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Background: Vγ9Vδ2 T-cells are antitumor immune effector cells that can detect metabolic dysregulation in cancer cells through phosphoantigen-induced conformational changes in the butyrophilin (BTN) 2A1/3A1 complex. In order to clinically exploit the anticancer properties of Vγ9Vδ2 T-cells, various approaches have been studied including phosphoantigen stimulation, agonistic BTN3A-specific antibodies, adoptive transfer of expanded Vγ9Vδ2 T-cells, and more recently bispecific antibodies. While Vγ9Vδ2 T-cells constitute a sizeable population, typically making up ~1-10% of the total T cell population, lower numbers have been observed with increasing age and in the context of disease., Methods: We evaluated whether bivalent single domain antibodies (VHHs) that link Vδ2-TCR specific VHHs with different affinities could support Vγ9Vδ2 T-cell expansion and could be incorporated in a bispecific engager format when additionally linked to a tumor antigen specific VHH., Results: Bivalent VHHs that link a high and low affinity Vδ2-TCR specific VHH can support Vγ9Vδ2 T-cell expansion. The majority of Vγ9Vδ2 T-cells that expanded following exposure to these bivalent VHHs had an effector or central memory phenotype and expressed relatively low levels of PD-1. Bispecific engagers that incorporated the bivalent Vδ2-TCR specific VHH as well as a tumor antigen specific VHH triggered antitumor effector functions and supported expansion of Vγ9Vδ2 T-cells in vitro and in an in vivo model in NOG-hIL-15 mice., Conclusion: By enhancing the number of Vγ9Vδ2 T-cells available to exert antitumor effector functions, these novel Vδ2-bivalent bispecific T cell engagers may promote the overall efficacy of bispecific Vγ9Vδ2 T-cell engagement, particularly in patients with relatively low levels of Vγ9Vδ2 T-cells., Competing Interests: NV. TG and HV own LAVA Therapeutics NV shares. DH, PY, VI-G, PH and HV are/were employed by LAVA Therapeutics NV. TG is scientific advisor to LAVA Therapeutics NV. The authors declare that this study received funding from Lava Therapeutics NV. LK, MJ and MV were funded by LAVA Therapeutics NV., (Copyright © 2024 King, de Jong, Veth, Lutje Hulsik, Yousefi, Iglesias-Guimarais, van Helden, de Gruijl and van der Vliet.)

Background: Despite considerable advancements in cancer immunotherapy, advanced melanoma still presents a substantial clinical challenge. In an effort to explore treatment options, we examined the immunotherapeutic potential of effector Vγ9Vδ2-T cells in vitro in a three-dimensional (3D) human organotypic melanoma-in-skin (Mel-RhS) model., Materials and Methods: Vγ9Vδ2-T cells were introduced into Mel-RhS via intradermal injection and cultured within the tissue microenvironment for up to 3 days., Results: Vγ9Vδ2-T cells remained viable for up to 3 days and were in close proximity to or within tumor nests. Upon Mel-RhS dissociation, a fraction was shown to be decorated by melanoma-associated chondroitin sulfate proteoglycan (MCSP), demonstrating their ability to actively navigate the tumor microenvironment and trogocytose cancer cells. Investigation into the apparent trogocytosis revealed an enhanced activated state of MCSP-decorated Vγ9Vδ2-T cells, evidenced by increased expression levels of 4-1BB, NKp44, programmed cell death protein-1 (PD-1), and programmed death-ligand 1 (PD-L1), compared with their MCSP - counterpart. These findings suggest that Vγ9Vδ2-T cells, upon successfully contacting melanoma cells, actively recognize and acquire MCSP from these malignant cells. Evidence of actual tumor cell elimination, although not significant, was only obtained after preincubation of Mel-RhS with pamidronate, a phosphoantigen-inducing agent, indicating the need for additional T cell receptor-mediated signaling for Vγ9Vδ2-T cells to reach their full oncolytic potential., Conclusions: This study highlights the viability and persistence of Vγ9Vδ2-T cells within the 3D microenvironment, their migratory and antitumor functionality, and the suitability of the model for testing T cell-based therapies, contributing both to the understanding of Vγ9Vδ2-T cell biology and their application in cancer immunotherapy., (© 2024 The Author(s).)

Background: CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach., Methods: To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d + tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography., Results: Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d + tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A'-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling., Conclusion: Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions., Competing Interests: Competing interests: DLH, PB and HJvdV are employees of LAVA Therapeutics, a company that develops bispecific gamma-delta T-cell engagers, and own LAVA Therapeutics shares and/or stock options. RL, TDdG and HJvdV are named inventors on international patent application WO 2016/122320 Al (‘Single domain antibodies targeting CD1d’) which partially relates to the work described in this paper. TDdG is a consultant for and a shareholder of LAVA Therapeutics. RL and MV were funded by a LAVA Therapeutics grant to Amsterdam UMC. JR has received funding from LAVA Therapeutics. DIG is a member of the scientific advisory board and a shareholder of Avalia Immunotherapies, a company working on NKT cell-based vaccines and is an inventor on patent applications WO 2021/127745 and WO 2021/127744 that describe effects of cross-linking CD1 molecules and WO2020/231274 that describes CD1d-dependent glycopeptide vaccines., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Vγ9Vδ2 T cells are effector cells with proven antitumor efficacy against a broad range of cancers. This study aimed to assess the antitumor activity and safety of a bispecific antibody directing Vγ9Vδ2 T cells to EGFR-expressing tumors. An EGFR-Vδ2 bispecific T-cell engager (bsTCE) was generated, and its capacity to activate Vγ9Vδ2 T cells and trigger antitumor activity was tested in multiple in vitro, in vivo, and ex vivo models. Studies to explore safety were conducted using cross-reactive surrogate engagers in nonhuman primates (NHP). We found that Vγ9Vδ2 T cells from peripheral blood and tumor specimens of patients with EGFR+ cancers had a distinct immune checkpoint expression profile characterized by low levels of PD-1, LAG-3, and TIM-3. Vγ9Vδ2 T cells could be activated by EGFR-Vδ2 bsTCEs to mediate lysis of various EGFR+ patient-derived tumor samples, and substantial tumor growth inhibition and improved survival were observed in in vivo xenograft mouse models using peripheral blood mononuclear cells (PBMC) as effector cells. EGFR-Vδ2 bsTCEs exerted preferential activity toward EGFR+ tumor cells and induced downstream activation of CD4+ and CD8+ T cells and natural killer (NK) cells without concomitant activation of suppressive regulatory T cells observed with EGFR-CD3 bsTCEs. Administration of fully cross-reactive and half-life extended surrogate engagers to NHPs did not trigger signals in the safety parameters that were assessed. Considering the effector and immune-activating properties of Vγ9Vδ2 T cells, the preclinical efficacy data and acceptable safety profile reported here provide a solid basis for testing EGFR-Vδ2 bsTCEs in patients with EGFR+ malignancies., (©2023 American Association for Cancer Research.)

