Blood metabolomics and impacted cellular mechanisms during transition into lactation in dairy cows that develop metritis.

The objective of this study was to identify metabolites associated with metritis and use them for identification of cellular mechanisms affected during transition into lactation. Holstein cows (n = 104) had blood collected in the prepartum period (d -14 ± 6 relative to calving), at calving (d 0), and at the day of metritis diagnosis (d 7 ± 2 after calving). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 52). Cows with metritis were paired with herdmates without metritis (n = 52) based on days in milk. The metabolome of plasma samples was evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Univariate analyses included t-tests and fold change analyses. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were used for partial least squares discriminant analysis coupled with permutational analysis using 2,000 permutations. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were also used for enriched pathway analyses and identification of cellular processes. Cows that developed metritis had affected cellular processes associated with lower amino acid metabolism in the prepartum period, greater lipolysis, cell death, and oxidative stress at calving and at metritis diagnosis, and greater leukocyte activation at calving, but lower immune cell activation at metritis diagnosis. In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.

Unraveling the immune and metabolic changes associated with metritis in dairy cows.

The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at -14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at -14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1ß. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

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 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.

Effect of adding clay with or without a Saccharomyces cerevisiae fermentation product on the health and performance of lactating dairy cows challenged with dietary aflatoxin B1.

The study was conducted to examine the effect of supplementing bentonite clay with or without a Saccharomyces cerevisiae fermentation product (SCFP; 19 g of NutriTek + 16 g of MetaShield, both from Diamond V, Cedar Rapids, IA) on the performance and health of dairy cows challenged with aflatoxin B1 (AFB1). Twenty-four lactating Holstein cows (64 ± 11 d in milk) were stratified by parity and milk production and randomly assigned to 1 of 4 treatment sequences. The experiment had a balanced 4 × 4 Latin square design with 6 replicate squares, four 33-d periods, and a 5-d washout interval between periods. Cows were fed a total mixed ration containing 36.1% corn silage, 8.3% alfalfa hay, and 55.6% concentrate (dry matter basis). Treatments were (1) control (no additives), (2) toxin (T; 1,725 µg of AFB1/head per day), (3) T + clay (CL; 200 g/head per day; top-dressed), and (4) CL+SCFP (CL+SCFP; 35 g/head per day; top-dressed). Cows were adapted to diets from d 1 to 25 (predosing period) and then orally dosed with AFB1 from d 26 to 30 (dosing period), and AFB1 was withdrawn from d 31 to 33 (withdrawal period). Milk samples were collected twice daily from d 21 to 33, and plasma was sampled on d 25 and 30 before the morning feeding. Transfer of ingested AFB1 into milk aflatoxin M1 (AFM1) was greater in T than in CL or CL+SCFP (1.65 vs. 1.01 and 0.94%, respectively) from d 26 to 30. The CL and CL+SCFP treatments reduced milk AFM1 concentration compared with T (0.45 and 0.40 vs. 0.75 µg/kg, respectively), and, unlike T, both CL and CL+SCFP lowered AFM1 concentrations below the US Food and Drug Administration action level (0.5 µg/kg). Milk yield tended to be greater during the dosing period in cows fed CL+SCFP compared with T (39.7 vs. 37.7 kg/d). Compared with that for T, plasma glutamic oxaloacetic transaminase concentration, indicative of aflatoxicosis and liver damage, was reduced by CL (85.9 vs. 95.2 U/L) and numerically reduced by CL+SCFP (87.9 vs. 95.2 U/L). Dietary CL and CL+SCFP reduced transfer of dietary AFB1 to milk and milk AFM1 concentration. Only CL prevented the increase in glutamic oxaloacetic transaminase concentration, and only CL+SCFP prevented the decrease in milk yield caused by AFB1 ingestion.

Effect of recombinant bovine somatotropin on leukocyte mRNA expression for genes related to cell energy metabolism, cytokine production, phagocytosis, oxidative burst, and adaptive immunity.

