Rumen-protected choline reduces hepatic lipidosis by increasing hepatic triacylglycerol-rich lipoprotein secretion in dairy cows.

Objectives were to determine the effects of supplementing rumen-protected choline (RPC) on hepatic composition and secretion of triacylglycerol-rich lipoprotein when cows were subjected to feed restriction to develop fatty liver. It was hypothesized that RPC reduces hepatic triacylglycerol by enhancing secretion of hepatic lipoprotein. Pregnant, nonlactating parous Holstein cows (n = 33) at mean (± standard deviation) 234 ± 2.2 d of gestation were blocked by body condition (3.79 ± 0.49) and assigned to receive 0 g/d (CON), 25.8 g/d choline ion from a RPC product containing 28.8% choline chloride (CC; treatment L25.8), or 25.8 g/d of choline ion from a RPC product containing 60.0% CC (H25.8). Cows were fed for ad libitum intake for the first 5 d and restricted to 41% of the net energy for lactation required for maintenance and pregnancy from d 6 to 13. Intake of metabolizable methionine was maintained at 18 g/d during feed restriction by supplying rumen-protected methionine. Hepatic tissue was sampled on d 6 and 13 and analyzed for triacylglycerol and glycogen, and mRNA expression of hepatic tissue was investigated. On d 14, cows were not fed and received a 10% solution of tyloxapol intravenously at 120 mg/kg of body weight to block hydrolysis of triacylglycerols in very low density lipoprotein (VLDL). Blood was sampled sequentially for 720 min and analyzed for concentration of triacylglycerol and total cholesterol. Lymph was sampled 6 h after tyloxapol infusion, and analyzed for concentrations of fatty acids, ß-hydroxybutyrate, glucose, triacylglycerol, and total cholesterol. A sample of serum collected at 720 min after tyloxapol was assayed for the metabolome composition. The area under the curve (AUC) of serum triacylglycerol, VLDL cholesterol, and total cholesterol were calculated. Orthogonal contrasts evaluated the effect of supplementing RPC (CON vs. [1/2 L25.8 + 1/2 H25.8]) and source of RPC (L25.8 vs. H25.8). Least squares means and standard errors of the means are presented in sequence as CON, L25.8, H25.8. During feed restriction, supplementation of RPC reduced hepatic triacylglycerol (9.0 vs. 4.1 vs. 4.5 ± 0.6%) and increased glycogen contents (1.9 vs. 3.5 vs. 4.1 ± 0.2%). Similarly, supplementation of RPC increased the expression of transcripts involved in the synthesis and assembly of lipoproteins (MTTP), cellular autophagy (ATG3), and inflammation (TNFA), and reduced the expression of transcripts associated with mitochondrial oxidation of fatty acids (HADHA, MLYCD) and stabilization of lipid droplets (PLIN2). After infusion of tyloxapol, RPC increased the AUC for serum triacylglycerol (21,741 vs. 32,323 vs. 28,699 ± 3,706 mg/dL × min) and VLDL cholesterol (4,348 vs. 6,465 vs. 5,740 ± 741 mg/dL × min) but tended to reduce the concentrations of triacylglycerol in lymph (16.7 vs. 13.8 vs. 11.9 ± 1.9 mg/dL). Feeding RPC tended to increase the concentrations of 89 metabolites in serum, after adjusting for false discovery, including 3 acylcarnitines, 1 AA-related metabolite, 11 bile acids, 1 ceramide, 6 diacylglycerols, 2 dihydroceramides, 1 glycerophospholipid, and 64 triacylglycerols compared with CON. Feeding 25.8 g/d of choline ion as RPC mediated increased hepatic triacylglycerol secretion to promote lipotropic effects that reduced hepatic lipidosis in dairy cows.

Effect of vitamin D source and amount on vitamin D status and response to endotoxin challenge.

The objectives were to test the effects of dietary vitamin D3 [cholecalciferol (CHOL)] compared with 25-hydroxyvitamin D3 [calcidiol (CAL)] on vitamin D status and response to an endotoxin challenge. Forty-five Holstein bull calves (5 ± 2 d of age) were blocked into weekly cohorts, fed a basal diet that provided 0.25 µg/kg body weight (BW) CHOL, and assigned randomly to 1 of 5 treatments: control [(CON) no additional vitamin D], 1.5 µg/kg BW CHOL (CHOL1.5), 3 µg/kg BW CHOL (CHOL3), 1.5 µg/kg BW CAL (CAL1.5), or 3 µg/kg BW CAL (CAL3). Calves were fed milk replacer until weaning at 56 d of age and had ad libitum access to water and starter grain throughout the experiment. Treatments were added daily to the diet of milk replacer until weaning and starter grain after weaning. Measures of growth, dry matter intake, and serum concentrations of vitamin D, Ca, Mg, and P were collected from 0 to 91 d of the experiment. At 91 d of the experiment, calves received an intravenous injection of 0.1 µg/kg BW lipopolysaccharide (LPS). Clinical and physiological responses were measured from 0 to 72 h relative to LPS injection. Data were analyzed with mixed models that included fixed effects of treatment and time, and random effect of block. Orthogonal contrasts evaluated the effects of (1) source (CAL vs. CHOL), (2) dose (1.5 vs. 3.0 µg/kg BW), (3) interaction between source and dose, and (4) supplementation (CON vs. all other treatments) of vitamin D. From 21 to 91 d of the experiment, mean BW of supplemented calves was less compared with CON calves, but the effect was predominantly a result of the CHOL calves, which tended to weigh less than the CAL calves. Supplementing vitamin D increased concentrations of 25-hydroxyvitamin D in serum compared with CON, but the increment from increasing the dose from 1.5 to 3.0 µg/kg BW was greater for CAL compared with CHOL (CON = 18.9, CHOL = 24.7 and 29.6, CAL = 35.6 and 65.7 ± 3.2 ng/mL, respectively). Feeding CAL also increased serum Ca and P compared with CHOL. An interaction between source and dose of treatment was observed for rectal temperature and derivatives of reactive metabolites after LPS challenge because calves receiving CHOL3 and CAL1.5 had lower rectal temperatures and plasma derivatives of reactive metabolites compared with calves receiving CHOL1.5 and CAL3. Supplementing vitamin D increased plasma P concentrations post-LPS challenge compared with CON, but plasma concentrations of Ca, Mg, fatty acids, glucose, ß-hydroxybutyrate, haptoglobin, tumor necrosis factor-α, and antioxidant potential did not differ among treatments post-LPS challenge. Last, supplementing vitamin D increased granulocytes as a percentage of blood leukocytes post-LPS challenge compared with CON. Supplementing CAL as a source of vitamin D to dairy calves was more effective at increasing serum 25-hydroxyvitamin D, Ca, and P concentrations compared with feeding CHOL. Supplemental source and dose of vitamin D also influenced responses to the LPS challenge.