Bispecific T cell engagers (bsTCEs) hold great promise for cancer treatment but face challenges due to the induction of cytokine release syndrome (CRS), on-target off-tumor toxicity, and the engagement of immunosuppressive regulatory T cells that limit efficacy. The development of Vγ9Vδ2-T cell engagers may overcome these challenges by combining high therapeutic efficacy with limited toxicity. By linking a CD1d-specific single-domain antibody (VHH) to a Vδ2-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vγ9Vδ2-T cells but also type 1 NKT cells to CD1d + tumors and triggers robust proinflammatory cytokine production, effector cell expansion, and target cell lysis in vitro. We show that CD1d is expressed by the majority of patient MM, (myelo)monocytic AML, and CLL cells and that the bsTCE triggers type 1 NKT and Vγ9Vδ2-T cell-mediated antitumor activity against these patient tumor cells and improves survival in in vivo AML, MM, and T-ALL mouse models. Evaluation of a surrogate CD1d-γδ bsTCE in NHPs shows Vγ9Vδ2-T cell engagement and excellent tolerability. Based on these results, CD1d-Vδ2 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML., Competing Interests: Declaration of interests J.M.R., V.I.G., F.S.v.d.B., B.W., R.C.R., A.E.P.A., T.R., P.W.H.I.P., and H.J.v.d.V. are employees of LAVA Therapeutics, a company that develops bispecific gamma-delta T cell engagers, and own LAVA Therapeutics shares and/or stock options. R.L., T.D.d.G., P.W.H.I.P., and H.J.v.d.V. are named inventors on international patent application WO2020/060,405 ("Dual acting CD1d immunoglobulin"), which partially relates to the work described in this paper. T.D.d.G. is a consultant for and a shareholder of LAVA Therapeutics. A.P.K. is a consultant for LAVA Therapeutics. R.L., M.J., M.V., and I.d.W. were funded by a LAVA Therapeutics grant to Amsterdam UMC., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The metabolites of choline have a central role in many mammalian biological processes, and choline supplementation to the periparturient dairy cow improves hepatic lipid metabolism. However, variability in responses to choline supplementation has highlighted a lack of understanding of choline absorption in the lactating dairy cow. Our objective was to determine net choline absorption by measuring net portal fluxes of choline and choline metabolites in cows receiving either dietary supplements of rumen-protected choline (RPC) or abomasal delivery of choline (ADC). We also evaluated markers for choline bioavailability by examining relationships between net portal absorption of choline and choline metabolites in plasma and milk. Five late-lactation Holstein cows were used in a 5×5 Latin square design, with 5-d treatment periods and a 2-d interval between periods. Treatments were (1) control (0g/d of choline), (2) 12.5g/d of choline fed as RPC, (3) 25g/d of choline fed as RPC, (4) 12.5g/d of choline provided as ADC, and (5) 25g/d of choline provided as ADC. At the end of each 5-d period, milk was sampled and 9 blood samples were collected simultaneously from an artery and portal vein at 30-min intervals. Plasma, milk, and feed ingredient concentrations of acetylcholine, betaine, free choline, glycerophosphocholine, lysophosphatidylcholine, phosphatidylcholine, phosphocholine, and sphingomyelin were quantified by hydrophilic interaction liquid chromatography-tandem mass spectrometry. With an increasing dose of ADC, the net portal flux of free choline increased and regression analysis indicated 61% net absorption of the infused dose. Among the choline metabolites, only concentrations of betaine, free choline, and phosphocholine increased in both arterial plasma (3.9, 1.9, and 0.4 times, respectively) and milk (2.5, 1.4, and 1.0 times, respectively) with 25g/d of ADC relative to the control. For RPC, the net portal flux of free choline was low relative to ADC (13%), which was similar to the relative difference observed in the concentrations and yields of milk free choline and betaine (averaged 21%). When evaluating markers for choline bioavailability, betaine was the leading candidate. Betaine in plasma and milk (alone or in combination with phosphocholine) was strongly associated with net free choline portal flux (coefficient of determination ranging from 0.64 to 0.79). In summary, free choline supply to the lactating dairy cow increases only specific choline metabolites in plasma and milk, which can be potential markers for choline bioavailability., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Aim: During 2008-2011 Australian Coding Standards mandated a causal relationship between diabetes and inpatient care as a criterion for recording diabetes as a comorbidity in hospital administrative datasets. We aim to measure the effect of the causality mandate on recorded diabetes and associated inter-hospital variations., Method: For patients with diabetes, all admissions between 2004 and 2013 to all New South Wales acute public hospitals were investigated. Poisson mixed models were employed to derive adjusted rates and variations., Results: The non-recorded diabetes incidence rate was 20.7%. The causality mandate increased the incidence rate four fold during the change period, 2008-2011, compared to the pre- or post-change periods (32.5% vs 8.4% and 6.9%). The inter-hospital variation was also higher, with twice the difference in the non-recorded rate between hospitals with the highest and lowest rates (50% vs 24% and 27% risk gap). The variation decreased during the change period (29%), while the rate continued to rise (53%). Admission characteristics accounted for over 44% of the variation compared with at most two per cent attributable to patient or hospital characteristics. Contributing characteristics explained less of the variation within the change period compared to pre- or post-change (46% vs 58% and 53%). Hospital relative performance was not constant over time., Conclusion: The causality mandate substantially increased the non-recorded diabetes rate and associated inter-hospital variation. Longitudinal accumulation of clinical information at the patient level, and the development of appropriate adoption protocols to achieve comprehensive and timely implementation of coding changes are essential to supporting the integrity of hospital administrative datasets., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Diagnostic data routinely collected for hospital admitted patients and used for case-mix adjustment in care provider comparisons and reimbursement are prone to biases. We aim to measure discrepancies, variations and associated factors in recorded chronic morbidities for hospital admitted patients in New South Wales (NSW), Australia. Of all admissions between July 2010 and June 2014 in all NSW public and private acute hospitals, admissions with over 24 hours stay and one or more of the chronic conditions of diabetes, smoking, hepatitis, HIV, and hypertension were included. The incidence of a non-recorded chronic condition in an admission occurring after the first admission with a recorded chronic condition (index admission) was considered as a discrepancy. Poisson models were employed to (i) derive adjusted discrepancy incidence rates (IR) and rate ratios (IRR) accounting for patient, admission, comorbidity and hospital characteristics and (ii) quantify variation in rates among hospitals. The discrepancy incidence rate was highest for hypertension (51% of 262,664 admissions), followed by hepatitis (37% of 12,107), smoking (33% of 548,965), HIV (27% of 1500) and diabetes (19% of 228,687). Adjusted rates for all conditions declined over the four-year period; with the sharpest drop of over 80% for diabetes (47.7% in 2010 vs. 7.3% in 2014), and 20% to 55% for the other conditions. Discrepancies were more common in private hospitals and smaller public hospitals. Inter-hospital differences were responsible for 1% (HIV) to 9.4% (smoking) of variation in adjusted discrepancy incidences, with an increasing trend for diabetes and HIV. Chronic conditions are recorded inconsistently in hospital administrative datasets, and hospitals contribute to the discrepancies. Adjustment for patterns and stratification in risk adjustments; and furthermore longitudinal accumulation of clinical data at patient level, refinement of clinical coding systems and standardisation of comorbidity recording across hospitals would enhance accuracy of datasets and validity of case-mix adjustment.

