Gene expression of hepatic gluconeogenic and fatty acid metabolism in early-lactation dairy cows as affected by dietary starch and monensin supplementation.

Our previously published companion papers demonstrated improved production performance and energetic metabolism in cows fed diets with increased propiogenic potential in early lactation. Study objectives were to further explore effects of dietary starch content and monensin on hepatic gene expression of key enzymes related to gluconeogenesis and fatty acid metabolism in early lactation. From d 1 to 21 postpartum, primiparous (n = 16) and multiparous (n = 33) Holstein cows were fed a high (HS; 26.2% starch, 34.3% neutral detergent fiber, 22.7% acid detergent fiber, 15.5% crude protein) or low (LS; 21.5% starch, 36.9% neutral detergent fiber, 25.2% acid detergent fiber, 15.4% crude protein) starch diet with a daily topdress containing either 0 (Con) or 450 mg/d monensin (Mon). Cows were randomly assigned to treatment. Liver biopsies were obtained from cows on d 7 postpartum for DNA and RNA quantification and mRNA expression analysis. In primiparous cows, Mon supplementation decreased CPT1A expression relative to controls, whereas in multiparous cows Mon increased its expression. Cows fed HS and Mon tended to have decreased HMGCS2 expression relative to cows fed HS and Con. In multiparous cows, Mon supplementation tended to increase PC and PCK1 expression relative to controls. Correlation analysis was performed for all gene expression variables. Overall, relationships were similar in directionality and magnitude between cows fed HS and LS and Con and Mon. However, for cows fed Con there was a positive relationship between HMGCS2 and PC and HMGCS2 and PCK1, whereas for cows fed Mon there was no relationship. There was a similar lack of relationship between HMGCS2 and PC for cows fed HS. Overall, results support changes in performance and energetic metabolism reported in our companion papers, indicating that cows fed diets of different starch content in early lactation with Mon supplementation throughout the transition period had alterations in hepatic gene expression consistent with increased hepatic propionate supply.

Effect of monensin on milk production efficiency and milk composition in lactating dairy cows fed modern diets.

Since the US Food and Drug Administration's approval of monensin in 2004, significant nutritional advances have been made to increase feed efficiency and milk fat production. Recent evidence suggests monensin's adverse effect on milk fat percentage may be absent when diets are formulated to address known diet-induced milk fat depression risk factors. Thus, study objectives were to evaluate effects of monensin level on dry matter intake (DMI), milk production and composition, and efficiency of high-producing cows fed diets formulated to optimize milk fat. Ninety-six lactating Holstein cows (36 primiparous, 60 multiparous; 106 ± 17 d in milk [DIM]) were balanced by parity, DIM, and milk production and were randomly assigned to 1 of 12 pens with 8 cows per pen. All cows received 11 g/t monensin for 5 wk after which pens received 1 of 4 dietary treatments (n = 3) formulated to provide 0 (CON), 11 (R11), 14.5 (R14.5), or 18 (R18) g/t monensin for 9 wk. The basal diet was 54% forage, 27% NDF, 29% starch, and 2.3% rumen unsaturated fatty acid load. Pen was the experimental unit and data were analyzed using the Fit Model Procedure of JMP. Effects of treatment, time, and treatment × time interaction were included as fixed effects and pen as a random effect. Least squares means were determined and linear and quadratic contrasts were tested. Dry matter intake tended to decrease linearly with increasing monensin dose. Milk yield, fat percentage, and protein percentage and yield were unaffected by treatment while fat yield was quadratically increased. Milk de novo and mixed fatty acid (FA) yields (g/d) increased quadratically with monensin whereas preformed FA linearly decreased during the experimental period. Energy-corrected milk (ECM) was quadratically increased by monensin. Milk urea nitrogen concentrations increased linearly with increasing monensin dose. Monensin linearly increased feed efficiency (ECM/DMI, 3.5% fat-corrected milk/DMI, and solids-corrected milk/DMI). Body weight gain did not differ between treatments. Estimated dietary energy tended to increase linearly with increasing monensin level. These data suggest monensin improves component-corrected milk production efficiency, estimated dietary energy, and does not negatively affect milk fat percentage or FA profile.

Effects of cashew nut shell extract supplementation on production, rumen fermentation, metabolism, and inflammatory biomarkers in transition dairy cows.

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), ß-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.

Effects of hindgut acidosis on production, metabolism, and inflammatory biomarkers in previously immune-activated lactating dairy cows.