Effect of source and amount of vitamin D on serum concentrations and retention of calcium, magnesium, and phosphorus in dairy cows.

The objectives of the experiment were to determine the effects of supplementing 2 amounts of 25-hydroxyvitamin D3 (calcidiol; CAL) compared with equal amounts of vitamin D3 (cholecalciferol; CHOL) on serum concentrations, absorptions, and retentions of Ca, Mg, and P in periparturient dairy cows. One hundred seventy-seven (133 parous and 44 nulliparous) pregnant Holstein cows were enrolled in the experiment. Cows were blocked by parity and previous lactation milk yield (parous) or genetic merit for energy-corrected milk yield (nulliparous) and assigned randomly to receive 1 or 3 mg/d of CAL or CHOL in a 2 × 2 factorial arrangement of treatments. Treatments were provided to individual cows as a top-dress to the prepartum diet from 250 d gestation until parturition. The prepartum diet had a dietary cation-anion difference of -128 mEq/kg of dry matter. All cows were fed a common postpartum diet containing 46 µg of vitamin D3/kg of dry matter without further supplementation of treatments. Concentrations of vitamin D metabolites, Ca, Mg, and P in serum were measured pre- and postpartum, in addition to total-tract digestibility and urinary excretion of Ca, Mg, and P in the prepartum period. Feeding 3 mg compared with 1 mg of CAL increased serum 25-hydroxyvitamin D3 (CAL1 = 94 vs. CAL3 = 173 ± 3 ng/mL). In comparison, the increment in serum 25-hydroxyvitamin D3 from feeding 3 mg compared with 1 mg of CHOL was small (CHOL1 = 58 vs. CHOL3 = 64 ± 3 ng/mL). Feeding CAL increased prepartum concentration of P in serum compared with CHOL (CHOL = 1.87 vs. CAL = 2.01 ± 0.02 mM), regardless of the amount fed, but neither source nor amount affected prepartum Ca or Mg in serum. Feeding CAL increased serum Ca and P for the first 11 d postpartum compared with CHOL (CHOL = 2.12 vs. CAL = 2.16 ± 0.01 mM serum Ca; CHOL = 1.70 vs. CAL = 1.78 ± 0.02 mM serum P) but the amount of vitamin D did not affect postpartum concentrations of Ca, Mg, and P in serum. Feeding CAL increased prepartum apparent digestibility of Ca compared with CHOL (CHOL = 26.6 vs. CAL = 33.5 ± 2.8%) but treatments did not affect Ca retention prepartum. Neither source nor amount of vitamin D affected Mg and P apparent digestibility, but CAL decreased the concentration of P excreted in urine during the prepartum period (CHOL = 1.8 vs. CAL = 0.8 ± 0.3 g/d). Calcidiol tended to increase the amount of Ca secreted in colostrum (CHOL = 9.1 vs. CAL = 11.2 ± 0.9 g/d) and Ca excreted in urine postpartum (CHOL = 0.4 vs. CAL = 0.6 ± 0.1 g/d) compared with CHOL. Collectively, feeding CAL at 1 or 3 mg/d compared with CHOL in the last 24 d of gestation is an effective way to increase periparturient serum P concentration and postpartum serum Ca of dairy cows fed a prepartum diet with negative DCAD.

Effect of prepartum source and amount of vitamin D supplementation on lactation performance of dairy cows.