Bioavailability of vitamin B12 is low in humans and animals. Improving vitamin B12 absorption is important for optimal performance in dairy cows and for increasing vitamin B12 concentrations in milk for human consumption. However, when supplemented in the diet, 80% of synthetic vitamin B12, cyanocobalamin (CN-CBL), is degraded in the rumen of dairy cows and only 25% of the amount escaping destruction in the rumen disappears from the small intestine between the duodenal and ileal cannulas. In pigs, vitamin B12 from milk is more efficiently absorbed than synthetic CN-CBL. The objective of this study was to determine the efficacy of casein hydrolysate and whey proteins as excipients for CN-CBL to increase portal-drained viscera (PDV) flux of the vitamin in lactating dairy cows. Four multiparous lactating Holstein cows (237 ± 17 DIM) equipped with a rumen cannula and catheters in the portal vein and a mesenteric artery were used in a randomized Youden square design. They were fed every 2 h to maintain steady digesta flow. On experimental days, they received a postruminal bolus of (1) CN-CBL alone (0.1 g), (2) CN-CBL (0.1 g) + casein hydrolysate (10 g), or (3) CN-CBL (0.1 g) + whey proteins (10 g). Starting 30 min after the bolus, blood samples were taken simultaneously from the 2 catheters every 15 min during the first 2 h and then every 2 h until 24 h postbolus. Milk yield, DMI, and vitamin B12 portal-arterial difference and PDV flux were analyzed using the MIXED procedure of SAS. Milk yield and DMI were not affected by treatments. The portal-arterial difference of vitamin B12 during the 24-h period following the bolus of vitamin was greater when the vitamin was given in solution with casein hydrolysate (2.9 ± 4.6 pg/mL) than alone (-17.5 ± 5.2 pg/mL) or with whey protein (-13.4 ± 4.2 pg/mL). The treatment effects were similar for the PDV flux. The present results suggest that CN-CBL given with casein hydrolysate increases vitamin B12 absorption as compared with CN-CBL given alone., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

We hypothesized that variable composition in finishing rations, more specifically; the proportion of potato-by-product (PBP) and rumen protected histidine (His) supplementation may influence growth and meat quality attributes. Two different diets were fed (1) finishing ration with corn and barley as grains (CB, n = 20) and (2) substitution of 10% corn, DM basis, with PBP (PBP, n = 20). Additionally, half of each dietary treatment received 50 g/hd/d rumen protected His (HS, n= 20) while the other half received no supplement (NS, n = 20). Inclusion of 10% PBP or HS did not affect growth or carcass traits. Color stability was analyzed using Hunter color values as well as AMSA visual appraisal in both longissimus thoracis (LT) and gluteus medius (GM) muscles. The LT, but not the GM, of CB steers was more color stable over a 9 d simulated retail display compared to those fed a PB diet. Steers receiving HS produced significantly (P < 0.05) more color stable LT and GM steaks., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

