availa Cu, and COPRO (Zinpro Corp., Eden Prairie, MN). Liver tissue was harvested on -30, -15, 10, and 30 d, and blood samples for biomarker analyses were obtained more frequently from -30 to 30 DIM. Short-term changes in blood ketones were measured via Precision Xtra (Abbott Diabetes Care, Alameda, CA) every other day from 1 to 15 d postpartum. Prepartal DM intake was lower in AAC cows. In contrast, a tendency for a diet by time (D × T) interaction resulted in greater postpartal DM intake of approximately 2 kg/d in cows fed AAC. Milk and milk protein yield had a D × T interaction because AAC cows produced approximately 3.3 kg/d more milk and 0.14 kg/d more protein during the first 30 DIM. Although blood glucose, fatty acids, and liver triacylglycerol were not affected by diet, the Precision Xtra ketones (1.44 vs. 2.18 mmol/L) and γ-glutamyltransferase (liver function biomarker) were lower in AAC than INO. Furthermore, feeding AAC increased (D × T) polymorphonuclear neutrophilic lymphocyte phagocytosis, antioxidant capacity postpartum, and overall concentration of liver tissue Co and Cu. Overall, the positive response in milk yield and milk protein in AAC cows might be partly explained by the beneficial effects of AAC on postpartal DM intake driven at least in part by better liver and immune function as a result of improved antioxidant status.

Corium molecular biomarkers reveal a beneficial effect on hoof transcriptomics in peripartal dairy cows supplemented with zinc, manganese, and copper from amino acid complexes and cobalt from cobalt glucoheptonate.

Supplying trace minerals in more bioavailable forms such as amino acid complexes (AAC) could help ameliorate the incidence of hoof disorders in peripartal dairy cows. The aim of this study was to evaluate the effects of supplementing metal AAC during the peripartal period on expression of 28 genes in corium tissue related to claw composition, oxidative stress, inflammation, chemotaxis, and transcriptional regulation. Forty-four multiparous Holstein cows received a common diet from -30 to 30 d relative to parturition and were assigned to receive an oral bolus containing either inorganic trace minerals (INO) or AAC (i.e., organic) Zn, Mn, Cu, and Co to achieve supplemental levels of 75, 65, 11, and 1 ppm, respectively, in the total diet dry matter. Inorganic trace minerals were provided in sulfate form, and AAC were supplied via Availa Zn, Availa Mn, Availa Cu, and COPRO (Zinpro Corp., Eden Prairie, MN). Locomotion score was recorded before enrollment and weekly throughout the experiment. Incidence of hoof health problems at 30 d in milk was evaluated before a hoof biopsy in a subset of cows (INO=10; AAC=9). Locomotion score did not differ between treatments in the prepartum or postpartum period. The incidence of heel horn erosion was lower in AAC cows, but the incidence of sole ulcers did not differ. Downregulation of KRT5, CTH, CALML5, and CYBB, and upregulation of BTD in AAC cows indicated a decrease in the need for activation of cellular pathways to regenerate corium tissue and increase biotin availability in the sole claw. These molecular changes in the sole could have been triggered by the lower incidence of heel erosion in response to AAC. Among the genes associated with oxidative stress, the AAC cows had greater expression of NFE2L2, a transcription factor that regulates the antioxidant response, and the antioxidant enzyme SOD1. Among genes associated with inflammation, AAC cows had greater expression of TLR4, and lower expression of TLR2, IL1B, and TNF compared with INO cows. Supplementation with metal AAC during the peripartal period affected the expression of genes involved in composition, oxidative stress, and inflammation status in the corium. The hoof biopsy procedure used in the present study should be further perfected and implemented in future lameness research to expand our understanding of hoof biology in dairy cows.

First-lactation performance in cows affected by digital dermatitis during the rearing period.

