Effects of peripartal yeast culture supplementation on lactation performance, blood biomarkers, rumen fermentation, and rumen bacteria species in dairy cows.

Feeding yeast culture fermentation products has been associated with improved feed intake and milk yield in transition dairy cows. These improvements in performance have been further described in terms of rumen characteristics, metabolic profile, and immune response. The objective of this study was to evaluate the effects of a commercial yeast culture product (YC; Culture Classic HD, Phibro Animal Health) on performance, blood biomarkers, rumen fermentation, and rumen bacterial population in dairy cows from -30 to 50 d in milk (DIM). Forty Holstein dairy cows were enrolled in a randomized complete block design from -30 to 50 DIM and blocked according to expected calving day, parity, previous milk yield, and genetic merit. At -30 DIM, cows were assigned to either a basal diet plus 114 g/d of ground corn (control; n = 20) or a basal diet plus 100 g/d of ground corn and 14 g/d of YC (n = 20), fed as a top-dress. Cows received the same close-up diet from 30 d prepartum until calving [1.39 Mcal/kg of dry matter (DM) and 12.3% crude protein (CP)] and lactation diet from calving to 50 DIM (1.60 Mcal/kg of DM and 15.6% CP). Blood samples and rumen fluid were collected at various time points from -30 to 50 d relative to calving. Cows fed YC compared with control showed a trend for increased energy-corrected milk (+3.2 kg/d). Lower somatic cell counts were observed in YC cows than in control. We detected a treatment × time interaction in nonesterified fatty acids (NEFA) that could be attributed to a trend for greater NEFA in YC cows than control at 7 DIM, followed by lower NEFA in YC cows than control at 14 and 30 DIM. In the rumen, YC contributed to mild changes in rumen fermentation, mainly increasing postpartal valerate while decreasing prepartal isovalerate. This was accompanied by alterations in rumen microbiota, including a greater abundance of cellulolytic (Fibrobacter succinogenes) and lactate-utilizing bacteria (Megasphaera elsdenii). These results describe the potential benefits of supplementing yeast culture during the late pregnancy through early lactation, at least in terms of rumen environment and performance.

Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows.

The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.

Response to adrenocorticotropic hormone or corticotrophin-releasing hormone and vasopressin in lactating cows fed an immunomodulatory supplement under thermoneutral or acute heat stress conditions.

Adrenal responsiveness was tested in nonpregnant, lactating Holstein dairy cows fed diets supplemented with OmniGen-AF (OG; Phibro Animal Health Corp., Teaneck, NJ), an immune modulator, and in nonsupplemented control (CON) cows following bolus infusions of a combination of corticotropin-releasing hormone (CRH; 0.3 µg/kg of BW) and arginine vasopressin (VP; 1.0 µg/kg of BW) or ACTH (0.1 IU/kg of BW) in 2 environments: thermoneutral [TN; temperature-humidity index (THI) <60] for 24 h/d and heat stress (HS; THI >68 for 17 h/d). Cows (506) were initially fed OG (n = 254) or CON (n = 252) diets for 44 d before selection of a subgroup of cows (n = 12; 6 OG, 6 CON) for the study. The 2 subgroups were balanced for parity, milk yield, and days in milk. All cows were transported to and housed in 2 environmentally controlled rooms at the University of Arizona Agricultural Research Complex (Tucson). Cows were given 3 d to acclimate to the rooms and then underwent 12 d of TN conditions and then 8 d of HS conditions for a total of 24 d on experiment. Cows were infused with CRH-VP on d 9 of TN and on d 1 of HS and with ACTH on d 10 of TN and on d 2 of HS. Hormone infusions took place at 1000 h (0 h) on each infusion day. Blood samples, taken in 30-min intervals, were first collected at 0800 h (-2 h) and were drawn until 1800 h (8 h). Before infusion, serum progesterone was elevated in OG cows compared with CON cows. Infusion of releasing factors (CRH-VP or ACTH) caused increases in serum cortisol and progesterone, but cortisol release was greater in CON cows than in OG cows during HS, whereas progesterone did not differ between the 2 treatments. Serum ACTH increased following infusion of releasing factors, but this increase was greater following CRH-VP infusion than ACTH infusion. Serum bovine corticosteroid-binding globulin also increased following infusion of releasing factors in both treatment groups, but this increase was greater during HS in cows fed OG. The free cortisol index (FCI) increased following CRH-VP and ACTH and was higher in HS than in TN for both OG and CON cows. However, the FCI response was blunted in OG cows compared with CON cows during HS. Heat stress enhanced the adrenal response to releasing factors. Additionally, the adrenal cortisol and FCI response to releasing factors was reduced during acute heat stress in cows fed OG. Collectively, these data suggest that OG supplementation reduced the adrenal responsiveness to factors regulating cortisol secretion during acute HS.