When fed to meet the metabolizable protein requirements of the National Research Council, dairy cows consume an excess of N, resulting in approximately 75% of dietary N being lost to the environment as urine and feces. Reductions in environmental N release could be attained through an improvement in N efficiency. The objective of this study was to determine if the predicted reduction in milk yield associated with feeding a low-protein diet to lactating dairy cows could be avoided by dietary supplementation with 1 or more ruminally protected (RP) AA. Fourteen multiparous and 10 primiparous Holstein cows, and 24 multiparous Holstein × Jersey crossbred cows were used in a Youden square design consisting of 8 treatments and 3 periods. The 8 dietary treatments were (1) a standard diet containing 17% crude protein [CP; positive control (PC)], (2) a 15% CP diet [negative control (NC)], (3) NC plus RP Met (+M), (4) NC plus RP Lys (+K), (5) NC plus RP Leu (+L), (6) NC plus RP Met and Lys (+MK), (7) NC plus RP Met and Leu (+ML), and (8) NC plus RP Met, Lys, and Leu (+MKL). Dry matter intake was not affected by treatment. Crude protein intake was lower for NC and RP AA treatments compared with the PC treatment. No detrimental effect was detected of the low-CP diet alone or in combination with AA supplementation on milk and fat yield. However, milk protein yield decreased for NC and +MKL diets, and lactose yield decreased for the +MKL compared with the PC diet. Milk urea N concentrations were lower for all diets, suggesting that greater N efficiency was achieved by feeding the low-protein diet. Minimal effects of treatments on arterial plasma essential AA concentrations were detected, with only Ile and Val being significantly lower in the NC than in the PC diet. Phosphorylation ratios of signaling proteins known to regulate mRNA translation were not affected by treatments. This study highlights the limitations of requirement models aggregated at the protein level and the use of fixed postabsorptive efficiency to calculate milk protein requirements. Milk protein synthesis regulation by signaling pathways in vivo is still poorly understood., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Twenty-four multiparous high-producing dairy cows (40.0±1.4kg/d) were used in a factorial design to evaluate effects of 2 environments [thermoneutral (TN) and heat stress (HS)] and a dose range of dietary rumen-protected niacin (RPN; 0, 4, 8, or 12g/d) on body temperature, sweating rate, feed intake, water intake, production parameters, and blood niacin concentrations. Temperature-humidity index values during TN never exceeded 68 (stress threshold), whereas temperature-humidity index values during HS were above 68 for 24h/d. The HS environment increased hair coat and skin, rectal, and vaginal temperatures; respiration rate; skin and hair coat evaporative heat loss; and water intake and decreased DMI (3.5kg/d), milk yield (4.1kg/d), 4% fat-corrected milk (2.7kg/d), and milk protein yield (181.7g/d). Sweating rate increased during HS (12.7g/m(2) per h) compared with TN, but this increase was only 10% of that reported in summer-acclimated cattle. Niacin supplementation did not affect sweating rate, dry-matter intake, or milk yield in either environment. Rumen-protected niacin increased plasma and milk niacin concentrations in a linear manner. Heat stress reduced niacin concentration in whole blood (7.86 vs. 6.89μg/mL) but not in milk. Reduced blood niacin concentration was partially corrected by dietary RPN. An interaction existed between dietary RPN and environment; dietary RPN linearly increased water intake in both environments, but the increase was greater during HS conditions. Increasing dietary RPN did not influence skin temperatures. During TN, supplementing 12g/d of RPN increased hair coat (unshaved skin; 30.3 vs. 31.3°C at 1600h) but not shaved skin (32.8 vs. 32.9°C at 1600h) temperature when compared with 0g/d at all time points, whereas the maximum temperature (18°C) of the room was lower than skin temperature. These data suggest that dietary RPN increased water intake during both TN and HS and hair coat temperature during TN; however, core body temperature was unaffected. Thus, encapsulated niacin did not improve thermotolerance of winter-acclimated lactating dairy cows exposed to moderate thermal stress in Arizona., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