The long-term effects of prepartum digital dermatitis (DD) on first-lactation performance were evaluated in a cohort of 719 pregnant heifers. All heifers were followed for a period of 6 mo until calving and classified on the basis of the number of DD events diagnosed during this period as type I, type II, or type III (no DD, one DD event, and multiple DD events, respectively). Health during the initial 60d in milk (DIM), reproductive and hoof health outcomes, and milk production were compared between the 3 heifer type groups. All logistic and linear models were adjusted for age, height, and girth circumference at enrollment, and the type of trace mineral supplementation during the prepartum period. Overall, cows experiencing DD during the rearing period showed worse production and health outcomes compared with healthy heifers during the first lactation. The percentages of assisted calvings, stillbirths, culled before 60 DIM, and diseased cows during the fresh period were numerically higher in type III cows compared with type I cows. However, none of these differences were statistically significant at the 95% confidence level. Significantly lower conception at first service [odds ratio (OR)=0.55, 95% confidence interval (95% CI): 0.33, 0.89] and increased number of days open (mean=24d, 95% CI: 5.2, 43) were observed in type III cows compared with type I cows. In relation to hoof health, a significantly increased risk of DD during the first lactation was found in type II and III cows (OR=5.16, 95% CI: 3.23, 8.29; and OR=12.5, 95% CI: 7.52, 21.1, respectively), as well as earlier occurrence of DD following calving (OR=59d, 95% CI=20, 96, and OR=74d, 95% CI: 37, 109). Compared with type I cows, statistically significant milk production losses during the initial 305 DIM of 199 and 335kg were estimated in type II and III cows, respectively. This difference was due to a greater rate of production decline (less persistence) after peak yield. No differences in monthly fat and protein percentages or somatic cell counts were observed between the heifer types. Given the long-term effects of DD on health, reproduction, and production, one of the priorities during the rearing period of dairy heifers should be efficient DD prevention and control programs. Such intensive intervention programs based on active long-term DD surveillance, mitigation of risk factors, and prompt treatment are expected to increase overall animal well-being and farm profitability by minimizing the effect of DD during the first lactation.

Effect of including canola meal and supplemental iodine in diets of dairy cows on short-term changes in iodine concentrations in milk.

The dietary requirement for iodine is based on thyroxine production, but data are becoming available showing potential improvements in hoof health when substantially greater amounts of I are fed. Feeding high amounts of I, however, can result in the milk having excessive concentrations of I. Canola meal contains goitrogenic compounds that reduce the transfer of I into milk. We hypothesized that including canola meal in diets would allow high supplementation rates of I without producing milk with unacceptable concentrations of I. Thirty midlactation Holstein cows were fed a diet with all supplemental protein from soybean meal (0% of diet dry matter as canola meal) or with all supplemental protein from canola meal (13.9% canola meal). A third treatment has a mix of soybean meal and canola meal (3.9% canola meal). Within canola-meal treatment, cows were fed 0.5 or 2.0mg of supplemental I per kilogram of diet dry matter from ethylenediamine dihydroiodide. Cows were maintained on the canola treatment for the duration of the experiment but were changed from one I treatment to the other after 13d of receiving the treatment. Milk I concentration before the treatments started (cows fed 0.5mg/kg of I) averaged 272µg/L and increased within 22h after cows were first fed diets with 2mg/kg of I. As inclusion rate of canola meal increased, the concentration of I in milk decreased linearly. After 12d of supplementation, milk from cows fed 0.5mg/kg of I had 358, 289, and 169µg of I/L for the 0, 3.9%, and 13.9% canola-meal treatments. For cows fed 2.0mg/kg of I, milk I concentrations were 733, 524, and 408µg/L, respectively. Concentrations of I in serum increased with increased I supplementation, but the effect of canola meal was opposite of what was observed for milk I. Cows fed the highest canola-meal diets had the highest serum I whether cows were fed 0.5 or 2.0mg/kg of I. Feeding dairy cows diets with 13.9% canola meal maintained milk I concentrations below 500µg/L when diets were supplemented with 2mg/kg of I.

The effect of digital dermatitis on hoof conformation.

Digital dermatitis (DD) is the most prevalent cause of lameness of infectious origin in cattle. However, little is known about the effects of DD on hoof conformation (HC) during the clinical disease. The objectives of the present study were to (1) evaluate the changes in HC observed in feet affected with clinical DD lesions and (2) investigate the temporal relationship between DD and heel horn erosion (HHE). A longitudinal study was carried out including a cohort of 644 Holstein heifers. Digital dermatitis, HC, and presence of HHE in the rear feet of each heifer were assessed during a period of 6 mo. A total of 1,979 feet evaluations were included in the data set, of which 157 corresponded to feet presenting DD lesions >20mm [mean (SD) size of 27.2 (8.2) mm]. Age, days of pregnancy, hip height, and girth circumference were also recorded at cow level. Significant HC changes were observed in DD-affected feet. Results standardized to a period of 90d of follow-up showed an increase in heel height [mean (95% CI) 3.4 (2.5, 4.4) and 2.8 (2.0, 3.7) mm] and claw angle [0.8 (0.2, 1.4) and 1.4 (0.7, 2.0) degrees] of the medial and lateral claws, respectively. In addition, an increase in depth of the interdigital cleft [3.2 (2.7, 3.7) mm] and on debris accumulation [14% (7, 21) of feet] was also observed. Feet affected with clinical DD lesions also experienced a 46% point increase in the presence of severe HHE. In the short term, HC changes returned to normal levels when clinical cure of DD was achieved after topical treatment. In conclusion, significant HC changes occur in heifers affected by clinical DD before lameness symptoms are detected. The transformation of the heel area in feet affected by DD likely promotes the creation of a local environment that favors the persistence of the disease and the occurrence of severe HHE. To avoid further hoof damage, active surveillance and early intervention to reduce HC changes are recommended to improve DD control programs. Successful restoration of HC can be achieved upon clinical cure of DD. The long-term effects in lifetime performance of the HC changes due to DD remain to be further investigated.