Effect of varying prepartum dietary cation-anion difference and calcium concentration on postpartum mineral and metabolite status and milk production of multiparous cows.

Eighty-two multiparous Holstein cows were enrolled 28 d before expected calving and assigned to 1 of 4 dietary treatments in a randomized block design experiment with a 2 × 2 factorial arrangement of treatments to determine the effect of feeding a neutral or acidogenic diet varying in Ca concentration on prepartum and postpartum intake, blood mineral and metabolite concentrations, and postpartum milk production. Prepartum diets were formulated to provide a dietary cation-anion difference (DCAD) of -21 (negative, NEG) or -2 (neutral, NEU) mEq/100 g of dry matter with either 1.3% or 1.8% Ca. After calving, cows remained on trial through 63 d in milk (DIM) and were fed a common lactation diet. Urine pH was lower for NEG compared with NEU and tended to be lower for 1.8% Ca compared with 1.3% Ca. Fractional excretion of Ca and Mg in urine was greater for NEG than for NEU. Prepartum plasma bicarbonate was lower and P was higher for NEG compared with NEU. Prepartum plasma P and blood urea nitrogen to creatinine ratio was higher for 1.3% compared with 1.8% Ca. Postpartum, concentrations of plasma total protein, albumin, blood urea nitrogen, Mg, and ionized Mg (iMg) were higher and Na was lower for NEU compared with NEG. An interaction of DCAD and Ca was observed for plasma creatinine, which was highest for cows fed NEU and 1.3% Ca compared with all other treatments. Interactions of DCAD and DIM were observed for plasma bicarbonate and iMg. Bicarbonate was higher at 3 DIM and lower at 14 DIM for NEU compared with NEG. Concentrations of iMg were higher at 1, 2, and 14 DIM for NEU compared with NEG. Interactions of Ca and DIM were observed for plasma Ca, Cl, and anion gap. Compared with cows fed 1.5% Ca, those fed 1.3% Ca had lower Ca and anion gap and higher Cl at 1 DIM and lower Cl and higher anion gap at 14 DIM. No differences were observed in body weight or body condition score due to DCAD or Ca. Prepartum dry matter intake (DMI) was lower for NEG compared with NEU and lower for 1.8% compared with 1.3% Ca. Postpartum DMI was not different among treatments. An interaction was observed for DCAD and DIM due to higher milk yield after 45 DIM for NEG compared with NEU. No differences were observed in milk component percentage or yield among treatments. There was an interaction of DIM and Ca for milk urea concentrations, which were higher at 5 wk and lower at 6 wk for 1.3% Ca compared with 1.8% Ca. These results suggest that feeding NEG prepartum alters plasma and urine mineral concentrations compared with feeding NEU and supports increased milk yield after 45 DIM. Feeding 1.8% Ca prepartum only improved plasma Ca at 1 DIM. Feeding either NEG or 1.8% Ca reduced DMI prepartum compared with NEU or 1.3% Ca.

Short communication: Blood mineral and gas concentrations of calves born to cows fed prepartum diets differing in dietary cation-anion difference and calcium concentration.

Eighty-two multiparous Holstein cows were fed diets differing in dietary cation-anion difference (DCAD) and Ca concentrations in a randomized block design experiment beginning 4 wk before anticipated calving to determine the effects on colostrum yield and quality and acid-base balance and mineral status of newborn calves. Treatments were arranged as a 2 × 2 factorial to provide 2 DCAD [-22 mEq/100 g of dry matter (NEG) or -3 mEq/100 g of dry matter (NEU)] and 2 supplemental Ca concentrations (1.3 or 1.8% of dry matter). After calving, cows were milked within 2 to 8 h and colostrum yield was recorded. Calves were fed 200 g of IgG of a commercial colostrum replacer within 4 h of birth. No differences were observed in birth weight or dystocia score among treatments, which averaged 42.7 kg and 1.12, respectively. Colostrum yield was not different among treatments and averaged 8.75 kg. Colostrum quality, as measured using a Brix refractometer, was not affected by DCAD but was higher for 1.3% compared with 1.8% Ca: 21.58% and 19.87%, respectively. Colostrum IgG concentrations were higher for NEG compared with NEU and for 1.3% compared with 1.8% Ca. No differences were observed in concentrations of serum IgG, Ca, P, K, Cl, anion gap, or whole-blood pH, partial pressure of O2, or SO2 of calves among treatments. Serum Mg and lactate concentrations were higher and CO2 tended to be lower for calves born to cows fed 1.3% compared with 1.8% Ca. Interactions of DCAD and Ca were observed for serum Na and Cl, which were higher for NEU-1.3% Ca and NEG-1.8% Ca compared with NEU-1.8% Ca and NEG-1.3% Ca. Whole-blood partial pressure of CO2, and HCO3 exhibited an interaction of DCAD and Ca and tended to be lower for NEU-1.3% Ca and NEG-1.8% Ca compared with NEU-1.8% Ca and NEG-1.3% Ca. Results of this trial indicate that feeding prepartum diets with 1.8% compared with 1.3% supplemental Ca reduced colostrum quality and serum concentrations of Mg and lactate in calves immediately after birth. Feeding NEG supported higher colostrum IgG concentrations. Blood mineral concentrations and blood gas balance tended to differ, but the effects were not consistent across DCAD and Ca.