Previous stressors and systemic inflammation may increase the intestine's susceptibility to hindgut acidosis (HGA). Therefore, our experimental objectives were to evaluate the effects of isolated HGA on metabolism, production, and inflammation in simultaneously immune-activated lactating cows. Twelve rumen-cannulated Holstein cows (118 ± 41 d in milk; 1.7 ± 0.8 parity) were enrolled in a study with 3 experimental periods (P). Baseline data were collected during P1 (5 d). On d 1 of P2 (2 d), all cows received an i.v. lipopolysaccharide (LPS) bolus (0.2 µg/kg of body weight; BW). During P3 (4 d), cows were randomly assigned to 1 of 2 abomasal infusion treatments: (1) control (LPS-CON; 6 L of H2O/d; n = 6) or (2) starch infused (LPS-ST; 4 kg of corn starch + 6 L of H2O/d; n = 6). Treatments were allocated into 4 equal doses (1.5 L of H2O or 1 kg of starch and 1.5 L of H2O, respectively) and administered at 0000, 0600, 1200, and 1800 h daily. Additionally, both treatments received i.v. LPS on d 1 and 3 of P3 (0.8 and 1.6 µg/kg of BW, respectively) to maintain an inflamed state. Effects of treatment, time, and their interaction were assessed. Repeated LPS administration initiated and maintained an immune-activated state, as indicated by increased circulating white blood cells (WBC), serum amyloid A (SAA), and LPS-binding protein (LBP) during P2 and P3 (29%, 3-fold, and 50% relative to P1, respectively) for both abomasal infusion treatments. Regardless of abomasal treatment, milk yield and dry matter intake were decreased throughout P2 and P3 but with lesser severity following each LPS challenge (54, 44, and 37%, and 49, 42, and 40% relative to baseline on d 1 of P2, d 1 and d 3 of P3, respectively). As expected, starch infusions markedly decreased fecal pH (5.56 at nadir vs. 6.57 during P1) and increased P3 fecal starch relative to LPS-CON (23.7 vs. 2.4% of dry matter). Neither LPS nor starch infusions altered circulating glucose, insulin, nonesterified fatty acids, or ß-hydroxybutyrate, although LPS-ST cows had decreased blood urea nitrogen throughout P3 (16% relative to LPS-CON). Despite the striking reduction in fecal pH, HGA had no additional effect on circulating WBC, SAA, or LBP. Thus, in previously immune-activated dairy cows, HGA did not augment the inflammatory state, as indicated by a lack of perturbations in production, metabolism, and inflammatory biomarkers.

Effects of hindgut acidosis on inflammation, metabolism, and productivity in lactating dairy cows fed a high-fiber diet.

Hindgut acidosis (HGA) may cause or contribute to the inflammatory state of transition dairy cows by compromising the intestinal barrier. Previous experiments isolating the effects of HGA on inflammatory metrics have generated inconsistent results, which may be explained by acclimation to low- versus high-starch diets. Thus, study objectives were to evaluate the effects of HGA in cows acclimated to a high-fiber diet. Ten rumen-cannulated Holstein cows (38 ± 5 kg/d milk yield; 243 ± 62 d in milk; 1.6 ± 1.1 parity; 663 ± 57 kg of body weight) were enrolled in a study with 2 experimental periods (P). Before P1, all cows were acclimated to a high-fiber, low-starch diet (50% neutral detergent fiber, 15% starch) for 17 d. During P1 (4 d), baseline data were collected for use as covariates. During P2 (7 d), cows were assigned to 1 of 2 abomasal infusion treatments: (1) control (CON; 1.5 L of H2O/infusion; n = 4) or (2) starch infused (ST; 1 kg of corn starch + 1.5 L of H2O/infusion; n = 6). All cows were infused with their respective treatments every 6 h daily at 0000, 0600, 1200, and 1800 h, such that ST cows received a total of 4 kg of corn starch/d. Starch infusions successfully induced HGA, as indicated by a marked decrease in fecal pH (1.2 units) relative to CON. However, in contrast to our assumptions, infusing starch had no deleterious effects on milk yield, energy-corrected milk, or voluntary dry matter intake during P2. Milk protein, lactose, their yields, fat yield, and somatic cell score remained unaffected by starch infusions, whereas milk fat content and urea nitrogen were decreased in ST relative to CON (8 and 17%, respectively). Overall, circulating glucose and ß-hydroxybutyrate concentrations remained similar between treatments, but starch infusions decreased nonesterified fatty acids on d 3 relative to CON. Blood urea nitrogen decreased throughout P2 in ST (38%) relative to CON. In contrast to our hypothesis, HGA did not alter circulating serum amyloid A or lipopolysaccharide binding protein, nor did it affect rectal temperature. In summary, HGA moderately altered metabolism but did not affect production or elicit an inflammatory response in lactating dairy cows previously acclimated to a high-fiber diet.

Effects of hindgut acidosis on production, metabolism, and inflammatory biomarkers in feed-restricted lactating dairy cows.

Study objectives were to evaluate the effects of hindgut acidosis (HGA) on production, metabolism, and inflammation in feed-restricted (FR) dairy cows. Twelve rumen-cannulated cows were enrolled in a study with 3 experimental periods (P). During P1 (5 d), baseline data were collected. During P2 (2 d), all cows were FR to 40% of their baseline feed intake. During P3 (4 d), cows remained FR and were assigned to 1 of 2 abomasal infusion treatments: (1) control (FR-CON; 6 L of H2O/d; n = 6) or (2) starch (FR-ST; 4 kg of corn starch + 6 L of H2O/d; n = 6). Respective treatments were partitioned into 4 equal doses (1 kg of corn starch/infusion) and were abomasally infused daily at 0000, 0600, 1200, and 1800 h. All 3 P were analyzed independently and the effects of treatment, time, and treatment × time were assessed using PROC MIXED, and P1 and P2 data were analyzed using the treatments cows were destined to be assigned to during P3. Hallmark production and metabolic responses to feed restriction were observed in both treatments, including decreased milk yield (39%) and energy-corrected milk (32%), circulating glucose (12%), insulin (71%), and increased circulating nonesterified fatty acids (3.2-fold) throughout both P2 and P3, relative to P1. However, despite a marked reduction in fecal pH (0.96 units), the aforementioned metrics were unaltered by HGA. During P3, starch infusions increased circulating ß-hydroxybutyrate, with the most pronounced increase occurring on d 2 (81% relative to FR-CON). Further, feed restriction decreased blood urea nitrogen during P2 (17% relative to P1) in both treatments, and this was exacerbated by starch infusions during P3 (31% decrease relative to FR-CON). In contrast to our hypothesis, neither feed restriction nor HGA increased circulating acute-phase proteins (serum amyloid A and lipopolysaccharide binding protein) relative to P1 or FR-CON, respectively. Thus, despite marked reductions in fecal pH, prior feed restriction did not appear to increase the susceptibility to HGA.