A randomized trial to evaluate the effect of a trace mineral premix on the incidence of active digital dermatitis lesions in cattle.

A balanced, parallel-group, single-blinded randomized efficacy study divided into 2 periods was conducted to evaluate the effect of a premix containing higher than typically recommended levels of organic trace minerals and iodine (HOTMI) in reducing the incidence of active digital dermatitis (DD) lesions acquired naturally and induced by an experimental infection challenge model. For the natural exposure phase of the study, 120 healthy Holstein steers 5 to 7 mo of age without signs of hoof disease were randomized into 2 groups of 60 animals. The control group was fed a standard trace mineral supplement and the treatment group was fed the HOTMI premix, both for a period of 60 d. On d 60, 15 steers free of macroscopic DD lesions were randomly selected from each group for the challenge phase and transported to an experimental facility, where they were acclimated and then challenged within a DD infection model. The same diet group allocation was maintained during the 60 d of the challenge phase. The primary outcome measured was the development of an active DD lesion greater than 20mm in diameter across its largest dimension. No lesions were identified during the natural exposure phase. During the challenge phase, 55% (11/20) and 30% (6/20) of feet were diagnosed with an active DD lesion in the control and treatment groups, respectively. Diagnosis of DD was confirmed by histopathologic demonstration of invasive Treponema spp. within eroded and hyperplastic epidermis and ulcerated papillary dermis. All DD confirmed lesions had dark-field microscopic features compatible with DD and were positive for Treponema spp. by PCR. As a secondary outcome, the average DD lesion size observed in all feet was also evaluated. Overall mean (standard deviation) lesion size was 17.1 (2.36) mm and 11.1 (3.33) mm for the control and treatment groups, respectively, with this difference being driven by acute DD lesions >20mm. A trend existed for the HOTMI premix to reduce the total DD infection rate and the average size of the experimentally induced lesions. Further research is needed to validate the effect of this intervention strategy in the field and to generate prevention and control measures aimed at optimizing claw health based on nutritional programs.

Immune response against Treponema spp. and ELISA detection of digital dermatitis.

The objective of this longitudinal study was to evaluate the immune response against Treponema spp. infection in dairy heifers affected with digital dermatitis (DD). In addition, the accuracy of an indirect ELISA detecting anti-Treponema IgG antibodies in identifying clinical DD status has been assessed. A cohort of 688 pregnant Holstein heifers was evaluated at least 3 times before calving during a period of 6 mo. Complete clinical assessment of DD presence on the back feet of each heifer and blood extraction were performed in a stand-up chute. Digital dermatitis cases were characterized by the M-stage classification system and size and level of skin proliferation. An ELISA was performed on blood serum samples obtained from a subcohort of 130 heifers. For description purposes, the animals were classified by the number of clinical cases experienced during the study period as type I (no clinical cases were observed), type II (only 1 acute clinical case diagnosed), and type III (at least 2 acute clinical cases diagnosed). Multivariable repeated-measures models were used to evaluate the immune response against Treponema spp. infection. A binormal Bayesian model for the ELISA data without cut-point values was used to assess the accuracy of the ELISA as a diagnostic tool. Animals that never experienced a DD event throughout the study kept a constant low level of antibody titer. A 56% increase in mean ELISA titer was observed in heifers upon a first clinical DD case diagnosis. After topical treatment of an acute DD case with oxytetracycline, the antibody titer decreased progressively in type II heifers, achieving mean levels of those observed in healthy cows after a mean of 223 d. Surprisingly, antibody titer was not increased in the presence of M1 (DD lesion <20mm in diameter surrounded by healthy skin) and M4.1 (DD lesion <20mm in diameter embedded in a circumscribed dyskeratotic or proliferative skin alteration) DD stages. Type III cows showed a slight increase in antibody levels. The presence of skin proliferation at first DD diagnosis was found to be associated with an odds ratio of 2.04 of becoming a type III heifer in relation to heifers presenting first lesions without skin proliferation. The ELISA validity was estimated by an area under the curve of 0.88. Predicted probabilities of infection are provided for a range of ELISA values and prevalence of infection. Early detection and treatment is essential to control DD and the ELISA can be used in understanding the immunopathology of DD and shows great promise for prescreening purposes during DD management programs in combination with traditional clinical inspection.