We previously reported that supplementation of rumen-protected choline (RPC) reduces the hepatic triacylglycerol concentration in periparturient dairy cows during early lactation. Here, we investigated the effect of RPC on the transcript levels of lipid metabolism-related genes in liver and adipose tissue biopsies, taken at wk -3, 1, 3, and 6 after calving, to elucidate the mechanisms underlying this RPC-induced reduction of hepatic lipidosis. Sixteen multiparous cows were blocked into 8 pairs and randomly allocated to either 1 of 2 treatments, with or without RPC. Treatments were applied from 3 wk before to 6 wk after calving. Both groups received a basal diet and concentrate mixture. One group received RPC supplementation, resulting in an intake of 14.4 g of choline per day, whereas controls received an isoenergetic mixture of palm oil and additional soybean meal. Liver and adipose tissue biopsies were taken at wk -3, 1, 3, and 6 to determine the mRNA abundance of 18 key genes involved in liver and adipose tissue lipid and energy metabolism. Milk samples were collected in wk 1, 2, 3, and 6 postpartum for analysis of milk fatty acid (FA) composition. The RPC-induced reduction in hepatic lipidosis could not be attributed to altered lipolysis in adipose tissue, as no treatment effect was observed on the expression of peroxisome proliferator-activated receptor γ, lipoprotein lipase, or FA synthase in adipose tissue, or on the milk FA composition. Rumen-protected choline supplementation increased the expression of FA transport protein 5 and carnitine transporter SLC22A5 in the liver, suggesting an increase in the capacity of FA uptake and intracellular transport, but no treatment effect was observed on carnitine palmitoyl transferase 1A, transporting long-chain FA into mitochondria. In the same organ, RPC appeared to promote apolipoprotein B-containing lipoprotein assembly, as shown by elevated microsomal triglyceride transfer protein expression and apolipoprotein B100 expression. Cows supplemented with RPC displayed elevated levels of glucose transporter 2 mRNA and a reduced peak in pyruvate carboxylase mRNA immediately after calving, showing that supplementation also resulted in improved carbohydrate metabolism. The results from this study suggest that RPC supplementation reduces liver triacylglycerol by improved FA processing and very-low-density lipoprotein synthesis, and RPC also benefits hepatic carbohydrate metabolism., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Twenty multiparous Holstein cows were used in four 5 × 5 Latin squares to determine the effects of feeding increasing amounts of distillers dried grains with solubles (DDGS) in diets with or without the supplementation (60 g/d) of a rumen-protected Lys (RPL) product (AminoShure-L, 38% l-Lys; Balchem Encapsulates, New Hampton, NY) on milk yield and composition and plasma concentration of AA. Dietary treatments were (1) control (CON; no DDGS), (2) 10% DDGS (10DG), (3) 20% DDGS (20DG), (4) 10% DDGS plus RPL (10DGRPL), and (5) 20% DDGS plus RPL (20DGRPL). Diets were formulated using the Cornell-Penn-Miner Dairy model (CPM v3.0; http://cahpwww.vet.upenn.edu/node/77) to provide a predicted decreasing supply of Lys (117, 99, and 91% of requirements) for the CON, 10DG, and 20DG diets, respectively. Addition of RPL to the 10DG and 20DG diets (unsupplemented diets) resulted in 2 additional treatments, 10DGRPL and 20DGRPL diets, respectively. The 10DGRPL and 20DGRPL diets met 110 and 100% of the Lys requirements, respectively. Periods lasted 21d, with the last 3d for data collection. Compared with cows fed the CON diet, cows fed diets with DDGS had a similar dry matter intake (DMI; 25.4 ± 0.88 kg/d), milk yield (30.7 ± 1.67 kg/d), and composition, except for protein percentage, which was higher (3.15 vs. 3.21 ± 0.05%) and resulted in higher (0.94 vs. 1.00 ± 0.05 kg/d) protein yield by cows fed diets containing 20% DDGS. Unexpectedly, despite diets being formulated based on predicted DMI of 23.3 kg/d and milk yield of 38.5 kg/d, cows had a greater DMI and lower milk yield across all treatments, which resulted in diets that were predicted by CPM Dairy to supply sufficient amounts of Lys (140, 118, and 104% of requirement for the CON, 10 DG, and 20 DG diet, respectively) and consequently, supplementation with RPL did not have an effect on milk production or composition. Plasma concentration of Lys decreased (11.8%) as DDGS inclusion increased. For other essential AA, plasma concentrations of cows fed diets with DDGS were lower for Arg, His, and Val and greater for Leu and Met compared with cows fed the CON diet. Supplementation with RPL failed to decrease the plasma concentration of other essential AA, which provides support that Lys was not limiting., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this experiment was to evaluate the effect of supplementing a metabolizable protein (MP)-deficient diet with rumen-protected (RP) Lys, Met, and specifically His on dairy cow performance. The experiment was conducted for 12 wk with 48 Holstein cows. Following a 2-wk covariate period, cows were blocked by DIM and milk yield and randomly assigned to 1 of 4 diets, based on corn silage and alfalfa haylage: control, MP-adequate diet (ADMP; MP balance: +9 g/d); MP-deficient diet (DMP; MP balance: -317 g/d); DMP supplemented with RPLys (AminoShure-L, Balchem Corp., New Hampton, NY) and RPMet (Mepron; Evonik Industries AG, Hanau, Germany; DMPLM); and DMPLM supplemented with an experimental RPHis preparation (DMPLMH). The analyzed crude protein content of the ADMP and DMP diets was 15.7 and 13.5 to 13.6%, respectively. The apparent total-tract digestibility of all measured nutrients, plasma urea-N, and urinary N excretion were decreased by the DMP diets compared with ADMP. Milk N secretion as a proportion of N intake was greater for the DMP diets compared with ADMP. Compared with ADMP, dry matter intake (DMI) tended to be lower for DMP, but was similar for DMPLM and DMPLMH (24.5, 23.0, 23.7, and 24.3 kg/d, respectively). Milk yield was decreased by DMP (35.2 kg/d), but was similar to ADMP (38.8 kg/d) for DMPLM and DMPLMH (36.9 and 38.5kg/d, respectively), paralleling the trend in DMI. The National Research Council 2001model underpredicted milk yield of the DMP cows by an average (±SE) of 10.3 ± 0.75 kg/d. Milk fat and true protein content did not differ among treatments, but milk protein yield was increased by DMPLM and DMPLMH compared with DMP and was not different from ADMP. Plasma essential amino acids (AA), Lys, and His were lower for DMP compared with ADMP. Supplementation of the DMP diets with RP AA increased plasma Lys, Met, and His. In conclusion, MP deficiency, approximately 15% below the National Research Council requirements from 2001, decreased DMI and milk yield in dairy cows. Supplementation of the MP-deficient diet with RPLys and RPMet diminished the difference in DMI and milk yield compared with ADMP and additional supplementation with RPHis eliminated it. As total-tract fiber digestibility was decreased with the DMP diets, but DMI tended to increase with RP AA supplementation, we propose that, similar to monogastric species, AA play a role in DMI regulation in dairy cows. Our data implicate His as a limiting AA in high-producing dairy cows fed corn silage- and alfalfa haylage-based diets, deficient in MP. The MP-deficient diets clearly increased milk N efficiency and decreased dramatically urinary N losses., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Fish oil is used as a ration additive to provide n-3 fatty acids to dairy cows. Fish do not synthesize n-3 fatty acids; they must consume microscopic algae or other algae-consuming fish. New technology allows for the production of algal biomass for use as a ration supplement for dairy cattle. Lipid encapsulation of the algal biomass protects n-3 fatty acids from biohydrogenation in the rumen and allows them to be available for absorption and utilization in the small intestine. Our objective was to examine the use of algal products as a source for n-3 fatty acids in milk. Four mid-lactation Holsteins were assigned to a 4×4 Latin square design. Their rations were supplemented with 1× or 0.5× rumen-protected (RP) algal biomass supplement, 1× RP algal oil supplement, or no supplement for 7 d. Supplements were lipid encapsulated (Balchem Corp., New Hampton, NY). The 1× supplements provided 29 g/d of docosahexaenoic acid (DHA), and 0.5× provided half of this amount. Treatments were analyzed by orthogonal contrasts. Supplementing dairy rations with rumen-protected algal products did not affect feed intake, milk yield, or milk component yield. Short- and medium-chain fatty acid yields in milk were not influenced by supplements. Both 0.5× and 1× RP algae supplements increased daily milk fat yield of DHA (0.5 and 0.6±0.10 g/d, respectively) compared with 1× RP oil (0.3±0.10 g/d), but all supplements resulted in milk fat yields greater than that of the control (0.1±0.10g/d). Yield of trans-18:1 fatty acids in milk fat was also increased by supplementation. Trans-11 18:1 yield (13, 20, 27, and 15±3.0 g/d for control, 0.5× RP algae, 1× RP algae, and 1× RP oil, respectively) was greater for supplements than for control. Concentration of DHA in the plasma lipid fraction on d 7 showed that the DHA concentration was greatest in plasma phospholipid. Rumen-protected algal biomass provided better DHA yield than algal oil. Feeding lipid-encapsulated algae supplements may increase n-3 content in milk fat without adversely affecting milk fat yield; however, preferential esterification of DHA into plasma phospholipid may limit its incorporation into milk fat., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Trans-10,cis-12 conjugated linoleic acid (CLA) inhibits milk fat synthesis in dairy ewes, but the effects under varying dietary metabolizable protein (MP) levels when energy-limited diets are fed have not been examined. The objectives of the study were to evaluate the response of lactating dairy ewes to CLA supplementation when fed diets limited in metabolizable energy (ME) and with either a low or high MP content. Twelve multiparous ewes in early lactation were randomly allocated to 1 of 4 dietary treatments: a high MP (110% of daily MP requirement) or low MP (93% of daily MP requirement) diet unsupplemented or supplemented with a lipid-encapsulated CLA to provide 2.4 g/d of trans-10,cis-12 CLA, in each of 4 periods of 25 d each in a 4×4 Latin square design. All diets were restricted to supply each ewe with 4.6 Mcal of ME/d (equivalent to 75% of ME requirement). Supplementation with CLA decreased milk fat percentage and yield by 33% and 24%, respectively, and increased milk, milk protein, and lactose yields by 16, 13, and 17%, respectively. Feeding the high MP diet increased the yields of milk, fat, protein, and lactose by 18, 15, 19, and 16%, respectively. Milk fat content of trans-10,cis-12 CLA (g/100g) was 0.09 and <0.01 for the CLA-supplemented and unsupplemented ewes, respectively. Ewes supplemented with CLA had a reduced yield (mmol/d) of fatty acids of C16, although the effect was greatest for C16. Plasma urea concentrations were lowest in ewes supplemented with CLA compared with those unsupplemented (6.5 vs. 7.4 mmol/L, respectively) and receiving low compared with high MP diets (5.6 vs. 8.3 mmol/L, respectively). In conclusion, dairy ewes fed energy-limited diets and supplemented with CLA repartitioned nutrients to increase yields of milk, protein, and lactose, with the response to CLA supplementation and additional MP intake being additive., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The effects of a dietary supplement of rumen-protected choline on feed intake, milk yield, milk composition, blood metabolites, and hepatic triacylglycerol were evaluated in periparturient dairy cows. Thirty-eight multiparous cows were blocked into 19 pairs and then randomly allocated to either one of 2 treatments. The treatments were supplementation either with or without (control) rumen-protected choline. Treatments were applied from 3 wk before until 6 wk after calving. Both groups received the same basal diet, being a mixed feed of grass silage, corn silage, straw, and soybean meal, and a concentrate mixture delivered through transponder-controlled feed dispensers. For all cows, the concentrate mixture was gradually increased from 0 kg/day (wk -3) to 0.9 kg of dry matter (DM)/d (day of calving) and up to 8.1 kg of DM/d on d 17 postcalving until the end of the experiment. Additionally, a mixture of 60 g of a rumen-protected choline supplement (providing 14.4 g of choline) and of 540 g of soybean meal or a (isoenergetic) mixture of 18 g of palm oil and 582 g of soybean meal (control) was offered individually in feed dispensers. Individual feed intake, milk yield, and body weight were recorded daily. Milk samples were analyzed weekly for fat, protein, and lactose content. Blood was sampled at wk -3, d 1, d 4, d 7, d 10, wk 2, wk 3, and wk 6 and analyzed for glucose, nonesterified fatty acids, and β-hydroxybutyric acid. Liver biopsies were taken from 8 randomly selected pairs of cows at wk -3, wk 1, wk 4, and wk 6 and analyzed for triacylglycerol concentration. We found that choline supplementation increased DM intake from 14.4 to 16.0 kg/d and, hence, net energy intake from 98.2 to 109.1 MJ/d at the intercept of the lactation curve at 1 day in milk (DIM), but the effect of choline on milk protein yield gradually decreased during the course of the study. Choline supplementation had no effect on milk yield, milk fat yield, or lactose yield. Milk protein yield was increased from 1.13 to 1.26 kg/d at the intercept of the lactation curve at 1 DIM, but the effect of choline on milk protein yield gradually decreased during the course of the study. Choline supplementation was associated with decreased milk fat concentration at the intercept of the lactation curve at 1 DIM, but the effect of choline on milk fat concentration gradually decreased as lactation progressed. Choline supplementation had no effect on energy-corrected milk yield, energy balance, body weight, body condition score, and measured blood parameters. Choline supplementation decreased the concentration of liver triacylglycerol during the first 4 wk after parturition. Results from this study suggest that hepatic fat export in periparturient dairy cows is improved by choline supplementation during the transition period and this may potentially decrease the risk for metabolic disorders in the periparturient dairy cow., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The feeding of conjugated linoleic acid (CLA) supplements to early-lactation dairy cows has been shown to decrease milk fat synthesis and possibly improve reproductive performance. However, previously reported studies used too few animals to clearly establish the effect of CLA on reproduction. Our objective was to combine data from these studies to evaluate the association of CLA with time to first ovulation and time to conception using methods of survival analysis and overall success of pregnancy by logistic regression. A database was compiled of individual animal data (n = 212) from 5 controlled studies in which CLA had been supplemented to early-lactation dairy cows. Survival analysis incorporated both semi-parametric models (Cox proportional hazards) and parametric models (log-normal). The probability of cows becoming pregnant increased in a nonlinear manner as trans-10, cis-12 CLA dose increased, with the optimal dose predicted to be 10.1 g/d. At the optimal dose, the probability of pregnancy was increased by 26% compared with those animals receiving no CLA (probability = 91% and 72%, respectively). Similarly, the log-normal model predicted that time to conception was decreased in a nonlinear manner with increasing trans-10, cis-12 CLA dose. The predicted optimal dose was 10.5 g of trans-10, cis-12 CLA/d and at this dose the median time to conception was decreased by 34 d when compared with those cows not receiving CLA (117 vs. 151 d in milk, respectively). The log-normal model was also the best-fit model for time to first ovulation. Overall, this multi-study analysis demonstrated a strong concordance between the nature of the dose response and the predicted optimal dose of trans-10, cis-12 CLA across the 3 reproductive variables evaluated. These results indicate that reproductive performance of dairy cows may be improved by feeding of CLA supplements during early lactation.