The objectives of this experiment were to determine the effects of supplementing 25-hydroxyvitamin D3 (calcidiol, CAL) compared with vitamin D3 (cholecalciferol, CHOL) at 1 or 3 mg/d in late gestation on production outcomes of dairy cows. One hundred thirty-three parous and 44 nulliparous pregnant Holstein cows were enrolled in the experiment. Cows were blocked by parity and previous lactation milk yield (parous) or genetic merit (nulliparous) and assigned randomly to receive 1 or 3 mg/d of CAL or CHOL in a 2 × 2 factorial arrangement of treatments (CAL1, CAL3, CHOL1, and CHOL3). Treatments were provided to individual cows as a top-dress to the prepartum diet from 250 d in gestation until parturition. The prepartum diet had a dietary cation-anion difference of -128 mEq/kg of dry matter. Production and disease were evaluated for the first 42 d in milk, and reproduction was evaluated to 300 d in milk. Incidence of postpartum diseases did not differ among treatments. Feeding CAL compared with CHOL increased yields of colostrum and colostrum fat, protein, and total solids, resulting in an increased amount of net energy for lactation secreted as colostrum (CHOL = 7.0 vs. CAL = 9.0 ± 0.7 Mcal). An interaction between source and amount was observed for milk yield: CAL3 increased milk yield compared with CHOL3 (CHOL3 = 34.1 vs. CAL3 = 38.7 ± 1.4 kg/d) but milk yield did not differ between CAL1 and CHOL1 (CHOL1 = 36.9 vs. CAL1 = 36.4 ± 1.4 kg/d). Concentrations of serum calcidiol on day of calving and average serum Ca from d 2 to 11 postpartum were positively associated with milk yield in the first 42 d in milk. Interactions between source and amount of vitamin D were also observed for pregnancy after first AI: the percentage of cows receiving CHOL1 and CAL3 that became pregnant was smaller than that of cows receiving CHOL3 and CAL1. However, pregnancy per AI and pregnancy by 300 d in milk did not differ among treatments. Overall, CAL3 increased milk yield compared with CHOL3, whereas in cows fed 1 mg/d (CAL1 and CHOL1), the source of vitamin D generally had no effect. The effect of CAL3 may be explained in part by serum CAL concentrations and postpartum serum Ca, which were associated with milk yield.

Effects of maternal choline supplementation on performance and immunity of progeny from birth to weaning.

The objectives were to investigate whether supplementation with rumen-protected choline (RPC) during late pregnancy in Holstein cows affects offspring immunity and growth, and whether effects are utero-placental, colostrum dependent, or both. A total of 105 multiparous Holstein cows were assigned randomly to a prepartum diet (1.54 Mcal of NEL/kg of DM, and 15.8% CP) without (control) or with added RPC (12.9 g/d of choline ion). Calves (n = 111) were blocked by sex and assigned randomly to colostrum from control cows or colostrum from RPC cows, resulting in 4 treatments in a 2 × 2 factorial arrangement: (1) calves born and fed colostrum from non-supplemented dams (NN; n = 33); (2) calves from non-supplemented dams and fed colostrum from RPC-fed cows (NC; n = 25); (3) calves from RPC-supplemented dams and colostrum from non-supplemented cows (CN; n = 28); and (4) calves from RPC-supplemented dams and colostrum from RPC-fed cows (CC; n = 25). Growth, intakes, and immunity of females were evaluated up to 56 d of age. Growth and intake of male calves was evaluated up to 35 d of age, and physiological and immune responses to intravenous LPS challenge were evaluated from 21 to 35 d of age. Effects of prenatal and colostrum treatments and interactions between treatments were analyzed using mixed models. Calves fed colostrum from RPC-supplemented dams had a 17.4% increase in apparent efficiency of absorption of IgG compared with calves fed colostrum from control dams (27.4 vs. 23.3%). Incidence of fever in the first 21 d of age tended to be less in females born from RPC-supplemented dams compared with females born from control dams (31 vs. 58%). Prenatal RPC females had increased hematocrit and concentrations of red blood cells, leukocytes, neutrophils, and lymphocytes in blood compared with prenatal females born from control dams. Compared with prenatal control females, prenatal RPC females had greater intake of milk replacer (704 vs. 748 ± 9.9 g/d) and starter (45.4 vs. 60.2 ± 5.9 g/d) during the first 21 d of age. In male calves, mean intake of DM was greater (1,074 vs. 976 ± 45 g/d) after the LPS challenge (0 to 8 d) by calves born from dams fed RPC compared with males born from control dams. Calves born from RPC-fed dams also had lower mean rectal temperature (39.0 vs. 39.2°C) and mean respiration rate (35.6 vs. 39.3 breaths/min) compared with males born from control dams. Moreover, serum concentrations of metabolites (i.e., ß-hydroxybutyric acid, fatty acids, and glucose), cytokines (i.e., tumor necrosis factor-α) and acute phase proteins (i.e., serum amyloid A) were consistent with less-severe inflammatory response to LPS in males born from dams fed RPC compared with control. Source of colostrum and interaction between prenatal and colostrum treatments had minimal effects on calf responses to LPS. Overall, maternal RPC supplementation during late gestation suggests a positive effect on immunity, in that colostrum from RPC-fed dams increased efficiency of IgG absorption and maternal supplementation with RPC during late gestation, regardless of colostrum source, attenuated responses to LPS.

Effect of source and amount of vitamin D on function and mRNA expression in immune cells in dairy cows.