Objectives of the current experiment were to evaluate the effects of treatment of periparturient dairy cows with recombinant bovine somatotropin (rbST) on mRNA expression in peripheral leukocytes for genes related to the somatotropic axis, cell energy metabolism, and innate and adaptive immune responses. Cows were enrolled in the experiment at 253 ± 3 d of gestation and assigned to an untreated control group (n = 98) or to receive 125 mg of rbST weekly from -21 to 21 d relative to calving (rbST125; n = 98). Data from a subsample of cows (control = 16, rbST125 = 16) are reported herein. Hemogram and polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of adhesion molecules were determined weekly, from -28 ± 3 to 24 ± 3 d relative to calving. Cows were vaccinated with ovalbumin at -21 ± 3, -7 ± 3, and 7 ± 3 d relative to calving. Serum IgG anti-ovalbumin and haptoglobin optical densities were determined weekly, from -28 ± 3 to 24 ± 3 d relative to calving. Leukocytes were isolated from whole blood on d -21 ± 3, -7 ± 3, and 7 ± 3 relative to calving to determine leukocyte mRNA expression for 66 genes. Cows in the rbST125 treatment had greater concentration of granulocytes 1 wk prepartum (control = 3.7 ± 0.4 vs. rbST125 = 4.6 ± 0.4 × 109 cells/L). Expression of CD18 by PMNL during the prepartum (control = 3,262 ± 280 vs. rbST125 = 3,926 ± 260 geometric mean fluorescence intensity) and percentage of PMNL positive for phagocytosis and oxidative burst 1 wk postpartum (control = 59.2 ± 2.8 vs. rbST125 = 67.6 ± 3.1%) were increased in rbST125 cows. Postpartum IgG anti-ovalbumin optical density was higher in rbST125 cows than control cows (control = 1.5 ± 0.1 vs. rbST125 = 1.9 ± 0.1 optical density). On d -7 relative to calving, leukocyte mRNA expression of IGF1R and JAK1 tended to be downregulated and expression of DEFB3 tended to be upregulated by rbST treatment. Expression of mRNA for STAT1, RELA, and NOD2 tended to be upregulated, expression of RAC2 was downregulated, and expression of JAK3, BLK, and TNFα tended to be downregulated on d 7 relative to calving by rbST treatment. Effects of treatment of periparturient cows with 125 mg of rbST on leukocyte gene expression were minute; thus, additional experiments are necessary to elucidate how rbST-induced increases in growth hormone and insulin-like growth factor-1 concentrations may modulate the immune response of periparturient cows.

Effects of 3 sequestering agents on milk aflatoxin M1 concentration and the performance and immune status of dairy cows fed diets artificially contaminated with aflatoxin B1.

This study examined whether adding 3 mycotoxin-sequestering agents to diets contaminated with aflatoxin B1 (AFB1) would reduce milk aflatoxin M1 (AFM1) concentration, and improve the performance and alter immune status of dairy cows. Fifteen lactating dairy cows were used in an experiment with an incomplete crossover design including four 28-d periods. Treatments included a control diet (C), a toxin diet (T; 1,725µg of AFB1/head per day; 75µg/kg), and diets containing the toxin and 20g/head per day of a proprietary mixture of Saccharomyces cerevisiae fermentation product containing a low (SEQ1) or high (SEQ2) dose of a chlorophyll-based additive, or a low dose of the chlorophyll-based additive and sodium bentonite clay (SEQ3). Sequestering agents were top-dressed on the total mixed ration (TMR) daily in each period, and AFB1 was dosed orally in gelatin capsules before the TMR was fed on d 21 to 25. Milk was sampled twice daily on d 20 to 28 and plasma was sampled on d 20 and 25. Sequestering agents did not affect milk AFM1 concentration during the toxin-dosing period. However, after AFB1 was withdrawn, the sequestering agents reduced the time required (24 vs. 48h) to reduce the milk AFM1 concentration below the Food and Drug Administration action level of 0.5µg/kg. Feeding T instead of C tended to reduce milk and fat-corrected milk yields, but feeding SEQ1 prevented these effects. Red blood cell count and hemoglobin concentration were reduced by feeding T instead of C, but not by feeding SEQ1, SEQ2, or SEQ3. The mean fluorescence intensity of antibody staining for 2 leukocyte adhesion molecules, L-selectin (CD62L) and ß-integrin (CD18), tended to be greatest when SEQ1 and SEQ3 were fed. Plasma acid-soluble protein concentration was decreased by feeding SEQ1, SEQ2, and SEQ3 instead of T. Sequestering agents had no effect on milk AFM1 concentration, but they reduced the time required to reduce milk AFM1 concentration to a safe level after withdrawal of AFB1 from the diet. Only SEQ1 prevented the adverse effects of AFB1 on milk and fat-corrected milk yields.