The effect of conjugated linoleic acid (CLA) supplements containing trans-10, cis-12 for reducing milk fat synthesis has been well described in dairy cows and sheep. Studies on lactating goats, however, remain inconclusive. Therefore, the current study investigated the efficacy of a lipid-encapsulated trans-10, cis-12 CLA supplement (LE-CLA) on milk production and milk fatty acid profile in dairy goats. Thirty multiparous Alpine lactating goats in late lactation were used in a 3 x 3 Latin square design (14-d treatment periods separated by 14-d intervals). Does were fed a total mixed ration of Bermuda grass hay, dehydrated alfalfa pellets, and concentrate. Does were randomly allocated to 3 treatments: A) unsupplemented (control), B) supplemented with 30 g/d of LE-CLA (low dose; CLA-1), and C) supplemented with 60 g/d of LE-CLA (high dose; CLA-2). Milk yield, dry matter intake, and milk protein content and yield were unaffected by treatment. Compared with the control, milk fat yield was reduced 8% by the CLA-1 treatment and 21% by the CLA-2 treatment, with milk fat content reduced 5 and 18% by the CLA-1 and CLA-2 treatments, respectively. The reduction in milk fat yield was due to decreases in both de novo fatty acid synthesis and uptake of preformed fatty acids. Milk fat content of trans-10, cis-12 CLA was 0.03, 0.09, and 0.19 g/100 g of fatty acids for the control, CLA-1, and CLA-2 treatments, respectively. The transfer efficiency of trans-10, cis-12 CLA from the 2 levels of CLA supplement into milk fat was not different between treatments and averaged 1.85%. In conclusion, trans-10, cis-12 CLA reduced milk fat synthesis in lactating dairy goats in a manner similar to that observed for lactating dairy cows and dairy sheep. Dose-response comparisons, however, suggest that the degree of reduction in milk fat synthesis is less in dairy goats compared with dairy cows and dairy sheep.