Effects of dietary antioxidant supplementation on metabolism and inflammatory biomarkers in heat-stressed dairy cows.

Heat-stress-induced inflammation may be ameliorated by antioxidant supplementation due to the purported effects of increased production of reactive oxygen species or oxidative stress on the gastrointestinal tract barrier. Thus, study objectives were to evaluate whether antioxidant supplementation [AGRADO Plus 2.0 (AP); EW Nutrition] affects metabolism and inflammatory biomarkers in heat-stressed lactating dairy cows. Thirty-two mid-lactation multiparous Holstein cows were assigned to 1 of 4 dietary-environmental treatments: (1) thermoneutral (TN) conditions and fed a control diet (TN-CON; n = 8), (2) TN and fed a diet with AP (10 g antioxidant; n = 8), (3) heat stress (HS) and fed a control diet (HS-CON; n = 8), or (4) HS and fed a diet with AP (HS-AP; n = 8). The trial consisted of a 23-d prefeeding phase and 2 experimental periods (P). Respective dietary treatments were top-dressed starting on d 1 of the prefeeding period and continued daily throughout the duration of the experiment. During P1 (4 d), baseline data were collected. During P2 (7 d), HS was artificially induced using an electric heat blanket (Thermotex Therapy Systems Ltd.). During P2, the effects of treatment, day, and treatment-by-day interaction were assessed using PROC MIXED of SAS (SAS Institute Inc.). Heat stress (treatments 3 and 4) increased rectal, vaginal, and skin temperatures (1.2°C, 1.1°C, and 2.0°C, respectively) and respiration rate (33 breaths per minute) relative to TN cows. As expected, HS decreased dry matter intake, milk yield, and energy-corrected milk yield (32%, 28%, and 28% from d 4 to 7, respectively) relative to TN. There were no effects of AP on body temperature indices or production. Milk fat, protein, and lactose concentrations remained unaltered by HS or AP; however, milk urea nitrogen was increased during HS regardless of AP supplementation (26% relative to TN). Circulating glucose remained unchanged by HS, AP, or time. Additionally, HS decreased circulating glucagon (29% from d 3 to 7 relative to TN), but there was no additional effect of AP. There was a tendency for nonesterified fatty acid concentrations to be increased in HS-AP cows throughout P2 (60% relative to TN-CON), whereas it remained similar in all other treatments. Blood urea nitrogen increased for both HS treatments from d 1 to 3 before steadily decreasing from d 5 to 7, with the overall increase being most pronounced in HS-CON cows (27% relative to TN-CON). Further, supplementing AP decreased blood urea nitrogen in HS-AP on d 3 relative to HS-CON (15%). Circulating serum amyloid A tended to be and lipopolysaccharide binding protein was increased by HS, but neither acute-phase protein was affected by AP. Overall, AP supplementation appeared to marginally alter metabolism but did not meaningfully alter inflammation during HS.

Effects of hindgut acidosis on metabolism, inflammation, and production in dairy cows consuming a standard lactation diet.

Postruminal intestinal barrier dysfunction caused by excessive hindgut fermentation may be a source of peripheral inflammation in dairy cattle. Therefore, the study objectives were to evaluate the effects of isolated hindgut acidosis on metabolism, inflammation, and production in lactating dairy cows. Five rumen-cannulated lactating Holstein cows (32.6 ± 7.2 kg/d of milk yield, 242 ± 108 d in milk; 642 ± 99 kg of body weight; 1.8 ± 1.0 parity) were enrolled in a study with 2 experimental periods (P). During P1 (4 d), cows were fed ad libitum a standard lactating cow diet (26% starch dry matter) and baseline data were collected. During P2 (7 d), all cows were fed the same diet ad libitum and abomasally infused with 4 kg/d of pure corn starch (1 kg of corn starch + 1.25 L of H2O/infusion at 0600, 1200, 1800, and 0000 h). Effects of time (hour relative to the first infusion or day) relative to P1 were evaluated using PROC MIXED in SAS (version 9.4; SAS Institute Inc.). Infusing starch markedly reduced fecal pH (5.84 vs. 6.76) and increased fecal starch (2.2 to 9.6% of dry matter) relative to baseline. During P2, milk yield, milk components, energy-corrected milk yield, and voluntary dry matter intake remained unchanged. At 14 h, plasma insulin and ß-hydroxybutyrate increased (2.4-fold and 53%, respectively), whereas circulating glucose concentrations remained unaltered. Furthermore, blood urea nitrogen increased at 2 h (23%) before promptly decreasing below baseline at 14 h (13%). Nonesterified fatty acids tended to decrease from 2 to 26 h (40%). Circulating white blood cells and neutrophils increased on d 4 (36 and 73%, respectively) and somatic cell count increased on d 5 (4.8-fold). However, circulating serum amyloid A and lipopolysaccharide-binding protein concentrations were unaffected by starch infusions. Despite minor changes in postabsorptive energetics and leukocyte dynamics, abomasal starch infusions and the subsequent hindgut acidosis had little or no meaningful effects on biomarkers of immune activation or production variables.