Effect of induced subclinical hypocalcemia on physiological responses and neutrophil function in dairy cows.

The objectives were to study the effects of induced subclinical hypocalcemia [SCH, blood ionized Ca (iCa(2+)) <1.0mM, without recumbency] on physiological responses and function of immune cells in dairy cows. Ten nonpregnant, nonlactating Holstein cows were blocked by lactation and assigned randomly to a normocalcemic (NC; intravenous infusion of 0.9% NaCl i.v. plus 43 g of oral Ca, as Ca sulfate and Ca chloride, at -1 and 11h) or an induced SCH [SCHI, 5% ethylene glycol tetraacetic acid (EGTA), a selective iCa(2+) chelator, intravenous infusion] treatment for 24h, using a crossover design. The sequence of treatments was either NC-SCHI or SCHI-NC, with a 6-d washout period. Ionized Ca was evaluated before, hourly during the infusion period, and at 48 and 72 h, to monitor concentrations and adjust the rate of infusion, maintaining blood iCa(2+) <1.0mM in SCHI throughout the 24-h infusion period. Additional measurements included heart and respiratory rates, rectal temperature, dry matter intake, rumen contractions, whole-blood pH, concentrations of glucose and K in whole blood, concentrations of total Ca, Mg, nonesterified fatty acids, ß-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic iCa(2+) in neutrophils and lymphocytes was quantified and neutrophil function was assayed in vitro. Infusion of a 5% EGTA solution successfully induced SCH in all SCHI cows, resulting in decreased blood iCa(2+) concentrations throughout the 24-h treatment period (0.77 ± 0.01 vs. 1.26 ± 0.01 mM iCa(2+)). Induction of SCH reduced dry matter intake on the day of infusion (5.3 ± 0.8 vs. 9.1 ± 0.8 kg/d) and rumen contractions (1.9 ± 0.2 vs. 2.7 ± 0.2 contractions/2 min) for the last 12h of infusion. Cows in SCHI had decreased plasma insulin concentration (1.44 ± 0.23 vs. 2.32 ± 0.23 ng/mL) evident between 6 and 18 h after the beginning of the infusion, accompanied by increased concentrations of glucose (4.40 ± 0.04 vs. 4.17 ± 0.04 mM). Plasma nonesterified fatty acids concentration was greater for SCHI than NC cows (0.110 ± 0.019 vs. 0.061 ± 0.014 mM). Neutrophils of cows in SCHI had a faster decrease in cytosolic iCa(2+) after stimulation with ionomycin (9.9 ± 1.0 vs. 13.6 ± 1.4 Fluo-4:Fura Red post-end ratio) in vitro. Furthermore, induction of SCH reduced the percentage of neutrophils undergoing phagocytosis (22.1 ± 2.1 vs. 29.3 ± 2.1%) and reduced the oxidative burst response after incubation of pathogenic bacteria (16.1 ± 1.7 vs. 24.2 ± 1.7%). Subclinical hypocalcemia compromised appetite, altered metabolism, and impaired function of immune cells in dairy cows.

Integrating uterine microbiome and metabolome to advance the understanding of the uterine environment in dairy cows with metritis.