An evaluation of an immunomodulatory feed ingredient in heat-stressed lactating Holstein cows: Effects on hormonal, physiological, and production responses.

Holstein cows (n = 30) were balanced by days in milk, milk production, and parity (91 ± 5.9 d in milk, 36.2 ± 2.5 kg/d, and 3.1 ± 1.4, respectively) and fed OmniGen-AF (OG; Phibro Animal Health, Teaneck, NJ), an immune stimulant, at 0 g/cow per d for control (CON) or 56 g/cow per d for OG for 52 d on a commercial dairy. At 52 d of the study cows were randomly selected (n = 12) from both groups (6 OG and 6 CON) and housed in environmentally controlled rooms at the Agricultural Research Complex for 21 d at the University of Arizona. Cows were subjected to 7 d of thermoneutral (TN) conditions, 10 d of heat stress (HS), and 4 d of recovery (REC) under TN conditions. Feed intake, milk production, and milk composition were measured daily. Rectal temperatures (RT) and respiration rates (RR) were recorded 3 times per day (600, 1400, and 1800 h). Blood samples were taken on d 7 (TN), 8 (HS), 10 (HS), 17 (HS), and 18 (TN) during the Agricultural Research Complex segment. Cows in HS had higher RR and RT and water intake and lower dry matter intake and milk yield than these measures in TN. There was a treatment × environment interaction with cows fed OG having lower RR and RT and higher dry matter intake during peak thermal loads than CON. However, milk yield did not differ between groups. Cows fed OG had lower milk fat percent than CON (3.7 vs 4.3%) during HS. The SCC content of milk did not differ between treatment groups but rose in both groups during the REC phase following HS. Plasma insulin and plasma glucose levels were not different between groups. However, plasma insulin in both groups was lower during acute HS, then rose across the HS period, and was highest during the REC phase. Plasma cortisol levels were highest in all cows on the first day of HS (d 8) but were lower in cows fed OG compared with CON. However, plasma ACTH concentrations were elevated in OG-fed animals at all times samples were collected. Plasma ACTH was also elevated in cows fed both OG and CON during HS. Feeding OG reduced plasma cortisol during acute but not chronic HS and increased basal plasma ACTH, suggesting that OG treatment may alter the hypothalamic pituitary adrenal axis.

A commercial immune modulating feed additive restores L-selectin and CCL5 expression following dexamethasone treatment of murine immune cells in a MyD88-dependent manner.

Bovine mastitis costs the dairy industry billions of dollars every year and presents a health challenge in dairy facilities. Immunosuppressive effects of the periparturient period increase the incidence of mastitis. During this time, cattle experience an elevation in circulating cortisol, which reduces polymorphonuclear cell function and ability to clear infection. OmniGen-AF (OMN; Phibro Animal Health, Teaneck, NJ) is an immunomodulatory feed additive that alters gene expression and is used to reduce rates of mastitis. We hypothesized that OMN restores gene expression during periods of immune stress through inhibiting the suppressive effects of glucocorticoid receptor signaling on Toll-like receptor signaling. To test our hypothesis, wild-type (WT) or MyD88 knockout mice were supplemented with OMN and challenged with lipopolysaccharide following dexamethasone (Dex) treatment. Polymorphonuclear cell and macrophage RNA was isolated from intraperitoneal lavages and analyzed for gene expression profiles. Treatment of mice with Dex suppressed expression of l-selectin and CCL5 as compared with phosphate-buffered saline treatment of WT mice. Expression of l-selectin and CCL5 was significantly reduced with Dex treatment in control-fed but not OMN-supplemented WT mice. The protective effect of OMN supplementation on l-selectin expression during Dex treatment was abolished in MyD88 knockout mice. These results suggest that OMN supplementation restores responses of certain genes suppressed by Dex in immune cells in a MyD88-dependent manner. Future research will determine the specific Toll-like receptors, transcription factors, and biochemical properties of OMN that restore gene expression in immunosuppressed cells.

Effects of prepartum dietary cation-anion difference on aspects of peripartum mineral and energy metabolism and performance of multiparous Holstein cows.