Objectives were to determine the effect of supplementing 2 sources of vitamin D, cholecalciferol (CH) or calcidiol (CA), at 1 (1mg) or 3 mg/d (3mg) prepartum on concentrations of vitamin D metabolites in plasma, measures of innate immune function, and leukocyte mRNA expression. Parous Holstein cows (n = 99) were assigned to a daily treatment administered as top-dress containing either 1 or 3 mg of CH (CH1 or CH3) or of CA (CA1 or CA3) from 250 d of gestation until calving. Plasma concentrations of vitamin D, immune cell population in blood, cell adhesion markers, and granulocyte phagocytosis and oxidative burst were evaluated pre- and postpartum. The mRNA expression in leukocytes was determined at 270 d of gestation and 3 d postpartum for genes involved in cell migration, pathogen recognition receptors, cell signaling, cytokines, antimicrobial mechanisms, oxidative burst, and Ca and vitamin D metabolism. Concentrations of vitamin D3 increased in cows fed CH, whereas those of 25-hydroxyvitamin D3 increased in cows fed CA. Percentage of granulocytes from total leukocytes differed with amount of vitamin D pre- (1mg = 24.5 vs. 3mg = 37.9%) and postpartum (1mg = 22.0 vs. 3mg = 31.0%), thus shifting mononuclear cells in the opposite direction pre- (1mg = 75.5 vs. 3mg = 62.1%) and postpartum (1mg = 78.0 vs. 3mg = 69.0%). Granulocytes displaying phagocytosis (1mg = 69.0 vs. 3mg = 62.9%) and intensity of phagocytosis prepartum (1mg = 7.46 vs. 3mg = 7.28) tended to be less in cows fed 3mg compared with 1mg. During prepartum, CA increased mRNA expression of genes related to cell adhesion and migration (CD44, ICAM1, ITGAL, ITGB1, LGALS8, SELL), pathogen recognition receptor (NOD2, TLR2, TLR6), cell signaling (FOS, JUN, NFKB2), cytokine signaling (IL1B, IL1R1, IL1RN), antimicrobial mechanisms (CTSB, LYZ), and Ca metabolism (ATP2B1, STIM1, TRPV5) compared with CH. Similarly, postpartum, CA increased mRNA expression of genes related to cell adhesion and migration (CXCR2, SELL, TLN1), cell signaling (AKT2), cytokines (CCL2, IL1R1, ILRN), antimicrobial mechanisms (DEFB3), oxidative burst (RAC2), and calcium metabolism (CALM3) compared with CH. Feeding additional vitamin D in the last 3 wk of gestation changed the profile of blood leukocytes and attenuated granulocyte phagocytosis during the transition period, whereas supplementing CA prepartum increased mRNA expression of genes involved in immune cell function, including genes related to pathogen recognition and antimicrobial effects of leukocytes.

Effect of duration of exposure to diets differing in dietary cation-anion difference on Ca metabolism after a parathyroid hormone challenge in dairy cows.

Objectives of the experiment were to determine the length of exposure to an acidogenic diet that would elicit changes in acid-base balance, mineral digestion, and response to parathyroid hormone (PTH)-induced changes in blood Ca and vitamin D3 in prepartum dairy cows. Nonlactating parous Holstein cows (n = 20) at 242 d of gestation were blocked by lactation (1 or >1) and pretreatment dry matter (DM) intake and, within block, they were randomly assigned to a diet with a dietary cation-anion difference (DCAD) of +200 mEq/kg of DM (DCAD +200) or an acidogenic diet with -150 mEq/kg of DM (DCAD -150). Water and DM intake were measured and blood was sampled daily. Urine was sampled every 3 h for 36 h, and then daily. During PTH challenges on d 3, 8, and 13, cows received i.v. PTH 1-34 fragment at 0.05 µg/kg of body weight every 20 min for 9 h to mimic the pulsatile release of endogenous PTH. Blood was sampled at 0 h, and hourly thereafter until 10 h, and at 12, 18, 24, 36, and 48 h relative to each challenge. Acid-base measures and concentrations of ionized Ca (iCa) in whole blood, and total Ca, Mg, P, and vitamin D metabolites in plasma were evaluated. On d 2 and 7, Ca, Mg, and P balances were evaluated. Cows fed DCAD -150 had smaller blood pH (7.431 vs. 7.389) and HCO3- (27.4 vs. 22.8 mM) compared with DCAD +200, and metabolic acidosis in DCAD -150 was observed 24 h after dietary treatments started. Concentrations of iCa begin to increase 24 h after feeding the acidogenic diet, and it was greater in DCAD -150 compared with DCAD +200 by 3 d in the experiment (1.23 vs. 1.26 mM). During the PTH challenges, cows fed DCAD -150 had greater concentration of iCa and area under the curve for iCa than those fed DCAD +200 (48.2 vs. 50.7 mmol/L × hour), and there was no interaction between treatment and challenge day. Concentration of 1,25-dihydroxyvitamin D3 in plasma did not differ during the PTH challenge, but change in 1,25-dihydroxyvitamin D3 relative to h 0 of the challenge was smaller in cows fed DCAD -150 than cows fed DCAD +200 (44.1 vs. 32.9 pg/mL). Urinary loss of Ca was greater in cows fed DCAD -150 compared with DCAD +200 (1.8 vs. 10.8 g/d); however, because digestibility of Ca increased in cows fed DCAD -150 (19.7 vs. 36.6%), the amount of Ca retained did not differ between treatments. Diet-induced metabolic acidosis was observed by 24 h after dietary treatment started, resulting in increases in concentration of iCa in blood observed between 1 and 3 d. Collectively, present results indicate that tissue responsiveness to PTH and changes in blood concentrations of iCa and digestibility of Ca are elicited within 3 d of exposure to an acidogenic diet. The increased apparent digestibility of Ca compensated for the increased urinary loss of Ca resulting in similar Ca retention.

Timing of initiation and duration of feeding rumen-protected choline affects performance of lactating Holstein cows.