BACKGROUND: Metritis is a prevalent uterine disease that affects the welfare, fertility, and survival of dairy cows. The uterine microbiome from cows that develop metritis and those that remain healthy do not differ from calving until 2 days postpartum, after which there is a dysbiosis of the uterine microbiome characterized by a shift towards opportunistic pathogens such as Fusobacteriota and Bacteroidota. Whether these opportunistic pathogens proliferate and overtake the uterine commensals could be determined by the type of substrates present in the uterus. The objective of this study was to integrate uterine microbiome and metabolome data to advance the understanding of the uterine environment in dairy cows that develop metritis. Holstein cows (n = 104) had uterine fluid collected at calving and at the day of metritis diagnosis. Cows with metritis (n = 52) were paired with cows without metritis (n = 52) based on days after calving. First, the uterine microbiome and metabolome were evaluated individually, and then integrated using network analyses. RESULTS: The uterine microbiome did not differ at calving but differed on the day of metritis diagnosis between cows with and without metritis. The uterine metabolome differed both at calving and on the day of metritis diagnosis between cows that did and did not develop metritis. Omics integration was performed between 6 significant bacteria genera and 153 significant metabolites on the day of metritis diagnosis. Integration was not performed at calving because there were no significant differences in the uterine microbiome. A total of 3 bacteria genera (i.e. Fusobacterium, Porphyromonas, and Bacteroides) were strongly correlated with 49 metabolites on the day of metritis diagnosis. Seven of the significant metabolites at calving were among the 49 metabolites strongly correlated with opportunistic pathogenic bacteria on the day of metritis diagnosis. The main metabolites have been associated with attenuation of biofilm formation by commensal bacteria, opportunistic pathogenic bacteria overgrowth, tissue damage and inflammation, immune evasion, and immune dysregulation. CONCLUSIONS: The data integration presented herein helps advance the understanding of the uterine environment in dairy cows with metritis. The identified metabolites may provide a competitive advantage to the main uterine pathogens Fusobacterium, Porphyromonas and Bacteroides, and may be promising targets for future interventions aiming to reduce opportunistic pathogenic bacteria growth in the uterus.

Flow cytometry panels for immunophenotyping dairy cattle peripheral blood leukocytes.

Many aspects of the bovine immune system remain poorly characterized, which poses an obstacle to improving dairy cow health. Herein, we describe two flow cytometry panels that included antibodies against CD8α, CD4, TCR-δ, CD172α, CD14, MHCII, CD21, CD62L, and CD11b. These panels were used to characterize the phenotype of leukocyte subpopulations from the peripheral blood of 30-day old Holstein calves and Holstein cows at 260 d of gestation and calving. No leukocyte subset differences were found between the pre- and post-partum cows. However, calf leukocytes presented a higher proportion of CD3+ lymphocytes, γδ T-cells, CD8+ γδ T-cells, and monocytes when compared with mature cows. Conversely, cow lymphocytes had a higher proportion of CD4+ and CD8+ T-cells, and B-cells than calf lymphocytes. The proportion of CD4+ T-cells and B-cells expressing CD62L was greater in calves than in cows, while cow B-cells expressed greater levels of CD11b than calf B-cells. In contrast, calf polymorphonuclear cells (PMN) and monocytes expressed greater levels of CD11b compared to cows. Moreover, calf monocytes expressed higher levels of MHCII compared with those of cows. Collectively, our data provides a resource to better understand the bovine immune system as well as immune-related diseases that affect dairy cattle.

Increasing serotonin bioavailability alters gene expression in peripheral leukocytes and lymphoid tissues of dairy calves.

Dairy calves are born with a naïve immune system, making the pre-weaning phase a critical window for immune development. In the U.S., 40-60% of dairy farms feed milk replacer to pre-weaned calves, which are devoid of bioactive factors with immunological roles. Serotonin is a bioactive factor with immunoregulatory properties naturally produced by the calf and present in milk. Human and rodent immune cells express the serotonin machinery, but little is known about the role of serotonin in the bovine immune system. Supplementing milk replacer with 5-hydroxytryptophan (serotonin precursor) or fluoxetine (reuptake inhibitor) increases serotonin bioavailability. We hypothesized that increased serotonin bioavailability promotes serotonergic signaling and modulates the expression of immune related genes in peripheral leukocytes and immune-related tissues of dairy calves. The present experiment targeted candidate genes involved in serotonin production, metabolism, transport, signaling and immune regulation. We established that bovine peripheral leukocytes express all known serotonin receptors, and can synthesize, uptake and degrade serotonin due to the expression of serotonin metabolism-related genes. Indeed, we showed that increasing serotonin bioavailability alters gene expression of serotonin receptors and immune-related genes. Further research will determine whether manipulation of the serotonin pathway could be a feasible approach to bolster dairy calves' immune system.