The objective of this study was to evaluate the mechanism of action through which conjugated linoleic acid (CLA) beneficially affects reproduction. Lactating Holstein cows (n = 45, 20 +/- 1 DIM) were assigned to 1 of 3 treatments: 70 g/d of Ca salts of tallow (control); 63 g/d of lipid-encapsulated CLA providing 7.1 g/d of cis-9, trans-11 CLA and 2.4 g/d of trans-10, cis-12 CLA (CLA 75:25); or 76 g/d of lipid-encapsulated CLA providing 7.1 g/d each of cis-9, trans-11 and trans-10, cis-12 CLA (CLA 50:50). Supplements were top-dressed for 37 d, milk production and DMI were recorded daily, and blood samples were taken 3 times per week. At 30 +/- 3 DIM, ovulation was synchronized in all cows with a modified Ovsynch protocol, and on d 15 of the cycle cows received an oxytocin injection; blood samples were obtained frequently to measure 13,14 dihydro, 15-keto PGF2alpha. On d 16 of the cycle cows received a PGF2alpha injection and ovarian follicular aspiration was performed 54 h later. Follicular fluid was analyzed for fatty acids, progesterone, and estradiol. Endometrial biopsies were taken before and again near the end of the supplementation period for fatty acid analysis. The CLA resulted in decreased milk fat content of 14.1 and 6.1% at wk 5 of treatment of CLA 50:50 and CLA 75:25, respectively. There were no differences in energy balance or plasma nonesterified fatty acids; however, plasma IGF-I was greater in cows supplemented with CLA 50:50. The CLA isomers were not detectable in endometrial tissue, but cis-9, trans-11 CLA tended to be greater in follicular fluid of supplemented cows. Response to the oxytocin challenge was not different among treatments. Progesterone during the early luteal phase and the estradiol:progesterone ratio in follicular fluid tended to be greater in cows supplemented with CLA 50:50. Overall, these results indicate that short periods of CLA supplementation do not alter uterine secretion of PGF2alpha. The mechanism through which CLA affects reproduction may involve improved ovarian follicular steroidogenesis and increased circulating concentrations of IGF-I.

Enrichment of milk fat with n-3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may be advantageous because of their beneficial effects on human health. In addition, these fatty acids play an important role in reproductive processes in dairy cows. Our objective was to evaluate the protection of EPA and DHA against rumen biohydrogenation provided by Ca salts of fish oil. Four Holstein cows were assigned in a Latin square design to the following treatments: 1) ruminal infusion of Ca salts of fish oil and palm fatty acid distillate low dose (CaFO-1), 2) ruminal infusion of Ca salts of fish oil and palm fatty acid distillate high dose (CaFO-2), 3) ruminal infusion of fish oil high dose (RFO), and 4) abomasal infusion of fish oil high dose (AFO). The high dose of fish oil provided approximately 16 and approximately 21 g/d of EPA and DHA, respectively, whereas the low dose (CaFO-1) provided 50% of these amounts. A 10-d pretreatment period was used as a baseline, followed by 9-d treatment periods with interceding intervals of 10 d. Supplements were infused every 6 h, milk samples were taken the last 3 d, and plasma samples were collected the last day of baseline and treatment periods. Milk fat content of EPA and DHA were 5 to 6 times greater with AFO, but did not differ among other treatments. Milk and milk protein yield were unaffected by treatment, but milk fat yield and DM intake were reduced by 20 and 15%, respectively, by RFO. Overall, results indicate rumen biohydrogenation of long chain n-3 fatty acids was extensive, averaging >85% for EPA and >75% for DHA for the Ca salts and unprotected fish oil supplements. Thus, Ca salts of fish oil offered no protection against the biohydrogenation of EPA and DHA beyond that observed with unprotected fish oil; however, the Ca salts did provide rumen inertness by preventing the negative effects on DM intake and milk fat yield observed with unprotected fish oil.

The trans-10, cis-12 conjugated linoleic acid (CLA) isomer inhibits milk fat synthesis, whereas milk yield and synthesis of other milk components generally remain unchanged in established lactation. However, in some CLA studies increases in milk yield, milk protein yield, or both have been observed in cows limited in energy, either in early lactation or when grazing pasture. Our objective was to evaluate the performance and monitor peripheral tissue responses to homeostatic signals regulating lipolysis and glucose uptake with CLA supplementation when cows were limited in metabolizable energy in combination with moderate or excess metabolizable protein supply. Holstein cows (n = 48; 112 +/- 5 d in milk; mean +/- SE) were provided ad libitum access to a diet that met energy and protein requirements for a 16-d standardization interval. Based on performance during this interval, the Cornell Net Carbohydrate and Protein System was used to design energy-limiting rations that provided 80% of metabolizable energy requirements, and these were fed throughout the treatment periods. Cows were randomly allocated to 4 treatments, in a 2-period crossover design. Treatments were 1) moderate metabolizable protein (MP) supply, 2) moderate MP supply + CLA, 3) excess MP supply, and 4) excess MP supply + CLA. Moderate and excess MP supply were at 88 and 117%, respectively, of the MP requirement established during the standardization period, as estimated by the Cornell Net Carbohydrate and Protein System. Each experimental period comprised 16 d, with crossover of CLA within each protein level. The lipid-encapsulated CLA supplement provided 12 g/d of trans-10, cis-12 CLA. Conjugated linoleic acid treatment reduced milk fat yield by 21% but increased milk yield and milk protein yield by 2.6 and 2.8%, respectively. Milk yield and content and yield of both milk protein and fat were unaltered by either protein treatment alone or in combination with CLA. Basal concentrations of glucose, insulin, and nonesterified fatty acids were unaffected by CLA supplementation. The fractional rate of glucose clearance in response to an insulin challenge and the nonesterified fatty acid response to an epinephrine challenge were also not altered by either CLA treatment or MP supply. Overall, the results demonstrate that CLA supplementation when cows are energy-limited allows for repartitioning of nutrients, resulting in increased yields of milk and milk protein, and this can occur without changes in whole-body glucose homeostasis and adipose tissue response to lipolytic stimuli.