Objectives were to evaluate the effects of altering timing of initiating and duration of supplementing rumen-protected choline (RPC) on lactation performance in dairy cows. The hypothesis was that RPC increases yields of milk and milk components, regardless of when supplementation is initiated, and that the effects of supplementing RPC starting prepartum and continuing post-transition would be additive. Cows at 241 ± 2.2 d of gestation were blocked by parity group (49 entering lactation 2, 50 entering lactation >2) and 305-d milk yield and, within block, assigned randomly to 1 of 4 treatments arranged as a 2 × 2 factorial with 2 levels of choline in transition, from 21 d pre- to 21 d postpartum, and 2 levels of choline in post-transition, from 22 to 105 d postpartum. The 2 levels of RPC supplemented were either 0 g/d or 12.9 g/d of choline ion fed as 60 g/d of an RPC product that was top-dressed onto the total mixed ration. Thus, treatments were as follows: NN (n = 25): no choline in transition or post-transition; NC (n = 25): no choline in transition and choline in post-transition; CN (n = 25): choline in transition and no choline in post-transition; CC (n = 24): choline in transition and in post-transition. Prepartum, treatments were supplemented (mean ± SD) for the last 18.8 ± 5.7 and 19.2 ± 5.0 d of gestation in treatments with 0 or 12.9 g/d of choline ion, respectively. Supplementing RPC prepartum did not affect dry matter intake (DMI), body weight (BW), or body condition score (BCS) in the last 3 weeks of gestation. Likewise, RPC did not affect the yield or the composition of colostrum. Supplementation with RPC during transition increased fat percent by 0.02 percentage units, fat yield by 0.16 kg/d, and energy-corrected milk (ECM) by 3.1 kg/d in the first 21 d postpartum, and increased fat yield by 0.10 kg/d and ECM by 2.4 kg/d from 22 to 105 d postpartum. Supplementing RPC during transition did not affect DMI postpartum, but it improved feed efficiency, and cows produced 0.11 kg/d more ECM per kg of DMI. Changes in BW and BCS during the first 21 d postpartum did not differ between treatments. Cows fed RPC during transition had more negative net energy balance and 0.1 unit smaller BCS in the first 105 d postpartum than non-supplemented cows. Supplementing RPC in post-transition did not influence productive performance in dairy cows, and choline supplementation during transition or post-transition did not affect measures of reproduction. Collectively, supplementing RPC to supply 12.9 g/d of choline ion benefited productive performance in dairy cows when supplementation occurred during the transition period, but no additional benefit was observed from supplementing RPC past 22 d postpartum.

Effect of prepartum energy intake and supplementation with ruminally protected choline on innate and adaptive immunity of multiparous Holstein cows.

Objectives were to evaluate the effect of prepartum energy intake and peripartal supplementation of ruminally protected choline (RPC) on select indicators of immune status in blood plasma and on lipopolysaccharide-stimulated blood cells ex vivo. At 47 ± 6 d before the expected calving date, 93 multiparous Holstein cows were assigned randomly to 1 of 4 dietary treatments in a 2 × 2 factorial arrangement. Cows were fed energy to excess [EXE; 1.63 Mcal of net energy for lactation (NEL)/kg of dietary dry matter (DM)] or to maintenance (MNE; 1.40 Mcal of NEL/kg of dietary DM) ad libitum throughout the nonlactating period. The RPC was fed at 0 or 60 g/d to supply 0 or 12.9 g/d of choline ions top-dressed for 17 ± 4.6 d prepartum through 21 d postpartum. After calving, cows were fed the same methionine-supplemented diet, apart from RPC supplementation. During the last 2 wk before calving and during the first 5 wk postpartum, blood was sampled repeatedly and analyzed for cell types, acute-phase proteins, tumor necrosis factor-α (TNFα), and neutrophil function. Samples of whole blood were collected at 3 and 14 DIM and stimulated with 1 µg/mL lipopolysaccharide (LPS) in vitro for 6 and 24 h. After 6 h of LPS exposure, peripheral blood leucocytes (PBL) were harvested, and relative transcript abundance for select cytokines were measured. Supernatant was analyzed for TNFα after 24 h of LPS exposure. The PBL from cows fed EXE diets during the whole dry period had increased transcripts for the proinflammatory cytokines CXCL8 and TNF, although the plasma concentrations of the acute-phase proteins haptoglobin and fibrinogen, and the killing activity of the blood neutrophils in the postpartum period, were not affected by feeding different energy levels prepartum. Feeding RPC to cows overfed energy prepartum modulated their inflammatory state, as evidenced by decreased IL6 in PBL and reduced mean fluorescence intensity of CD14 during the postpartum period, compared with cows not fed RPC. Feeding RPC also decreased TNFα protein production, abundances of IL1B, CXCL8, and TNF transcripts, and mean fluorescence intensity of CD80 of PBL stimulated by LPS, regardless of prepartum energy intake. In contrast, proportions of blood neutrophils undergoing phagocytosis and oxidative burst were increased at 17 d postpartum in cows supplemented with RPC. Collectively, these data indicate that transition cows supplemented with RPC experienced less inflammation, which may partially explain increased milk production in cows supplemented with RPC.

Effects of rumen-protected choline on the inflammatory and metabolic status and health of dairy cows during the transition period.