Comparative therapeutic effects of orally administered 1,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(3) on type-1 diabetes in non-obese diabetic mice fed a normal-calcaemic diet.

Frequent injections of the hormonal form of vitamin D(3), 1,25 dihydroxyvitamin D(3) (1,25D3) reportedly inhibits autoimmune type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by correcting some of the abnormalities in antigen-presenting cells which contribute the development of pathogenic T cell responses. This route of administration greatly elevates the levels of these compounds in the bloodstream for hours after treatment, which requires mice to be fed diets formulated to contain much reduced levels of Ca to avoid the toxic effects of hypercalcaemia. In the current work, we demonstrate that feeding 1,25D3 or its synthetic precursor, 1alpha(OH) vitamin D(3) (1alphaD3), as part of a T1D supportive chow diet containing normal levels of Ca, is an effective means of reducing the incidence of disease in NOD mice, but the doses required for protection elicited hypercalcaemia. However, T1D protection elicited by D3 analogue feeding appears, at least partially, to have an immunological basis, as splenic T cells from treated mice had a decreased capacity to adoptively transfer disease. Protection is associated with an increased proportion of T cells with CD4+ forkhead box P3+ regulatory phenotype within the islet infiltrate of treated animals. The 1alphaD3 precursor is converted rapidly to the active 1,25D3 isoform in vivo. However, feeding the 1alphaD3 analogue elicited stronger T1D protection than the 1,25D3 compound, but also induced more severe hypercalcaemia. In future, the dietary supplementation of novel low-calcaemic D3 analogues may enable their continuous delivery at levels that inhibit T1D development in susceptible humans consuming normal levels of Ca.

The effect of feeding calcium- and phosphorus-deficient diets to broiler chickens during the starting and growing-finishing phases on carcass quality.

There is considerable data on the effect of reducing inorganic Ca and P in broiler finisher diets on carcass quality. However, there is limited information on the effect of reducing dietary Ca and P during the different phases of growout. Two experiments were conducted from 0 to 35 d in floor pens. In both experiments, at least 4 replicates per treatment (50 chicks per replicate) were used. Corn-soybean meal and soybean oil-based diets deficient in Ca and P were fed. During the starter phase (ST), from 0 to 18 d, chicks were fed a 23% CP diet containing 0.60% Ca and 0.47% total P (tP). During the grower-finisher phase (GF), from 19 to 35 d, birds were fed a 19% CP diet containing 0.30% Ca and 0.37% tP. A combination of 1,000 phytase units/kg of Natuphos phytase and 5 microg/kg of 1alpha-hydroxycholecalciferol (P + 1alpha) was supplemented to some of the feed during the ST and GF. Diets containing adequate Ca and P were also fed during the ST (0.90% Ca and 0.68% tP) and GF (0.80% Ca and 0.67% tP). The level of tibia ash and the incidence of bone disease were measured at 18 and 35 d. At the end of the experiments, birds were processed and evaluated for muscle hemorrhages and broken bones. In both experiments, broilers fed diets that were not P + 1alpha supplemented demonstrated poor bone mineralization, considerable leg problems, and a high incidence of broken bones after processing. Broilers fed P + 1alpha throughout had more broken clavicles and femurs compared with birds fed the adequate diets. Day-18 tibia ash was significantly correlated to broken tibias and femurs during processing. Day-35 tibia ash was better correlated to bloody breast meat than to broken bones. It is concluded that carcass quality depends on the levels of Ca and P fed and the age of the bird. Tibia ash, traditionally used as an indication of bone strength, was better correlated to the incidence of bloody breasts.

Improvements in nitrogen-corrected apparent metabolizable energy of peanut meal in response to phytase supplementation.