Effect of zinc source (zinc sulfate or zinc hydroxychloride) on relative abundance of fecal Treponema spp. in lactating dairy cows.

Previous research revealed a potential effect of dietary trace mineral source on both ruminal and fecal microbiota. However, the effect of Zn source, specifically, has not previously been considered. Based on reported postruminal solubility, we hypothesized that Zn hydroxychloride would decrease Treponema spp. fecal excretion relative to cows fed Zn sulfate. To test this hypothesis, lactating Holstein cows (n = 24; 685 ± 9 kg of body weight; 159 ± 8 d in milk; parity 3 ± 0.2) were randomly assigned to 1 of 2 dietary treatments: control (75 mg/kg Zn from ZnSO4) or Zn hydroxychloride (HYD; 75 mg/kg IntelliBond Z; Micronutrients USA LLC). Single fecal grab samples were collected on d 1 before dietary treatments and on d 27 after dietary treatments were applied. Fecal microbial DNA was extracted and sequenced to establish taxonomy using a universal primer for the 16S rRNA gene. Supplementation of HYD decreased the relative abundance of Treponema 2 by 3-fold (14.7% vs. 4.9%). Poor sequencing resolution at the species level limited inference of Treponema spp. toward management or gut health implications of HYD supplementation. However, the inclusion of pathogenic species among Treponema spp. indicates a potential implication of HYD feeding to reduce environmental exposure of the dairy cow to Treponema spp.

Invited review: The influence of immune activation on transition cow health and performance-A critical evaluation of traditional dogmas.

The progression from gestation into lactation represents the transition period, and it is accompanied by marked physiological, metabolic, and inflammatory adjustments. The entire lactation and a cow's opportunity to have an additional lactation are heavily dependent on how successfully she adapts during the periparturient period. Additionally, a disproportionate amount of health care and culling occurs early following parturition. Thus, lactation maladaptation has been a heavily researched area of dairy science for more than 50 yr. It was traditionally thought that excessive adipose tissue mobilization in large part dictated transition period success. Further, the magnitude of hypocalcemia has also been assumed to partly control whether a cow effectively navigates the first few months of lactation. The canon became that adipose tissue released nonesterified fatty acids (NEFA) and the resulting hepatic-derived ketones coupled with hypocalcemia lead to immune suppression, which is responsible for transition disorders (e.g., mastitis, metritis, retained placenta, poor fertility). In other words, the dogma evolved that these metabolites and hypocalcemia were causal to transition cow problems and that large efforts should be enlisted to prevent increased NEFA, hyperketonemia, and subclinical hypocalcemia. However, despite intensive academic and industry focus, the periparturient period remains a large hurdle to animal welfare, farm profitability, and dairy sustainability. Thus, it stands to reason that there are alternative explanations to periparturient failures. Recently, it has become firmly established that immune activation and the ipso facto inflammatory response are a normal component of transition cow biology. The origin of immune activation likely stems from the mammary gland, tissue trauma during parturition, and the gastrointestinal tract. If inflammation becomes pathological, it reduces feed intake and causes hypocalcemia. Our tenet is that immune system utilization of glucose and its induction of hypophagia are responsible for the extensive increase in NEFA and ketones, and this explains why they (and the severity of hypocalcemia) are correlated with poor health, production, and reproduction outcomes. In this review, we argue that changes in circulating NEFA, ketones, and calcium are simply reflective of either (1) normal homeorhetic adjustments that healthy, high-producing cows use to prioritize milk synthesis or (2) the consequence of immune activation and its sequelae.

Effects of dietary microbial feed supplement on production efficacy in lactating dairy cows.

This study evaluated the effects of a microbial feed supplement (MFS; Galaxis, Ascus Biosciences Inc.) comprising 2 native rumen microbes on performance parameters in mid-lactation dairy cows. Forty-six lactating primiparous and multiparous Holstein cows [629 ± 62 kg of body weight, mean ± standard deviation (SD); parity 1.64 ± 0.49; 119 ± 38 days in milk; 45.11 ± 3.81 and 52.73 ± 4.77 kg/d of milk yield for primiparous and multiparous, respectively] were enrolled in a study containing 3 experimental periods (P). During all periods, enrolled cows were fed the same base total mixed ration (TMR) ad libitum once daily. During P1 (7 d), baseline data were obtained for covariate analysis. At the beginning of P2 (60 d), cows were assigned to 1 of 2 dietary treatment groups in a randomized complete block design to balance for milk yield (MY), parity, and days in milk: (1) a control diet (CON; base TMR; n = 23), or (2) a control diet supplemented with 5 g/d of MFS (MFS; n = 23). Sample size was determined based on previous, unpublished results involving this MFS; a 3-kg difference between groups with a SD of 3.5 kg could be detected with sufficient power (0.81) using a total sample size of 46 cows. Treatment was top-dressed and hand-mixed into the top one-third of the TMR. During P3 (7 d), no treatment was administered, and all cows were fed the base TMR. When analyzing all cows in the data set, MFS had little to no effect on performance. However, modeling revealed that the fixed effect of covariate milk production level had a significant effect on the response of MY and ECM, and further investigation of the data revealed that treatment effectiveness in P2 correlated with milk production during P1. Cows were retrospectively categorized into 2 milk production groups (MPG) balanced for parity: MPG1 (i.e., <53 kg/d of ECM during P1; n = 34) or MPG2 (i.e., ≥53 kg/d of ECM during P1; n = 12). Energy-corrected milk was increased by 4.4% in MFS-administered MPG1 cows compared with CON cows during P2. Although there were no significant effects of MFS on production variables for MPG2 cows, MY tended to be decreased by 3.9% in MFS-administered cows compared with CON cows. Further investigation is needed to understand production level response differences and the effect of supplemented native rumen microbes on animal health and productivity.