Analysis of foot health records from 17 confinement dairies.

Foot health records are useful in monitoring the degree of lameness within dairy herds and, perhaps more importantly, providing insight into the underlying factors causing lameness. A database containing the incidence of foot lesions on large confinement dairy operations is largely unavailable but could prove useful to demonstrate the importance of collecting and analyzing foot lesion data to reduce lameness. Our objective was to merge foot lesion records from several dairy herds and establish a database to demonstrate how to use such data to better understand when and why foot lesions occur as an important means to manage lameness in dairy herds. The database consisted of 12 mo of records from 17 dairies (14 freestall, 1 combination dirt lot and freestall, 2 dirt lot) representing 58,155 cows from herds ranging in size from 631 to 9,355 animals in 9 states from the United States and 2 herds located in the Southern Hemisphere. Data were partitioned and analyzed as 2 separate data sets: (1) herds recording only lame events (cows lame when examined; n=8), and (2) herds recording both lame and routine trim events (n=9). Data were analyzed using PROC FREQ (SAS Institute Inc., Cary, NC) and significance was determined using Chi-square. White line disease, sole ulcer, toe ulcer, digital dermatitis, and foot rot comprised 93 and 40% (excluding routine trim with no lesion, 55%) of lesions for herds recording only lame events and those recording lame and trim events, respectively. Ratio of infectious to noninfectious lesions decreased with increasing lactation number in both data sets. Digital dermatitis and foot rot were greatest in the first 60 d in milk and differed across lactation number. Noninfectious lesions were greatest following summer heat stress, whereas infectious lesions were greatest during the coolest quarter of the year. In conclusion, analysis of the foot health data from these dairies demonstrates that (1) infectious lesions of the foot skin and soft tissues predominate in early lactation and during cooler months of the year, and (2) noninfectious lesions predominate during the 3 mo following summer heat stress and their distribution follows a typical lactation curve.

Effects of cobalt supplementation and vitamin B12 injections on lactation performance and metabolism of Holstein dairy cows.

The objective of this study was to determine lactation performance and metabolism of primiparous and multiparous dairy cows fed different levels and sources (inorganic and organic) of Co or given weekly vitamin B(12) injections. Forty-five primi- and multiparous cows at 60 d prepartum were blocked by expected calving date, and randomly assigned to 1 of 5 treatments in a randomized complete block design with treatments starting at 60 d prepartum. The 5 treatments were (1) no supplemental dietary Co (control, CON), (2) 25mg/d of supplemental dietary Co from Co carbonate (CoCarb), (3) 25mg/d of supplemental dietary Co from Co glucoheptonate (LCoGH), (4) 75 mg/d of supplemental dietary Co from Co glucoheptonate (HCoGH), and (5) CON diet plus weekly 10mg i.m. of vitamin B(12) injections (IB12). Cows remained on their respective treatment until 150 d after calving. Cobalt concentrations (mg/kg of dry matter) in the lactating diets were 1.0, 1.9, 2.3, and 5.1 for CON/IB12, CoCarb, LCoGH, and HCoGH, respectively. Dry matter intake, body weight, and body condition score were not affected by treatment. The LCoGH treatment tended to have greater milk yield than CoCarb, and CON had similar milk yields to the mean of LCoGH and HCoGH. Cobalt supplementation or the use of vitamin B(12) injections did not influence plasma or liver measures of energy metabolism. Injections of vitamin B(12) increased plasma, liver, and milk vitamin B(12) contents. Dietary Co addition did not affect plasma vitamin B(12) concentrations; however, it did increase milk vitamin B(12) concentrations throughout lactation and liver vitamin B(12) at calving with no effect of source or level of Co. Folate status of cows in the study was low and possibly limited the effect of improved vitamin B(12) status on lactation performance. Overall, Co supplementation (inorganic and organic) or vitamin B(12) injections improved measures of vitamin B(12) status, but not lactation performance compared with CON possibly due to Co being above requirements in the CON diet.