A study was conducted to investigate the effect of phytase on the AMEn of peanut meal. One hundred twenty Ross x Ross broiler chicks of mixed sex were fed one of 4 experimental diets from 5 to 15 d of age. Diets used were Diet 1, a low P corn-soybean based basal diet; Diet 2, a 50% basal + 50% peanut meal diet; Diet 3, the basal diet supplemented with 24,000 phytase units (FTU) of Natuphos 5000 phytase/kg; and Diet 4, a phytase-supplemented 50% basal + 50% peanut meal diet. Chromic oxide was added to the basal diet at 0.1% as an indigestible marker. Apparent metabolizable energy was determined by substituting peanut meal at the expense of the basal diet. Other parameters measured included the phytate content of the diets as well as phytate P disappearance. Phytase significantly improved phytate P disappearance for both the corn and soybean meal basal diet (23.8 to 93.7%) as well as the 50% basal + 50% peanut meal diet (16.7 to 89.5%). Phytase increased the AMEn of peanut meal on a DM basis by approximately 9%, from 3,209 to 3,559 kcal/kg.

The effect of maternal dietary vitamin D3 supplementation on performance and tibial dyschondroplasia of broiler chicks.

A series of experiments was conducted to investigate the effects of maternal dietary vitamin D3 supplementation at 4 different times during the laying cycle, on the performance and bone quality of broiler chicks fed a diet that induced tibial dyschondroplasia (TD) or an adequate diet. Ross x Ross broiler breeder hens were fed a corn-soy diet with various levels of vitamin D3 from 24 to 66 wk of age. Eggs were collected at 39, 44, 53, and 64 wk of age and hatched. Chicks from hens fed 250 IU of D3/kg (low maternal D3 or LMD3) and 2,000 IU of D3/ kg (high maternal D3 or HMD3) levels were placed in battery brooders and fed the diets from 0 to 16 d. At 16 d, the chicks were weighed and killed; the left tibias were used for bone ash determinations, and the right tibias were used to score the incidence and severity of TD (0, 1, 2, or 3, where 3 is the most severe). Body weight gain and feed intake were significantly lower for the LMD3 chicks at wk 44 and 64, although there was no difference in weight at hatch. For the first 2 hatches (wk 39 and 44), the LMD3 and HMD3 chicks demonstrated high average TD scores (2.03 and 1.57 vs. 2.05 and 1.75 for the LMD3 vs. HMD3 chicks, respectively) and high average incidences of severe TD (50 and 35% vs. 45 and 34% for LMD3 vs. HMD3 levels, respectively). However, results from the last 2 hatches (wk 53 and 64) showed that HMD3 chicks, compared with LMD3 chicks, had reduced average TD scores (1.39 and 1.47 vs. 1.01 and 0.44 for LMD3 vs. HMD3 levels, respectively) and severe TD incidence (36 and 40% vs. 17 and 8% for the LMD3 vs. HMD3 levels, respectively). In this experiment, as egg production declined toward the end of the laying cycle, hens fed the HMD3 might have been able to deposit sufficient quantities of vitamin D3 in the egg to maintain excellent body weight gain at 16 d of age and reduce the incidence and severity of TD. Hens fed the LMD3 diet were unable to produce similar improvements.

Calcium requirements of the modern broiler chicken as influenced by dietary protein and age.

Two experiments were conducted to examine the calcium requirements of broiler chickens fed corn-soybean meal diets. Experiment 1 used a 6 x 2 x 2 factorial arrangement and was conducted with broilers in floor pens during the grower phase (19 to 42 d). Diets were mixed with 6 levels of dietary Ca (0.325, 0.4, 0.475, 0.55, 0.625, and 0.9%) and 17 or 23% CP and fed to males and females separately. Experiment 2 was a 6 x 2 factorial design conducted using Petersime battery brooders during the starter phase (0 to 16 d). The same 6 levels of dietary Ca used in experiment 1 were fed separately to each sex, but only at the 23% level of CP. The diets used in both experiments were formulated to contain 0.45% nonphytin phosphorus. In experiment 1, grower chickens did not demonstrate significant body weight gain (BWG) or feed conversion ratio (FCR) response (g of feed per g of gain) to the different levels of Ca at either level of protein. The percentage tibia ash did not respond to increasing Ca levels beyond 0.625% Ca at either protein level. In experiment 2, BWG increased linearly up to 0.55 and 0.625% dietary Ca for males and females, respectively. Feed conversion ratio decreased linearly with increasing dietary Ca up to 0.625% Ca, and tibia ash was highest at 0.9% Ca for both sexes. These results suggest that the current NRC Ca requirements for the broiler starter (1.0%) are sufficient for maximum bone ash, but that Ca requirements for grower birds (0.9%) may be excessive for optimum BWG, FCR, and tibia ash.