The effects of zinc amino acid complex on biomarkers of gut integrity, inflammation, and metabolism in heat-stressed ruminants.

Study objectives were to evaluate the effects of replacing 40 mg/kg of dietary Zn from Zn sulfate (ZS) with Zn amino acid complex (ZA; Zinpro Corporation, Eden Prairie, MN) on inflammation and intestinal integrity in heat-stressed and pair-fed (PF) ruminants. Forty Holstein steers (173.6 ± 4.9 kg) were randomly assigned to 1 of 5 dietary-environmental treatments: (1) thermoneutral (TN) ad libitum with 75 mg/kg of dry matter (DM) ZS (ZSCON); (2) TN pair-fed with 75 mg/kg DM ZS (ZSPF); (3) TN pair-fed with 40 mg/kg DM ZA and 35 mg/kg DM ZS (ZAPF); (4) heat stress (HS) ad libitum with 75 mg/kg DM ZS (ZSHS); and (5) HS ad libitum 40 mg/kg DM ZA and 35 mg/kg DM ZS (ZAHS). Before study initiation, calves were fed their respective diets for 21 d. Following the pre-feeding phase, steers were transferred into environmental chambers and were subjected to 2 successive experimental periods. During period 1 (5 d), all steers were fed their respective diets ad libitum and housed in TN conditions (20.2 ± 1.4°C, 30.4 ± 4.3% relative humidity). During period 2 (6 d), ZSHS and ZAHS steers were exposed to cyclical HS conditions (27.1 ± 1.5°C to 35.0 ± 2.9°C, 19.3 ± 3.5% relative humidity), whereas the ZSCON, ZSPF, and ZAPF steers remained in TN conditions and were fed ad libitum or pair-fed relative to their ZSHS and ZAHS counterparts. Overall, steers exposed to HS had markedly increased rectal temperature (0.83°C), respiration rate (26 breaths per min), and skin temperature (8.00°C) relative to TN treatments. Rectal temperature from ZAHS steers was decreased (0.24°C) on d 4 to 6 of HS relative to ZSHS steers. Regardless of diet, HS decreased DMI (18%) relative to ZSCON steers. Circulating glucose from HS and PF steers decreased (16%) relative to ZSCON steers. Heat stress and nutrient restriction increased circulating nonesterified fatty acids 2- and 3-fold, respectively, compared with ZSCON steers. Serum amyloid A increased ~2-fold in PF relative to ZSCON and HS steers. We detected no treatment effect on blood pH; however, ZAHS steers had increased HCO3 relative to ZSHS. Relative to ZSHS, ZAHS steers had increased jejunum villi height (25%), a tendency for increased ileum villi height (9%), and decreased duodenal villi width (16%). In summary, ZA supplementation has some beneficial effects on thermal indices, intestinal architecture characteristics, and biomarkers of leaky gut in heat-stressed steers, indicative of an ameliorated heat load, and thus may be a nutritional strategy to minimize negative consequences of HS.

Evaluating effects of zinc hydroxychloride on biomarkers of inflammation and intestinal integrity during feed restriction.

Objectives were to evaluate effects of supplemental zinc hydroxychloride (HYD; Micronutrients, Indianapolis, IN) on gut permeability, metabolism, and inflammation during feed restriction (FR). Holstein cows (n = 24; 159 ± 8 d in milk; parity 3 ± 0.2) were enrolled in a 2 × 2 factorial design and randomly assigned to 1 of 4 treatments: (1) ad libitum fed (AL) and control diet (ALCON; 75 mg/kg Zn from zinc sulfate; n = 6); (2) ad libitum fed and HYD diet (ALHYD; 75 mg/kg Zn from HYD; n = 6); (3) 40% of ad libitum feed intake and control diet (FRCON; n = 6); or (4) 40% of ad libitum feed intake and HYD diet (FRHYD; n = 6). Prior to study initiation, cows were fed their respective diets for 21 d. The trial consisted of 2 experimental periods (P) during which cows continued to receive their respective dietary treatments. Period 1 (5 d) served as the baseline for P2 (5 d), during which cows were fed ad libitum or restricted to 40% of P1 feed intake. In vivo total-tract permeability was evaluated on d 4 of P1 and on d 2 and 5 of P2, using the paracellular permeability marker chromium (Cr)-EDTA. All cows were euthanized at the end of P2 to assess intestinal architecture. As anticipated, FR cows lost body weight (∼46 kg), entered into calculated negative energy balance (-13.86 Mcal/d), and had decreased milk yield. Circulating glucose, insulin, and glucagon decreased, and nonesterified fatty acids and ß-hydroxybutyrate increased in FR relative to AL cows. Relative to AL cows, FR increased lipopolysaccharide-binding protein, serum amyloid A (SAA), and haptoglobin (Hp) concentrations (2-, 4-, and 17-fold, respectively); and peak SAA and Hp concentrations were observed on d 5. Circulating SAA and Hp from FRHYD tended to be decreased (47 and 61%, respectively) on d 5 relative to FRCON. Plasma Cr area under the curve increased (32%) in FR treatments on d 2 and tended to be increased (17%) on d 5 of P2 relative to AL treatments. No effects of diet were observed on Cr appearance. Relative to AL cows, FR increased jejunum villus width and decreased jejunum crypt depth and ileum villus height and crypt depth. Relative to FRCON, ileum villus height tended to increase in FRHYD cows. Feed restriction tended to decrease jejunum and ileum mucosal surface area, but the decrease in the ileum was ameliorated by dietary HYD. In summary, FR induced gut hyperpermeability to Cr-EDTA, and feeding HYD appeared to benefit some key metrics of barrier integrity.