Abomasal infusion studies have shown that trans-10, cis-12 conjugated linoleic acid (CLA) decreases milk fat synthesis. However, supplements of CLA must avoid rumen biohydrogenation for this technology to be applied to ruminants. Rumen protection methods would reduce CLA metabolism in the rumen and increase its supply to the small intestine. Our objective was to compare the efficacy of 2 forms of rumen-protected CLA at inducing milk fat depression. Three mid to late lactation Holstein cows each fitted with a rumen fistula were used in a 3 x 3 Latin square design. Treatments were: 1) control, 2) calcium salts of CLA (Ca-CLA), and 3) formaldehyde-protected CLA (FP-CLA). Supplements were designed to provide 10 g/d of trans-10, cis-12 CLA and were administered intraruminally once per day to ensure exact delivery of amount. Both CLA treatments substantially reduced milk fat yield and content compared with control, with the reductions in milk fat yield averaging 34% for the Ca-CLA treatment and 44% for the FP-CLA treatment. In contrast, milk yield, milk protein yield, and dry matter intake were unaltered by CLA treatment. Efficiency of transfer of trans-10, cis-12 CLA from the supplement into milk fat was 3.2 and 7.0% for Ca-CLA and FP-CLA, respectively. These values are much lower than transfer efficiencies reported for abomasally infused CLA, suggesting that much of the trans-10, cis-12 CLA present in the 2 formulations was biohydrogenated in the rumen. Overall, the extent of the reduction in milk fat yield indicates that both protection formulations are acceptable methods for the formulation of CLA supplements to induce milk fat depression in lactating dairy cows.

This study identified suitable predictors of ruminal pH and identified relationships between ruminal pH and animal measures for diets based on fresh pasture. Animal and dietary variables (121 treatment means from six countries) were collated from 23 studies of lactating dairy cows fed pasture. Mean daily ruminal pH ranged from 5.6 to 6.7 across studies. Within studies, a low ruminal pH was associated with higher (P < 0.05; r2 > 0.40) microbial N flow from the rumen, total and individual volatile fatty acid concentrations, milk and milk component yields, and dry matter intake, and with lower (P < 0.05; r2 > 0.30) concentrations of milk fat, fat:protein, and acetate:propionate. Large variation between studies meant that these ruminal and production variables could not be used to make reliable predictions of ruminal pH in future pasture-based studies or feeding scenarios. Ruminal pH was positively related (P < 0.05; r2 < 0.15) to forage neutral detergent fiber (NDF) and NDF content within study, and negatively related (P = 0.001; r2 = 0.14) to nonstructural carbohydrate across studies. No single dietary variable, or group of variables, could be used to make a reliable prediction of ruminal pH. Estimates of effective fiber for diets containing only pasture were made using the Cornell Net Carbohydrate and Protein System ruminal pH equation. Mean effectiveness of fiber in pasture was 43% of NDF, and ranged from 17 to 78% across studies. High flows of microbial nitrogen, milk, milk fat yield, and dry matter intake suggested that the performance of cows fed high quality pasture was not limited when mean ruminal pH decreased to 5.8.

Many models of digestion assume steady-state conditions and do not account for diurnal variation in the rumen environment. This experiment examined the relationships between diurnal pH, pasture digestion, and microbial protein synthesis. Four dual-flow continuous culture fermenters were used to test the effect of increasing time at suboptimal pH on parameters of digestion. Fermentation of high quality pasture was controlled at pH 5.4 (suboptimal) for four intervals during each 24-h period (0, 4, 8, and 12 h) according to a 4 x 4 Latin square design. During the remainder of each day, pH was controlled at 6.3 (optimal). Samples were collected during the last 3 d of each of the four 9-d experimental periods. A negative quadratic relationship was observed between time at suboptimal pH and apparent digestibility of organic matter and dry matter. The largest reduction in digestibility of organic matter, dry matter, and neutral detergent fiber was exhibited after 4 h at suboptimal pH. A negative linear relationship was found between time at suboptimal pH and microbial N flow, with the greatest decline in microbial N flow occurring at 12 h at suboptimal pH. These results suggest that the period of time that pH is below optimal may be more critical for digestion than the relationship between mean daily pH and optimal pH. Modeling non-steady-state ruminal conditions to account for diurnal variation in the ruminal environment may improve the prediction of digestion, especially fiber.

The ruminal pH of dairy cows fed high quality pasture is often below values recommended to optimize digestion. Four continuous culture fermenters were used to determine the pH required for the optimal digestion of pasture. High quality pasture was fermented at four controlled levels of pH (5.4, 5.8, 6.2, and 6.6) according to a 4 x 4 Latin square design. Automatic infusion of 5 M NaOH and 5 M HCl controlled pH to +/- 0.1. Digesta samples were collected during the last 3 d of each of the four 9-d experimental periods. Digestion and synthesis of microbial protein were largely insensitive to pH across a broad range of pH (5.8 to 6.6), but a large reduction in both occurred when pH was 5.4. The digestibility of pasture dry matter and synthesis of microbial protein were optimized at pH 6.35 and 6.13, respectively. The proportions of individual volatile fatty acids were not changed as pH increased. Digestion of high quality pasture in continuous culture was comparable to that predicted by the Cornell Net Carbohydrate and Protein System between pH 6.2 and 6.6. However, the model underpredicted organic matter and fiber digestibility between pH 5.4 and 5.8, compared with values obtained in continuous culture. This suggests that when ruminal pH is less than 6.2, the model may over-predict the production response to supplementation of high quality pasture with an effective fiber source.

For a quick analytical identification of histones a two-dimensional electrophoretic system has been developed. First the proteins are separated on cellulose acetate strips in alkaline buffer. Then they are reelectrophoresed in a second dimension on polyacrylamide gels either with sodium dodecyl sulfate or in the presence of urea at pH 2.7. The technique is demonstrated with yeast and calf thymus histones.