Phytase and 1alpha-hydroxycholecalciferol supplementation of broiler chickens during the starting and growing/finishing phases.

Supplemental 1alpha-hydroxycholecalciferol (1alpha-OHD3) has been shown to have qualitatively similar and quantitatively additive effects to exogenous phytase. Two experiments were conducted from 0 to 35 d in floor pens to determine the additive effect of phytase and 1alpha-OHD3 when supplemented to Ca- and P-deficient diets. In both experiments, at least 4 replicates per treatment (50 chicks per replicate) were used. Corn-soybean-meal-and soybean-oil-based diets were fed and birds were raised in a house impervious to ultraviolet light. During the starter phase (ST), from 0 to 18 d, chicks were fed a 23% CP diet containing 0.60% Ca and 0.47% total P (tP). During the grower/finisher phase (GF), from 19 to 35 d, birds were fed a 19% CP diet containing 0.30% Ca and 0.37% tP. A combination of 1,000 phytase units/kg of Natuphos phytase and 5 microg/kg of 1alpha-OHD3 (P+1A) was supplemented to some of the feed during the ST and GF. Diets containing adequate Ca and P were also fed during the ST (0.90% Ca, 0.68% tP) and GF (0.80% Ca, 0.67% tP). Performance characteristics and the incidence of rickets and tibial dyschondroplasia were measured at 18 and 35 d. In experiment 1, unsupplemented chicks performed well but had considerable leg problems. Chicks fed P+1A during the ST or GF did not perform as well as birds fed P+1A throughout. Birds fed P+1A throughout performed as well birds fed the adequate diets without any indication of leg problems. In experiment 2, unsupplemented birds performed similarly to unsupplemented birds in experiment 1. However, chicks fed the supplements or the control diets did not perform as well or accumulate as much bone ash as birds in experiment 1, although the diets were formulated identically in both experiments. Diets with as little as 0.30% Ca and 0.37% tP appear to be adequate for broilers older than 18 d if supplemented with the correct amounts of phytase and 1alpha-OHD3. However, there are unknown variables that may limit the potential of broilers in terms of bone mineralization and bone pathology, even when adequate diets are fed.

Effects of calcium and nonphytate phosphorus concentrations on phytase efficacy in broiler chicks.

Phytase supplementation over a range of different levels of dietary Ca and nonphytate phosphorus (NPP) was investigated by comparing surface response curves from regression equations generated with (experiment 1) and without (experiment 2) phytase using various performance and bone quality parameters. Cobb x Cobb broiler chicks were raised from 0 to 16 d in 2 experiments using corn-soybean meal based diets. Experiment 1 used a 4 x 4 factorial arrangement with diets formulated to contain combinations of 4 levels of Ca: 0.38, 0.58, 0.78, and 0.98% and 4 levels of NPP: 0.2, 0.3, 0.4, and 0.5%. Experiment 2 used a composite rotatable design in which rations were formulated to contain dietary Ca levels of 0.38, 0.47, 0.68, 0.89, and 0.98% and NPP levels of 0.20, 0.24, 0.35, 0.46, and 0.50%. An extra point was included in the design to contain the lowest Ca and lowest NPP levels (0.38% Ca and 0.20% NPP). All combinations of Ca and NPP were fed with 657 phytase units/kg Natuphos 5000 phytase, plus 4 combinations (0.38% Ca and 0.20% NPP, 0.47% Ca and 0.24% NPP, 0.68% Ca and 0.35% NPP, and 0.89% Ca and 0.46% NPP) were fed without phytase to determine the suitability of comparing multiple regression response surfaces for particular variables among experiments. Comparison of surfaces, with and without phytase, showed that growth and bone quality responses to phytase were greatest at low NPP levels and high Ca levels, and these decreased when the Ca level was reduced or when the NPP level was increased. A third experiment confirmed that phytase elicits a greater response at higher Ca levels and lower NPP levels (0.86% Ca and 0.20% NPP) versus low Ca levels and low NPP levels (0.47% Ca and 0.24% NPP). The data demonstrated why it is impossible to determine a single NPP equivalency value for phytase supplements.