Evaluating acute inflammation's effects on hepatic triglyceride content in experimentally induced hyperlipidemic dairy cows in late lactation.

Inflammation appears to be a predisposing factor and key component of hepatic steatosis in a variety of species. Objectives were to evaluate effects of inflammation [induced via intravenous lipopolysaccharide (LPS) infusion] on metabolism and liver lipid content in experimentally induced hyperlipidemic lactating cows. Cows (765 ± 32 kg of body weight; 273 ± 35 d in milk) were enrolled in 2 experimental periods (P); during P1 (5 d), baseline data were obtained. At the start of P2 (2 d), cows were assigned to 1 of 2 treatments: (1) intralipid plus control (IL-CON; 3 mL of saline; n = 5) or (2) intralipid plus LPS (IL-LPS; 0.375 µg of LPS/kg of body weight; n = 5). Directly following intravenous bolus (saline or LPS) administration, intralipid (20% fat emulsion) was intravenously infused continuously (200 mL/h) for 16 h to induce hyperlipidemia during which feed was removed. Blood samples were collected at -0.5, 0, 4, 8, 12, 16, 24, and 48 h relative to bolus administration, and liver biopsies were obtained on d 1 of P1 and at 16 and 48 h after the bolus. By experimental design (feed was removed during the first 16 h of d 1), dry matter intake decreased in both treatments on d 1 of P2, but the magnitude of reduction was greater in LPS cows. Dry matter intake of IL-LPS remained decreased on d 2 of P2, whereas IL-CON cows returned to baseline. Milk yield decreased in both treatments during P2, but the extent and duration was longer in LPS-infused cows. Administering LPS increased circulating LPS-binding protein (2-fold) at 8 h after bolus, after which it markedly decreased (84%) below baseline for the remainder of P2. Serum amyloid A concentrations progressively increased throughout P2 in IL-LPS cows (3-fold, relative to controls). Lipid infusion gradually increased nonesterified fatty acids and triglycerides in both treatments relative to baseline (3- and 2.5-fold, respectively). Interestingly, LPS infusion blunted the peak in nonesterified fatty acids, such that concentrations peaked (43%) higher in IL-CON compared with IL-LPS cows and heightened the increase in serum triglycerides (1.5-fold greater relative to controls). Liver fat content remained similar in IL-LPS relative to P1 at 16 h; however, hyperlipidemia alone (IL-CON) increased liver fat (36% relative to P1). No treatment differences in liver fat were observed at 48 h. In IL-LPS cows, circulating insulin increased markedly at 4 h after bolus (2-fold relative to IL-CON), and then gradually decreased during the 16 h of lipid infusion. Inducing inflammation with simultaneous hyperlipidemia altered the characteristic patterns of insulin and LPS-binding protein but did not cause fatty liver.

Effects of a Saccharomyces cerevisiae fermentation product on heat-stressed dairy cows.

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP) on body temperature indices, metabolism, acute phase protein response, and production variables during heat stress (HS). Twenty multiparous lactating Holstein cows (body weight = 675 ± 12 kg; days in milk = 144 ± 5; and parity = 2.3 ± 0.1) were used in an experiment conducted in 2 replicates (10 cows/replicate). Cows were randomly assigned to 1 of 2 dietary treatments: control diet (CON; n = 10) or the CON diet supplemented with 19 g/d of SCFP (n = 10; NutriTek, Diamond V, Cedar Rapids, IA). Cows were fed their respective diets for 21 d before initiation of the study. The experiment consisted of 2 periods: thermoneutral (period 1; P1) and heat stress (period 2; P2). During P1 (4 d), cows were fed ad libitum and housed in thermoneutral conditions for collecting baseline data. During P2 (7 d), HS was artificially induced using an electric heat blanket (EHB; Thermotex Therapy Systems Ltd., Calgary, AB, Canada). Cows were fitted with the EHB for the entirety of P2. Rectal temperature, respiration rate, and skin temperature were obtained twice daily (0600 and 1800 h) during both periods. Overall, HS increased rectal temperature, skin temperature, and respiration rate (1.4°C, 4.8°C, and 54 breaths/min, respectively) relative to P1, but no dietary treatment differences were detected. Compared with P1, HS decreased dry matter intake and milk yield (36 and 26%, respectively), and the reductions were similar between dietary treatments. Relative to P1, HS increased milk fat content and milk urea nitrogen (17 and 30%, respectively) and decreased milk protein and lactose contents (7 and 1.4%, respectively). Overall, HS increased (52%) plasma cortisol concentrations of CON, but circulating cortisol did not change in SCFP-fed cows. Heat stress increased circulating lipopolysaccharide binding protein and serum amyloid A (SAA; 2- and 4-fold, respectively), and SCFP supplementation tended to decrease peak SAA (∼33%) relative to CON cows. Overall, although HS did not influence circulating white blood cells and neutrophils, SCFP increased circulating white blood cells and neutrophils by 9 and 26%, respectively, over CON in P2. In conclusion, HS initiated an acute phase protein response and feeding SCFP blunted the cortisol and SAA concentrations and altered some key leukocyte dynamics during HS.