The objectives of this study were to determine the effect of decreasing dietary cation-anion difference [DCAD; (Na+ + K+) - (Cl- + S2-)] of the prepartum diet on aspects of mineral metabolism, energy metabolism, and performance of peripartum dairy cows. Multiparous Holstein cows (n = 89) were enrolled between 38 and 31 d before expected parturition and randomized to treatments in a completely randomized design (restricted to balance for previous 305-d mature equivalent milk production, parity, and body condition score) at 24 d before expected parturition. Treatments consisted of a low-K ration without anion supplementation [CON; n = 30, DCAD = +18.3 mEq/100 g of dry matter (DM)]; partial anion supplementation to a low-K ration (MED; n = 30, DCAD = +5.9 mEq/100 g of DM); and anion supplementation to a low-K ration to reach a targeted average urine pH between 5.5 and 6.0 (LOW; n = 29, DCAD = -7.4 mEq/100 g of DM). Cows were fed a common postpartum diet and data collected through 63 d in milk. Urine pH (CON = 8.22, MED = 7.89, and LOW = 5.96) was affected quadratically by decreasing prepartum DCAD. A linear relationship between urine pH and urine Ca:creatinine ratio was observed (r = -0.81). Plasma Ca concentrations in the postpartum period (d 0 to 14; CON = 2.16, MED = 2.19, and LOW = 2.27 mmol/L) were increased linearly with decreasing prepartum DCAD. A treatment by parity (second vs. third and greater) interaction for postpartum plasma Ca concentration suggested that older cows had the greatest response to the low DCAD diet and older cows fed LOW had decreased prevalence of hypocalcemia after calving. A quadratic effect of decreasing DCAD on prepartum DMI was observed (CON = 13.6, MED = 14.0, and LOW = 13.2 kg/d). Milk production in the first 3 wk postpartum was increased linearly with decreasing DCAD (CON = 40.8, MED = 42.4, and LOW = 43.9 kg/d) and DMI in this period also tended to linearly increase (CON = 20.2, MED = 20.9, and LOW = 21.3 kg/d). Overall, effects on intake and milk yield analyzed over wk 1 to 9 postpartum were not significant. This study demonstrates that feeding lower DCAD diets prepartum improves plasma Ca status in the immediate postpartum period and results in increased DMI and milk production in the 3 wk after parturition. Compared with no anion supplementation or lower levels of anion supplementation, greater improvements were observed with the lower DCAD feeding strategy, in which an average urine pH of 5.5 to 6.0 was targeted.

Effect of nutritional immunomodulation and heat stress during the dry period on subsequent performance of cows.

Heat stress in dairy cows during the dry period impairs milk yield in the next lactation. Feeding OmniGen-AF (OG; Phibro Animal Health Corp., Teaneck, NJ) to lactating cows during heat stress may increase dry matter intake (DMI) and lowers respiration rate (RR) and rectal temperature (RT), but the effects in dry cows are not known. We hypothesized that OG supplementation before, during, and after the dry period (approximately 160 d total) would overcome the effects of heat stress and improve cow performance in the next lactation. Cows were randomly assigned to OG or control (placebo) treatments for the last 60 d in milk (DIM), based on mature-equivalent milk yield in the previous lactation. Cows were dried off 45 d before expected calving and randomly assigned to heat stress (HT) or cooling (CL) treatments. Thus, cows received dietary supplementation during late lactation before they were exposed to either CL or HT. After dry-off, treatment groups included heat stress with placebo (HT, only shade, 56 g/d of placebo, n = 17), HT with OG supplementation (HTOG, 56 g/d of OG, n = 19), cooling with placebo (CL, shade, fans, and soakers, 56 g/d of placebo, n = 16), and CL with OG supplementation (CLOG, 56 g/d of OG, n = 11). After parturition, all cows were kept under the same CL system and management, and all cows continued to receive OG or control treatment until 60 DIM. Cooling cows during the dry period reduced afternoon RT (CL vs. HT; 38.9 ± 0.05 vs. 39.3 ± 0.05°C) and RR (CL vs. HT; 45 ± 1.6 vs. 77 ± 1.6 breaths/min). Respiration rate was also decreased by OG supplementation under HT conditions (HTOG vs. HT; 69.7 ± 1.6 vs. 77.2 ± 1.6 breaths/min). An interaction was observed between OG supplementation and HT; HTOG cows tended to have lower morning RT compared with HT cows. During the dry period, OG reduced DMI relative to control cows. Birth weight was greater in calves from CL cows (CL vs. HT; 40.6 ± 1.09 vs. 38.7 ± 1.09 kg). No differences were detected among treatments in hematocrit, total protein, and body condition score. Cows offered CLOG, CL, and HTOG treatments had greater body weight during the dry period (794.9 ± 17.9, 746.8 ± 16.7, and 762.9 ± 14.9 kg, respectively) than HT cows (720 ± 16.2 kg). Gestation length was approximately 4 d longer for CL cows compared with HT cows. Cows offered CLOG, CL, and HTOG treatments produced more milk (41.3 ± 1.6, 40.7 ± 1.6, and 40.5 ± 1.6 kg/d, respectively) than HT treatment (35.9 ± 1.6 kg/d). Body weight after parturition and DMI were evaluated up to 60 DIM and averaged 661.5 ± 15.8 and 19.4 ± 0.7 kg/d, respectively, with no differences observed among treatments. These results confirm that exposure of dry cows to heat stress negatively affects milk yield in the subsequent lactation. Active cooling of dry cows and OG supplementation can reduce the negative effects of heat stress in the dry period on subsequent performance.