Comparison of peanut meal and soybean meal as protein supplements for laying hens.

Peanut protein is severely limiting in threonine and has been used to create threonine deficiency in animals. The availability of purified threonine at low cost raises the possibility of economically using peanut meal (PNM) and threonine combinations in poultry diets. An experiment was conducted to compare corn and PNM based diets to corn and soybean meal (SBM) based diets at three protein levels (16, 18.5, and 21%) in diets for 22-to-34-wk-old commercial Leghorns. Birds were housed two per cage with four cages per replicate and six replicates per treatment. Feed consumption, egg production, and feed per dozen eggs were almost identical for PNM (93.8 g/hen per d, 92.2 eggs per 100 hens/d, and 1.22 kg/dozen) and SBM (93.7 g/hen per d, 92.2 eggs per 100 hens/d, and 1.22 kg/dozen). Dietary protein level had no consistent effect on any of these parameters but did significantly improve body weight gains and egg weights (1.2 to 2.5 g/egg). PNM-fed hens laid slightly smaller eggs during the first 6 wk (P<0.05), but there were no egg size differences during the last 6 wk of the experiment (P>0.14). PNM-fed hens laid eggs with better interior quality at 26 and 30 wk of age. After 2 wk of storage, Haugh units remained better for eggs from hens fed PNM than SBM when kept refrigerated (4 degrees C; P<0.05) or at room temperature (20 degrees C; P<0.10). Egg specific gravity was slightly lower for hens fed PNM. It is concluded that PNM is an excellent ingredient for laying hen diets.

The influence of temperature on the threonine and tryptophan requirements of young broiler chicks.

Two experiments were conducted to investigate the influence of a warm environment (35 degrees C) on the threonine and tryptophan requirements of young broiler chicks from 7 to 18 or 21 d of age. Seven hundred forty (experiment 1) and one thousand eight (experiment 2) 1-d-old Cobb x Cobb straight-run broiler chicks were raised in wire-floored battery brooders in moderate temperature rooms (33 to 34 degrees C). For the first 7 d, all chicks were fed a standard corn-soybean-meal-based crumbled starter diet. On d 7, six chicks each (experiment 1) and eight chicks each (experiment 2) were randomly assigned to individual pens. In experiment 1, chicks were fed a corn-peanut meal basal diet supplemented with six levels of threonine (0.630, 0.651, 0.673, 0.715, 0.758, or 0.800% of the diet). In experiment 2, chicks were fed a corn-corn gluten meal based basal diet supplemented with six levels of tryptophan (0.090, 0.115, 0.140, 0.165, 0.190, or 0.215% of the diet). Each dietary treatment was repeated with three pens in each room and three rooms at each temperature. Three rooms were set at a moderate temperature of 25 degrees C, and the other three rooms were set at a warmer temperature of 35 degrees C. Body weight gain, feed consumption, and feed conversion ratio were measured. The threonine requirements of young broiler chicks were 0.733 +/- 0.016% (R2 = 0.59) and 0.752 +/- 0.046% (R2 = 0.25) for body weight gain, and 0.744 +/- 0.016% (R2 = 0.67) and 0.722 +/- 0.016% (R2 = 0.47) for feed conversion ratio at 25 and 35 degrees C, respectively (broken-line linear model). The tryptophan requirements of young broiler chicks were 0.151 +/- 0.004% (R2 = 0.85) and 0.144 +/- 0.003% (R2 = 0.89) for body weight gain, 0.144 +/- 0.003% (R2 = 0.88) and 0.142 +/- 0.003% (R2 = 0.88) for feed consumption, and 0.146 +/- 0.005% (R2 = 0.76) and 0.127 +/- 0.002% (R2 = 0.94) for feed conversion ratio at 25 and 35 degrees C, respectively. On average, the threonine and tryptophan requirements of broiler chicks at 35 degrees C were very similar to those kept at 25 degrees C.