The objectives of this study were to evaluate the effects of rumen-protected choline (RPC) supplementation from 21 d pre- to 21 d postpartum on markers of metabolic status and inflammatory response, concentrations of liposoluble vitamins, and plasma total Ca in parous Holstein cows. The hypotheses were that supplementing RPC during the transition period would reduce hepatic triacylglycerol accumulation postpartum and attenuate markers of inflammatory response following parturition, and collectively, such responses were expected to benefit health of dairy cows. Parous cows at 241 d of gestation were blocked by parity group and 305-d milk yield, and within block, they were assigned randomly to receive either 0 g/d [no choline in transition (NT), n = 55] or 12.9 g/d choline ion [choline in transition (CT), n = 58] from 21 d pre- to 21 postpartum. The RPC product was individually top-dressed onto the total mixed ration once daily. Prepartum, treatments were supplemented (mean ± standard deviation) for the last 18.8 ± 5.7 and 19.2 ± 5.0 d of gestation in NT and CT, respectively. Supplementing RPC prepartum did not affect concentrations of plasma metabolites and inflammatory markers during the last 3 wk of gestation. Postpartum, cows fed RPC had greater hepatic concentration of hepatic triacylglycerol (NT = 3.4 vs. CT = 4.4%) and tended to have increased concentration of ß-hydroxybutyrate (NT = 0.48 vs. CT = 0.53 mM) in plasma. In spite of the increased hepatic triacylglycerol in cows fed RPC, treatment did not affect the concentrations of the inflammatory marker tumor necrosis factor-α or of the positive acute phase proteins, haptoglobin and fibrinogen. Supplementing choline tended to increase the concentration of plasma triacylglycerol by 0.69 mg/dL in the first 21 d postpartum and reduced the incidence of subclinical hypocalcemia by 20.9 percentage units compared with NT. Supplementing transition cows with RPC did not affect the concentrations of liposoluble vitamins in the first 7 d postpartum or the incidence of individual diseases or morbidity in early lactation. The inability of supplemental choline to reduce hepatic triacylglycerol might have been a consequence of the increased productive performance without additional dry matter intake.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Lactation performance and energy metabolism.

The objectives of this experiment were to evaluate the effects of feeding diets with 2 dietary cation-anion difference (DCAD) levels and supplemented with either cholecalciferol (CH) or calcidiol (CA) during late gestation on lactation performance and energetic metabolism in dairy cows. The hypothesis was that combining a prepartum acidogenic diet with calcidiol supplementation would benefit peripartum Ca metabolism and, thus, improve energy metabolism and lactation performance compared with cows fed an alkalogenic diet or cholecalciferol. Holstein cows at 252 d of gestation were blocked by parity (28 nulliparous and 51 parous cows) and milk yield within parous cows, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial, with 2 levels of DCAD (positive, +130, and negative, -130 mEq/kg) and 2 sources of vitamin D, CH or CA, fed at 3 mg per 11 kg of diet dry matter (DM). The resulting treatment combinations were positive DCAD with CH (PCH), positive DCAD with CA (PCA), negative DCAD with CH (NCH), or negative DCAD with CA (NCA), which were fed for the last 21 d of gestation. After calving, cows were fed the same lactation diet. Body weight and body condition were evaluated prepartum and for the first 49 d postpartum. Blood was sampled thrice weekly prepartum, and on d 0, 1, 2, 3, and every 3 d thereafter until 30 d postpartum for quantification of hormones and metabolites. Lactation performance was evaluated for the first 49 d postpartum. Feeding a diet with negative DCAD reduced DM intake in parous cows by 2.1 kg/d, but no effect was observed in nulliparous cows. The negative DCAD reduced concentrations of glucose (positive = 4.05 vs. negative = 3.95 mM), insulin (positive = 0.57 vs. negative = 0.45 ng/mL), and insulin-like growth factor-1 (positive = 110 vs. negative = 95 ng/mL) prepartum. Treatments did not affect DM intake postpartum, but CA-supplemented cows tended to produce more colostrum (PCH = 5.86, PCA = 7.68 NCH = 6.21, NCA = 7.96 ± 1.06 kg) and produced more fat-corrected milk (PCH = 37.0, PCA = 40.1 NCH = 37.5, NCA = 41.9 ± 1.8 kg) and milk components compared with CH-supplemented cows. Feeding the negative DCAD numerically increased yield of fat-corrected milk by 1.0 kg/d in both nulliparous and 1.4 kg/d in parous cows. Minor differences were observed in postpartum concentrations of hormones and metabolites linked to energy metabolism among treatments. Results from this experiment indicate that replacing CH with CA supplemented at 3 mg/d during the prepartum period improved postpartum lactation performance in dairy cows.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Vitamin D, mineral, and bone metabolism.