Effect of feeding a negative dietary cation-anion difference diet for an extended time prepartum on postpartum serum and urine metabolites and performance.

Forty-five multiparous Holstein cows and 15 springing Holstein heifers were used in a randomized block design trial to determine the effect of length of feeding a negative dietary anion-cation difference (DCAD) diet prepartum on serum and urine metabolites, dry matter (DM) intake, and milk yield and composition. After training to eat through Calan doors (American Calan Inc., Northwood, NH), cows within parity were assigned randomly to 1 of 3 treatments and fed a negative-DCAD diet for 3 (3 W), 4 (4 W), or 6 wk (6 W) before predicted calving. Actual days cows were fed negative-DCAD diets was 19.2 ± 4.1, 27.9 ± 3.1, and 41.5 ± 4.1d for 3 W, 4 W, and 6 W, respectively. Before the trial, all cows were fed a high-forage, low-energy diet. During the trial, cows were fed a diet formulated for late gestation (14.6% CP, 42.3% NDF, 20.5% starch, 7.1% ash, and 0.97% Ca) supplemented with Animate (Prince Agri Products Inc., Quincy, IL), with a resulting DCAD (Na + K - Cl - S) of -21.02 mEq/100g of DM. After calving, cows were fed a diet formulated for early lactation (18.0% CP, 36.4% NDF, 24.2% starch, 8.1% ash, and 0.94% Ca) for the following 6 wk with a DCAD of 20.55 mEq/100g of DM. Urine pH was not different among treatments before calving and averaged 6.36. No differences were observed in prepartum DM intake, which averaged 11.4, 11.5, and 11.7 kg/d for 3 W, 4 W, and 6 W, respectively. Prepartum serum total protein, albumin, and Ca concentrations, and anion gap were within normal limits but decreased linearly with increasing time cows were fed a negative-DCAD diet. No differences were observed in serum metabolite concentrations on the day of calving. Postpartum, serum total protein and globulin concentrations increased linearly with increasing length of time the negative-DCAD diet was fed. No differences were observed in postpartum DM intake, milk yield, or concentration of fat or protein among treatments: 19.1 kg/d, 40.6 kg/d, 4.30%, and 2.80%; 19.6 kg/d, 41.5 kg/d, 4.50%, and 2.90%; and 18.6 kg/d, 41.0 kg/d, 4.30%, and 2.73% for 3 W, 4 W, and 6 W, respectively. Results of this trial indicate that no differences existed in health or milk production or components in cows fed a negative-DCAD diet for up to 6 wk prepartum compared with those fed a negative-DCAD diet for 3 or 4 wk prepartum.

Effects of OmniGen AF feed supplementation on glucocorticoids, blood leukocyte messenger RNA abundance, and energy metabolism in overstocked Holstein dairy cows.

The physiological consequences of overstocking require more investigation, and no research has explored whether dietary supplements could mitigate the anticipated negative physiological effects. OmniGen AF (OG, Phibro Animal Health Corporation, Teaneck, NJ, USA) is a nutritional supplement that has been shown to support the immune system of cattle following internal and environmental stressors. This study aimed to determine if a 45-day period of OG feed supplementation would influence whole blood leukocyte messenger RNA abundance, energy metabolism and glucocorticoid concentration, during a two-week period of overstocking. Two stocking density treatments (control: one headlock and lying stall per cow; overstocked: 0.5 headlocks and 0.5 lying stalls per cow) and two diet treatments (control: no added supplement; and OG: 56 g/cow per day) were investigated. Four pens of 15 cows were fed their assigned diet (two pens per diet; control stocking density) for 45 days after which each stocking density treatment was applied for a 14-day period using a cross-over design; this study design was replicated twice. During each 14-day period, blood was collected on day four to measure whole blood leukocyte messenger RNA abundance (cluster of differentiation 80, interleukin 8 receptor-beta, interleukin 10 receptor-beta and L-selectin) and fecal samples were collected every two days to measure fecal cortisol metabolite concentration (11,17-dioxoandrostanes). At the end of each 14-day period, eight cows from each pen were selected for an intravenous glucose tolerance test; glucose, insulin and non-esterified fatty acids were measured. There were no effects of diet or stocking density on leukocyte messenger RNA abundance. Fecal cortisol metabolite concentrations were highest for overstocked cows on the control diet on day four of the stocking density treatment; however, by day 10, overstocked cows fed OG had the highest fecal cortisol metabolite concentrations. Overstocked cows, regardless of diet, had an attenuated insulin response during the glucose tolerance test, represented by a lower area under the curve estimate. Cows fed OG but not overstocked, had a lower non-esterified fatty acid nadir during the glucose challenge, compared to all the other treatments. In conclusion, overstocking prompts a physiological stress response and alters energy metabolism by decreasing the insulin response to an intravenous glucose challenge. Feeding OG during overstocking delayed the increase in fecal cortisol metabolites by several days; however, it is unclear if this altered glucocorticoid response benefited the cow, as OG had no effect on insulin responses or immune parameters.