Effects of maintaining eucalcemia following immunoactivation in lactating Holstein dairy cows.

Periparturient hypocalcemia is a common metabolic disorder and it is ostensibly associated with negative health and production outcomes. Acute infection also markedly decreases circulating Ca, but the reasons for and consequences of it on physiological and immunological parameters are unknown. Objectives were to evaluate the effects of maintaining eucalcemia on production, metabolic, and immune variables following an intravenous lipopolysaccharide (LPS) challenge. Twelve multiparous lactating Holstein cows (717 ± 20 kg of body weight; 176 ± 34 d in milk; parity 3 ± 0.2) were enrolled in a study containing 2 experimental periods (P); during P1 (3 d), cows consumed feed ad libitum and baseline values were obtained. At the initiation of P2 (4 d), cows were randomly assigned to 1 of 2 treatments: (1) LPS administered (LPS-Con; 0.5 µg/kg of body weight LPS; n = 6) or (2) LPS administered + eucalcemic clamp (LPS-Ca; 0.5 µg/kg of body weight LPS; Ca infusion; n = 6). Cows were fasted for the first 12 h during P2. After LPS administration, ionized Ca was determined every 15 min for 6 h and every 30 min for an additional 6 h and intravenous Ca infusion was adjusted in LPS-Ca cows to maintain eucalcemia. Blood ionized Ca was decreased 23% for the first 12 h postbolus in LPS-Con cows, and by design, Ca infusion prevented hypocalcemia. To maintain eucalcemia for the 12 h, 13.7 g of Ca was infused. The total Ca deficit (including Ca not secreted into milk) accumulated over the 12 h was 10.4 and 20.2 g for the LPS-Con and LPS-Ca treatments, respectively. Mild hyperthermia (0.8°C) occurred for ∼6 h post-LPS administration relative to P1. From 6 to 7 h postbolus rectal temperature from LPS-Ca cows was increased (0.6°C) relative to LPS-Con cows. On d 1 of P2, milk yield decreased (61%) in both treatments relative to P1. Relative to LPS-Con cows, milk yield decreased (15%) in LPS-Ca cows during P2. Overall, circulating LPS-binding protein continuously increased postbolus, and at 24 h LPS-binding protein levels in LPS-Ca cows were increased (80%) relative to LPS-Con cows. During P2, serum amyloid A increased (4-fold) in both treatments relative to P1. Administering LPS initially decreased circulating neutrophils, then cell counts progressively increased with time. Calcium infusion decreased neutrophil counts (40%) from 9 to 12 h postbolus relative to LPS-Con cows. Neutrophil function, as assessed by oxidative burst and myeloperoxidase production, did not differ due to treatment. In summary, maintaining eucalcemia (via intravenous Ca infusion) during an immune challenge appeared to intensify inflammation and adversely affect lactation performance.

Energetic metabolism, milk production, and inflammatory response of transition dairy cows fed rumen-protected glucose.

Objectives were to evaluate the effects of rumen-protected glucose (RPG) supplementation on milk production, post-absorptive metabolism, and inflammatory biomarkers in transition dairy cows. Fifty-two multiparous cows were blocked by previous 305-d mature-equivalent milk (305ME) yield and randomly assigned to 1 of 2 iso-energetic and iso-nitrogenous treatments: (1) control diet (CON; n = 26) or (2) a diet containing RPG (pre-fresh 5.3% of dry matter and 6.0% of dry matter postpartum; n = 26). Cows received their respective dietary treatments from d -21 to 28 relative to calving, and dry matter intake was calculated daily during the same period. Weekly body weight, milk composition, and fecal pH were recorded until 28 d in milk (DIM), and milk yield was recorded through 105 DIM. Blood samples were collected on d -7, 3, 7, 14, and 28 relative to calving. Data were analyzed using repeated measures in the MIXED procedure (SAS Institute Inc., Cary, NC) with previous 305ME as a covariate. Fecal pH was similar between treatments and decreased (0.6 units) postpartum. Dry matter intake pre- and postpartum were unaffected by treatment, as was milk yield during the first 28 or 105 DIM. Milk fat, protein, and lactose concentration were similar for both treatments. Blood urea nitrogen and plasma glucose concentrations were unaffected by treatment; however, results showed increased concentration of circulating insulin (27%), lower nonesterified fatty acids (28%), and lower postpartum ß-hydroxybutyrate (24%) in RPG-fed cows. Overall, circulating lipopolysaccharide-binding protein and haptoglobin did not differ by treatment, but at 7 DIM, RPG-fed cows had decreased lipopolysaccharide-binding protein and haptoglobin concentrations (31 and 27%, respectively) compared with controls. Supplemental RPG improved some biomarkers of post-absorptive energetics and inflammation during the periparturient period, changes primarily characterized by increased insulin and decreased nonesterified fatty acids concentrations, with a concomitant reduction in acute phase proteins without changing milk production and composition.