Pregnant Holstein cows, 28 nulliparous and 51 parous, were blocked by parity and milk yield and randomly allocated to receive diets that differed in dietary cation-anion difference (DCAD), +130 or -130 mEq/kg, and supplemented with either calcidiol or cholecalciferol at 3 mg/11 kg of dry matter from 255 d of gestation until parturition. Blood was sampled thrice weekly prepartum, and on d 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 postpartum to evaluate effects of the diets on vitamin D, mineral and bone metabolism, and acid-base status. Blood pH and concentrations of minerals, vitamin D metabolites, and bone-related hormones were determined, as were mineral concentrations and losses in urine and colostrum. Supplementing with calcidiol increased plasma concentrations of 25-hydroxyvitamin D3, 3-epi 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 compared with supplementing with cholecalciferol. Cows fed the diet with negative DCAD had lesser concentrations of vitamin D metabolites before and after calving than cows fed the diet with positive DCAD, except for 25-hydroxyvitamin D2. Feeding the diet with negative DCAD induced a compensated metabolic acidosis that attenuated the decline in blood ionized Ca (iCa) and serum total Ca (tCa) around calving, particularly in parous cows, whereas cows fed the diet with positive DCAD and supplemented with calcidiol had the greatest 1,25-dihydroxyvitamin D3 concentrations and the lowest iCa and tCa concentrations on d 1 and 2 postpartum. The acidogenic diet or calcidiol markedly increased urinary losses of tCa and tMg, and feeding calcidiol tended to increase colostrum yield and increased losses of tCa and tMg in colostrum. Cows fed the diet with negative DCAD had increased concentrations of serotonin and C-terminal telopeptide of type 1 collagen prepartum compared with cows fed the diet with positive DCAD. Concentrations of undercarboxylated and carboxylated osteocalcin and those of adiponectin did not differ with treatment. These results provide evidence that dietary manipulations can induce metabolic adaptations that improve mineral homeostasis with the onset of lactation that might explain some of the improvements observed in health and production when cows are fed diets with negative DCAD or supplemented with calcidiol.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Health and reproductive responses.

The objectives of the experiment were to evaluate the effects of feeding diets with distinct dietary cation-anion difference (DCAD) levels and supplemented with 2 sources of vitamin D during the prepartum transition period on postpartum health and reproduction in dairy cows. The hypotheses were that feeding acidogenic diets prepartum would reduce the risk of hypocalcemia and other diseases, and the benefits of a negative DCAD treatment on health would be potentiated by supplementing calcidiol compared with cholecalciferol. Cows at 252 d of gestation were blocked by parity (28 nulliparous and 52 parous cows) and milk yield within parous cows, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial, with 2 levels of DCAD, positive (+130 mEq/kg) or negative (-130 mEq/kg), and 2 sources of vitamin D, cholecalciferol or calcidiol, fed at 3 mg for each 11 kg of diet dry matter. The resulting treatment combinations were positive DCAD with cholecalciferol (PCH), positive DCAD with calcidiol (PCA), negative DCAD with cholecalciferol (NCH), and negative DCAD with calcidiol (NCA), which were fed from 252 d of gestation to calving. After calving, cows were fed the same lactation diet supplemented with cholecalciferol at 0.70 mg for every 20 kg of dry matter. Blood was sampled 7 d before parturition, and at 2 and 7 d postpartum to evaluate cell counts and measures of neutrophil function. Postpartum clinical and subclinical diseases and reproductive responses were evaluated. Feeding a diet with negative DCAD eliminated clinical hypocalcemia (23.1 vs. 0%) and drastically reduced the incidence and daily risk of subclinical hypocalcemia, and these effects were observed in the first 48 to 72 h after calving. The diet with negative DCAD tended to improve the intensity of oxidative burst activity of neutrophils in all cows prepartum and increased the intensity of phagocytosis in parous cows prepartum and the proportion of neutrophils with killing activity in parous cows postpartum (58.5 vs. 67.6%). Feeding calcidiol improved the proportion of neutrophils with oxidative burst activity (60.0 vs. 68.7%), reduced the incidences of retained placenta (30.8 vs. 2.5%) and metritis (46.2 vs. 23.1%), and reduced the proportion of cows with multiple diseases in early lactation. Combining the negative DCAD diet with calcidiol reduced morbidity by at least 60% compared with any of the other treatments. Cows with morbidity had lower blood ionized Ca and serum total Ca concentrations than healthy cows. Treatments did not affect the daily risk of hyperketonemia in the first 30 d of lactation. Despite the changes in cow health, manipulating the prepartum DCAD did not influence reproduction, but feeding calcidiol tended to increase the rate of pregnancy by 55%, which reduced the median days open by 19. In conclusion, feeding prepartum cows with a diet containing a negative DCAD combined with 3 mg of calcidiol benefited health in early lactation.

Effects of supplementation with ruminally protected choline on performance of multiparous Holstein cows did not depend upon prepartum caloric intake.