Search for muon-neutrino to electron-neutrino transitions in MINOS.

This Letter reports on a search for nu(mu) --> nu(e) transitions by the MINOS experiment based on a 3.14x10(20) protons-on-target exposure in the Fermilab NuMI beam. We observe 35 events in the Far Detector with a background of 27+/-5(stat)+/-2(syst) events predicted by the measurements in the Near Detector. If interpreted in terms of nu(mu) --> nu(e) oscillations, this 1.5sigma excess of events is consistent with sin2(2theta(13)) comparable to the CHOOZ limit when |Delta m2|=2.43x10(-3) eV2 and sin2(2theta(23))=1.0 are assumed.

An immunomodulatory feed additive enhances in vitro viral vaccine recall antigen responses in dairy heifers.

Enhancing immunological responses to vaccination is an important goal in many herd health management systems. OmniGen-AF®(OG) is an immunomodulatory feed additive that has been shown to enhance innate immune function in ruminants and its effects on adaptive immunity require additional study. The objective of this study was to evaluate post-vaccine antibody titers and circulating cellular memory development in heifers fed OG and administered a commercially available modified-live bovine respiratory disease (BRD) vaccine. Twenty-four Holstein heifers were assigned to one of two diets for 170 days: Control TMR (CON; n = 11), or TMR plus OG (TRT; 9 g/100 kg BW/day; n = 13). Samples for hematology, serology, and cellular assays were collected on D-110, 0, 21, 42, and 60 of the trial. Heifers were administered two priming doses of a modified-live BRD vaccine, with a third dose given on D0. There were no significant differences in total WBC and absolute number or the percentage of circulating lymphocytes, monocytes, neutrophils, RBC, or platelets on D-110 through D21. On D42 and D60, CON had significantly higher numbers of lymphocytes. On D0, mean serum neutralizing (SN) titer to BHV-1 was significantly higher for CON compared to TRT. SN titers were not significantly different between CON and TRT at any other time point for BHV-1, BVDV type 1, or BVDV type 2. TRT mounted a significantly stronger recall proliferative response to 0.5 multiplicity of infection (MOI) of BHV-1, BVDV type 1 and BVDV type 2 on D42 and D60; 0.25 MOI of BVDV type 1 on D21 and D42; and 0.25 MOI BVDV type 2 on D42 compared to CON. IL-4 production induced by 0.5 and 1.0 MOI BHV-1 (D42 and D60); 0.25 MOI of BVDV type 1 (D21); and 0.25 and 0.5 MOI of BVDV type 2 (D60) were significantly higher for TRT than CON. IL-17 production induced by 0.25 MOI of BVDV type 1 was significantly higher on D60 for TRT compared to CON. IFN-gamma and IL-10 were not significantly different between treatments. These data indicate feeding OG has a beneficial effect on responses to vaccine antigens in Holstein dairy heifers.

Effects of an immunomodulatory feed additive on intramammary infection prevalence and somatic cell counts in a dairy herd experiencing major health issues.

A trial was conducted to determine if feeding OmniGen-AF® (OG) to 22 late lactation cows 60 days prior to and during the early dry period, a time of increased susceptibility to mastitis, could reduce disease incidence in a dairy herd experiencing major health issues. Treated cows (n = 11) consumed a ration containing OG [9 g/100 kg of body weight/day] beginning 60 days before dry-off, during the dry period, and through 30 days in milk (DIM). Control cows received the same ration during the dry period through 30 DIM only. Body weights, body condition scores (BCS), intramammary infection (IMI) prevalence, new IMI rates, somatic cell counts (SCC), milk yield, and adverse health events were measured. No differences were found between treatments for body weight or BCS. Adverse health event data at calving showed no differences between treatments except for percentage of cows with hyperketonemia, which was lower among treated cows (63.6% vs 100%). Prevalence of IMI from calving through 30 DIM for treated cows (6.1%) was lower than controls (11.05%); likewise, new IMI rate during this time for treated cows (0.61%) was lower than controls (5.81%). The SCC from calving through 30 DIM for treated cows (215,000/ml) was lower than controls (493,000/ml). Average production/day at the first DHIA test (~33 DIM) showed that treated cows produced more milk (39.9 kg) than controls (35.34 kg). In conclusion, feeding OG 60 days prior to dry-off reduced hyperketonemia and mastitis, lowered SCC, and numerically increased milk yield in a dairy herd experiencing major health issues.