The acute phase protein orosomucoid 1 is upregulated in early lactation but does not trigger appetite-suppressing STAT3 signaling via the leptin receptor.

Dairy cows consume inadequate amounts of feed in early lactation and during conditions and diseases such as excessive fatness, heat stress, and infectious diseases. Affected cows often experience increases in plasma concentrations of acute phase proteins consistent with the negative effect of inflammation on appetite. The acute phase protein orosomucoid 1 (ORM1), also known as alpha-1-acid glycoprotein, was recently reported to reduce appetite in the mouse through its ability to bind the full-length leptin receptor (Ob-Rb) and activate appetite-suppressing signal transducer and activator of transcription 3 (STAT3) signaling. These observations raise the possibility that ORM1 exerts appetite-suppressing effects in dairy cattle during periods of increased inflammatory tone. The applicability of this model was assessed in 2 ways. First, we asked whether ORM1 is regulated during periods of inadequate appetite such as the transition from late pregnancy to early lactation and periods of increased inflammatory tone. Plasma ORM1 was invariant in late pregnancy but increased 2.5-fold between parturition and d 7 of lactation. Gene expression studies showed that liver was the major source of this elevation with little contribution by adipose tissue or mammary gland. Additional studies showed that plasma ORM1 was not increased further by excessive fatness or by reproductive dysfunction in early lactation and was completely unresponsive to inflammatory stimuli such as heat stress or intravascular administration of the endotoxin lipopolysaccharide during established lactation. Second, we tested the ability of ORM1 to trigger STAT3 signaling through Ob-Rb using Chinese hamster ovary K1 (CHO-K1) cells transfected with a STAT3 expression plasmid. In this configuration, CHO-K1 cells did not express Ob-Rb and were incapable of leptin-induced STAT3 phosphorylation. Leptin responsiveness was conferred by co-transfecting with bovine Ob-Rb, with leptin causing increases of 5.7-fold in STAT3 phosphorylation and 2.1-fold in the expression of the STAT3-dependent gene, SOCS3. In contrast, neither bovine or human ORM1 triggered STAT3 phosphorylation irrespective of dose and period of incubation tested. In summary, bovine ORM1 is not increased during periods of increased inflammatory tone except in early lactation and is incapable of Ob-Rb-dependent STAT3 signaling. Overall, these data are inconsistent with ORM1 mediating the appetite-suppressing effects of inflammation in cattle through Ob-Rb.

Validating a heat stress model: The effects of an electric heat blanket and nutritional plane on lactating dairy cows.

The efficacy of an electric heat blanket (EHB) has previously been confirmed as an alternative method to evaluate heat stress (HS). However, a pair-feeding design has not been used with the EHB model. Therefore, study objectives were to determine the contribution of the nutritional plane to altered metabolism and productivity during EHB-induced HS. Multiparous Holstein cows (n = 18; 140 ± 10 d in milk) were subjected to 2 experimental periods (P); during P1 (4 d), cows were in thermoneutral conditions with ad libitum feed intake. During P2 (4 d), cows were assigned to 1 of 2 treatments: (1) thermoneutral conditions and pair-fed (PF; n = 8) or (2) EHB-induced HS with ad libitum feed intake (n = 10). Overall, the EHB increased rectal temperature, vaginal temperature, skin temperature, and respiration rate (1.4°C, 1.3°C, 0.8°C, and 42 breaths/min, respectively) relative to PF cows. The EHB reduced dry matter intake (DMI; 47%) and, by design, PF cows had a similar pattern and extent of decreased DMI. Milk yield decreased in EHB and PF cows by 27.3% (12.1 kg) and 13.4% (5.4 kg), respectively, indicating that reduced DMI accounted for only ∼50% of decreased milk synthesis. Milk fat content tended to increase (19%) in the EHB group, whereas in the PF cows it remained similar relative to P1. During P2, milk protein and lactose contents tended to decrease or decreased (1.3 and 2.2%, respectively) in both EHB and PF groups. Milk urea nitrogen remained unchanged in PF controls but increased (34.2%) in EHB cows relative to P1. The EHB decreased blood partial pressure of CO2, total CO2, HCO3, and base excess levels (17, 16, 17, and 81%, respectively) compared with those in PF cows. During P2, the EHB and PF cows had similar decreases (4%) in plasma glucose content, but no differences in circulating insulin were detected. However, a group by day interaction was detected for plasma nonesterified fatty acids; levels progressively increased in PF controls but remained unaltered in the EHB cows. Blood urea nitrogen increased in the EHB cows (61%) compared with the PF controls. In summary, utilizing the EHB model indicated that reduced nutrient intake explains only about 50% of the decrease in milk yield during HS, and the postabsorptive changes in nutrient partitioning are similar to those obtained in climate-controlled chamber studies. Consequently, the EHB is a reasonable and economically feasible model to study environmental physiology of dairy cows.