Objectives were to evaluate the effect of prepartum energy intake on performance of dairy cows supplemented with or without ruminally protected choline (RPC; 0 or 17.3 g/d of choline chloride; 0 or 60 g/d of ReaShure, Balchem Corp., New Hampton, NY). At 47 ± 6 d before the expected calving date, 93 multiparous Holstein cows were assigned randomly to 1 of 4 dietary treatments in a 2 × 2 factorial arrangement. Cows were fed energy to excess [EXE; 1.63 Mcal of net energy for lactation/kg of dry matter (DM)] or to maintenance (MNE; 1.40 Mcal of net energy for lactation/kg of DM) in ad libitum amounts throughout the nonlactating period. The RPC was top-dressed for 17 ± 4.6 d prepartum through 21 d postpartum (PP). After calving, cows were fed the same methionine-balanced diet, apart from RPC supplementation, through 15 wk PP. Liver was biopsied at -14, 7, 14, and 21 d relative to parturition. Cows fed EXE or MNE diets, respectively, consumed 40 or 10% more Mcal/d than required at 15 d before parturition. Cows fed the MNE compared with the EXE diet prepartum consumed 1.2 kg/d more DM postpartum but did not produce more milk (41.6 vs. 43.1 kg/d). Thus, PP cows fed the EXE diet prepartum were in greater mean negative energy balance, tended to have greater mean concentrations of circulating insulin, fatty acids, and ß-hydroxybutyrate, and had greater triacylglycerol in liver tissue (8.3 vs. 10.7% of DM) compared with cows fed the MNE diet prepartum. Cows fed RPC in transition tended to produce more milk (43.5 vs. 41.3 kg/d) and energy-corrected milk (44.2 vs. 42.0 kg/d) without increasing DM intake (23.8 vs. 23.2 kg/d) during the first 15 wk PP, and tended to produce more milk over the first 40 wk PP (37.1 vs. 35.0 kg/d). Energy balance of cows fed RPC was more negative at wk 2, 3, and 6 PP, but mean circulating concentrations of fatty acids and ß-hydroxybutyrate did not differ from those of cows not fed RPC. Despite differences in energy balance at 2 and 3 wk PP, mean concentration of hepatic triacylglycerol did not differ between RPC treatments. Feeding RPC reduced the daily prevalence of subclinical hypocalcemia from 25.5 to 10.5%, as defined by concentrations of total Ca of <8.0 mg/dL in serum in the first 7 d PP. Pregnancy at first artificial insemination tended to be greater for cows fed RPC (41.3 vs. 23.6%), but the proportion of pregnant cows did not differ by 40 wk PP. Heifers born from singleton calvings from cows fed RPC tended to experience greater daily gain between birth and 50 wk of age than heifers from cows not supplemented with RPC. Feeding RPC for approximately 38 d during the transition period tended to increase yield of milk for 40 wk regardless of amount of energy consumed during the pregnant, nonlactating period.

Assessment of serum 25-hydroxyvitamin D concentrations of beef cows and calves across seasons and geographical locations.

Vitamin D is critical for the growth and development of calves and positively contributes to immune function of cattle. Serum 25-hydroxyvitamin D (25(OH)D) concentrations above 20 ng/mL have traditionally been considered adequate for growth and development of cattle, but recent evidence has indicated that concentrations below 30 ng/mL are insufficient for immunity. Because little information is available regarding vitamin D status of beef cattle, the objective of this study was to evaluate vitamin D status of beef cow-calf herds on pasture as affected by season and location. Serum samples were collected from 43 cow-calf pairs plus an additional 54 calves in herds located in Florida, Idaho, and Minnesota in the spring calving season. Samples were collected again over the summer months from animals in the Florida and Minnesota herds. Effects of subcutaneous injection of vitamins A, D, and E also were investigated in a subset of calves from the Idaho herd. All cows sampled had serum 25(OH)D concentrations above 30 ng/mL at the time of calving in the spring. The average serum 25(OH)D concentrations of cows rose from near 60 ng/mL in the spring to 75 ng/mL in the summer ( < 0.001). Most calves, on the other hand, had serum 25(OH)D concentrations below 20 ng/mL. The calves in the Florida and Minnesota herds similarly rose from averages of 10 to 15 ng/mL at birth to near 50 ng/mL by the end of summer. Serum 25(OH)D of severely deficient calves increased from 3 ng/mL in nonsupplemented calves to 11 ng/mL at 48 h after birth if given a bolus supplementation of 40,000 IU of vitamin D via subcutaneous injection of a vitamin A, D, and E supplement at birth ( < 0.001). Vitamin D supplementation of cows late in pregnancy has been shown to increase serum 25(OH)D of calves; however, beef cattle generally receive very little supplemental vitamin D, as was the case for the cows studied here. The lower serum 25(OH)D of cows in spring compared with summer and the prevalence of vitamin D deficiency of calves observed here indicate that increased vitamin D supplementation of cows over the winter months or vitamin D supplementation of newborn calves would be beneficial.

Manual medicine diversity: research pitfalls and the emerging medical paradigm.

Recent studies published in leading medical journals have concluded that chiropractic treatment is not particularly helpful for relieving asthma and migraine symptoms because even though study participants showed notable improvement in symptoms, those subjects who received sham manual medicine treatments also showed improvement. Yet the sham treatment received by control groups in these studies is reminiscent in many ways of traditional osteopathic manipulation. This seems to represent not only a failure to recognize the value of many manual medicine techniques but also an ignorance of the broad spectrum of manual medicine techniques used by various practitioners, from osteopathic physicians to chiropractors to physical therapists. Such blind spots compromise research methodology with regard to manual medicine studies, which could, in turn, diminish the role of manual medicine in clinical practice. Osteopathic manipulative treatment provides an excellent model for recognizing and integrating the full range of manual medicine techniques into research and clinical applications because of the wide range of techniques employed. The potential exists for these techniques to contribute much to medical research and clinical practice--provided that osteopathic physicians and other manual medicine practitioners work to alleviate ignorance about the efficacy of various forms of manipulation.

The abdominal brain and enteric nervous system.

Conventional medical treatment for neurologic disorders such as epilepsy, migraine, and autism focuses on the brain. Although standard medical treatment is often helpful, the underlying causes of these disorders are not well understood. Furthermore, some individuals respond poorly or not at all to regular medicine. Evidence is accumulating in the medical literature that the enteric nervous system (ENS)-that part of the nervous system associated with the alimentary canal-also plays a role in these disorders. Historically, the concept of an autonomous abdominal nervous system was advocated by Byron Robinson, Johannis Langley, and Edgar Cayce. The work of these three prominent historical figures is considered along with modem view-points on the abdominal nervous system. Complementary therapies that address the nervous system of the abdomen have potential as useful adjuncts to conventional treatment for certain neurologic disorders.