A comparison of susceptibility to photodynamic treatment between endothelial and tumor cells in vitro and in vivo.

BACKGROUND: Photodynamic therapy (PDT) is being widely used for treatment of cancer and non-malignant diseases. The mechanisms of phototoxicity to solid tumor are not yet completely understood. Knowledge of the inherent sensitivity of endothelial cells in comparison to tumor cells would be helpful to predict tumor response to PDT, and thereby optimize treatment protocols. METHODS AND RESULTS: The intrinsic sensitivity of rodent endothelial and tumor cells to PDT was studied using an in vitro clonogenicity assay that strictly controlled for light and photosensitizer exposure, as well as cellular photosensitizer and oxygen concentration. Taking into consideration cell size, ploidy and glutathione content, no significant difference in sensitivity to phototoxicity was observed between tumor and endothelial cells. Electron microscopy studies were also conducted to examine endothelial and tumor cells for differential cellular damage following interstitial PDT of rat prostate tumor. No evidence for selective damage to endothelial cells was demonstrated. CONCLUSIONS: Rodent tumor cells and endothelial cells are equally susceptible to Photofrin-mediated PDT damage. Sufficient photosensitizer accumulated in solid tumor seems to be one of the key factors for PDT effectiveness.

Nuclear magnetic resonance spectroscopy and sensitizer-adduct measurements of photodynamic therapy-induced ischemia in solid tumors.

Dunning R3327-AT prostate carcinomas growing in Fischer X Copenhagen rats were treated with interstitial photodynamic therapy (PDT--15 mg/kg Photofrin II 4 hours before illumination with 630-nm light via four parallelly implanted optical fibers) at different light intensities. Forty to 60 minutes after treatment, 31P-nuclear magnetic resonance spectra of tumors in anesthetized animals were obtained at 2.35 Tesla using surface coil localization. Areas under resonance peaks were normalized to the area under the peak of a phosphorus standard positioned at a fixed distance on the opposite side of the surface coil. Tumor concentrations of phosphomonoesters and phosphodiesters showed no change after tumor light doses up to 3000 J. Phosphocreatine, alpha-adenosine triphosphate (ATP), beta-ATP, and gamma-ATP signals decreased and inorganic phosphate signals increased with increasing light doses. The intratumor pH did not change significantly at these short times after PDT. In other R3327-AT and R3327-H tumor-bearing animals, [3H]misonidazole was administered 30 minutes prior to PDT treatments of both tumors. Twenty-four hours later, the tumors were resected in toto, and levels of retained [3H]misonidazole were determined in lased tumor specimens by liquid scintillation procedures. The amount of [3H]misonidazole activity in tumor tissue (covalently bound after hypoxic reduction) increased with light doses up to 3000 J. Sensitizer-adduct formation was found to correlate with the ratio of the concentration of inorganic phosphate to that of beta-ATP, both of which are presumed measures of tumor oxygenation status. These measurements have high-lighted the heterogenous nature of the oxygenation status of these experimental tumors. The precision of each assay for estimating tumor oxygenation is discussed.

Characteristics of the metabolism-induced binding of misonidazole to hypoxic mammalian cells.

[14C]Misonidazole (MISO) becomes bound to macromolecules of mammalian cells upon hypoxic incubation. Intracellular enzyme processes are implicated since the temperature dependence for this process showed an activation energy of 33.5 kcal/mol. The sensitizer bound to both hypoxic and aerobic cells was associated with the macromolecular fraction and the soluble fraction in the proportion, 23 and 77%, respectively. The initial rate of binding of [14C]MISO to the macromolecular (acid-insoluble) fraction of hypoxic EMT-6 mouse tumor and V-79 hamster cells increased proportionally with the square root of extracellular concentration of MISO up to at least 5mM. High concentrations of dimethyl sulfoxide (an effective OH radical scavenger), allopurinol (an effective inhibitor of xanthine oxidase), and diamide (a chemical which can deplete cellular levels of glutathione) had little or no effect on this metabolism-induced binding process. The addition of high concentrations of exogenous cysteamine to hypoxic cell cultures resulted in almost complete inhibition of binding. Extracellular bovine albumin at high concentration in hypoxic cell cultures had little effect on the production of adducts to cell macromolecules and only small amounts of [14C]MISO were found to bind to the extra-cellular bovine albumin. This result suggests that MISO preferentially binds to molecules within the cell in which it is metabolically activated. In experiments where cells labeled under hypoxic conditions with [14C]MISO were subsequently permitted to proliferate in aerobic monolayers, a half-life of the acid-insoluble addition products of approximately 55 hr was measured. A large number of [14C]MISO adducts (approximately 10(9)/cell) can be generated in hypoxic cells without any evidence of cytotoxicity, and they are slowly cleared from cells. These are favorable characteristics as regards the development of this technique as a marker for hypoxic cells in solid tumors.

Treatment of Dunning R3327-AT rat prostate tumors with photodynamic therapy in combination with misonidazole.

Fischer X Copenhagen rats bearing Dunning R3327-AT tumors were treated with hematoporphyrin derivative and red light (630 nm from an argon-driven dye laser) alone or in combination with the hypoxic cell radiosensitizer, misonidazole (MISO). In vitro studies had suggested that hypoxia might significantly decrease the cytotoxicity of photodynamic therapy (PDT) and labeling with [14C]MISO had revealed a significant fraction of viable hypoxic cells in this tumor. PDT alone resulted in a growth delay of 8.8 days but no tumor cures were observed. The administration of MISO (i.p. at 0.5 mg/g) 33 min prior to PDT resulted in an average growth delay of 15.2 days and tumor cures (local control at 33 days) in 20% of animals treated. If MISO at a similar dosage was administered 30 min after PDT an average growth delay of 16.3 days was measured and tumor cure was observed in 70% of the animals treated. These results suggest that the hypoxic cell fraction in R3327-AT tumors might be a limitation to their curability by PDT. The addition of MISO and/or other hypoxic cell cytotoxic agents to PDT procedures may provide an effective means of treating PDT-resistant hypoxic cells in solid tumors.

Tolyporphin: a natural product from cyanobacteria with potent photosensitizing activity against tumor cells in vitro and in vivo.

Tolyporphin (TP), a porphyrin extracted from cyanobacteria, was found to be a very potent photosensitizer of EMT-6 tumor cells grown both in vitro as suspensions or monolayers and in vivo in tumors implanted on the backs of C.B17/Icr severe combined immunodeficient mice. Thus, during photodynamic treatment (PDT) of EMT-6 tumor cells in vitro, the photokilling effectiveness of TP measured as the product of the reciprocal of D50 (the light dose necessary to kill 50% of cells) and the concentration of TP is approximately 5000 times higher than that of Photofrin II (PII), the only PDT photosensitizer thus far approved for clinical trials. TP almost exclusively localizes in the perinuclear region and specifically in the endoplasmic reticulum (ER), as shown by microspectrofluorometry on single living EMT-6 cells costained with the ER and/or Golgi fluorescent vital probes, 3,3'-dihexyloxacarbocyanine iodide and N-[4,4-difluoro-(5,7-dimethyl-BODIPY)-1-pentanoyl]-D-erythro-sphin gosine (Molecular Probes, Eugene, OR). As a result, the singlet oxygen-mediated photodynamic activity of TP induces an effective inactivation of the acyl CoA:cholesterol-O-acyltransferase, a sensitive marker of ER membrane integrity and alterations of the nuclear membrane. In vivo, with the EMT-6 mouse tumor model, an exceptional effectiveness is also observed as compared to that of PII and other second generation photosensitizers of the pheophorbide class, which are themselves much more potent than PII. The outstanding PDT activity of TP observed in vivo may be due to its unique biodistribution properties, in particular much less extraction by the liver, resulting in a higher delivery to other tissues, including tumor.

Single-photon emission computed tomography and positron-emission tomography assays for tissue oxygenation.

Radiotherapy prescription can now be customized to target the major mechanism(s) of resistance of individual tumors. In that regard, functional imaging techniques should be exploited to identify the dominant mechanism(s). Tumor biology research has identified several mechanisms of tumor resistance that may be unique to radiation treatments. These fall into 3 broad areas associated with (1) tumor hypoxic fraction, (2) tumor growth rate, (3) and the intrinsic radiosensitivity of tumor clonogens. Imaging research has markers in various stages of development for quantifying relevant information about each of these mechanisms, and those that measure tumor oxygenation and predict for radioresistance are the most advanced. Positron-emission tomography (PET) measurement of oxygen 15 has yielded important information, particularly about brain tissue perfusion, metabolism, and function. Indirect markers of tumor hypoxia have exploited the covalent binding of bioreductive intermediates of azomycin-containing compounds whose uptakes are inversely proportional to intracellular oxygen concentrations. Pilot clinical studies with single-photon emission computed tomography (SPECT) and PET detection of radiolabeled markers to tumor hypoxia have been reported. Recently, other studies have attempted to exploit the reduction properties of both technetium and copper chelates for the selective deposition of radioactive metals in hypoxic tissues. A growing number of potentially useful isotopes are now available for labeling several novel chemicals that could have the appropriate specificity and sensitivity. Preclinical studies with "microSPECT" and "microPET" will be important to define the optimal radiodiagnostic(s) for measuring tissue oxygenation and for determining the time after their administration for optimal hypoxic signal acquisition. Radiolabeled markers of growth kinetics and intrinsic radiosensitivity of cells in solid tumors are also being developed. We conclude that radiation oncology is uniquely positioned to benefit from functional imaging markers that identify important mechanisms of tumor radioresistance, since several strategies for overcoming these individual mechanisms have already been identified.