Bovine Plasma Research Articles

In vivo / in vitro Correlation of Pharmacokinetics of Gentamicin, Vancomycin, Teicoplanin and Doripenem in a Bovine Blood Hemodialysis Model.

Background: Elimination of a drug during renal replacement therapy is not only dependent on flow rates, molecular size and protein binding, but is often influenced by difficult to predict drug membrane interactions. In vitro models allow for extensive profiling of drug clearance using a wide array of hemofilters and flow rates. We present a bovine blood based in vitro pharmacokinetic model for intermittent renal replacement therapy. Methods: Four different drugs were analyzed: gentamicin, doripenem, vancomicin and teicoplanin. The investigated drug was added to a bovine blood reservoir connected to a hemodialysis circuit. In total seven hemofilter models were analyzed using commonly employed flow rates. Pre-filter, post-filter and dialysate samples were drawn, plasmaseparated and analyzed using turbidimetric assays or HPLC. Protein binding of doripenem and vancomycin was measured in bovine plasma and compared to previously published values for human plasma. Results: Clearance values were heavily impacted by choice of membrane material and surface as well as by dialysis parameters such as blood flow rate. Gentamicin clearance ranged from a minimum of 90.12 ml/min in a Baxter CAHP-170 diacetate hemofilter up to a maximum of 187.90 ml/min in a Fresenius medical company Fx80 polysulfone model (blood flow rate 400 ml/min, dialysate flow rate 800 ml/min). Clearance of Gentamicin vs Vancomicin over the F80s hemofilter model using the same flow rates was 137.62 mL vs 103.25 ml/min. Doripenem clearance with the Fx80 was 141.25 ml/min. Conclusion: Clearance values corresponded very well to previously published data from clinical pharmacokinetic trials. In conjunction with in silico pharmacometric models. This model will allow precise dosing recommendations without the need of large scale clinical trials.

Quantification of bovine plasma amino acids via liquid chromatography–electrospray ionization-mass spectrometry: Comparison of underivatized and precolumn derivatized methods

The objectives of this experiment were to evaluate and compare underivatized (UND) and precolumn derivatized (DER) methods for quantification of bovine plasma AA by isotope dilution ratio via liquid chromatography-electrospray ionization (ESI)-single quadrupole mass spectrometry. Linearity of the mass-to-charge ratio signal and area signal sensitivity of 12C were evaluated for each AA with 5-point standard curves (range: 1.1–500 µM). Plasma from lactating dairy cows was isolated by centrifugation and deproteinized using 1 N perchloric acid with a final concentration of 0.5 N. Deproteinized plasma was filtered and injected into a 50 × 2-mm column (Imtakt) or extracted, derivatized, and injected into a 250 × 3-mm column (EZ:faast, Phenomenex) and analyzed via liquid chromatography-ESI-single quadrupole mass spectrometry. Coefficients of variation and recovery rates were evaluated using 4 replicates of pooled plasma samples spiked with each AA at concentrations of 10, 20, and 50 µM. In addition, a subset of 24 plasma samples was used to directly compare methods using linear regression, correlation coefficient (r), concordance correlation coefficient (CCC), and Bland-Altman plot test. Both methods showed linearity within the dynamic range analyzed for all essential AA (coefficient of determination, R2 ≥ 0.995) and most other AA, although the UND samples had poor linearity (R2 ≤ 0.990) or peak resolution problems for Asp, Gly, Tyr, and Ser. Moreover, area signal sensitivity for 12C AA was greater for DER samples than for UND samples [range: 2.2× (Pro) to 309.5× (Ala)]. Both methods had recovery rates ranging from 85.7 to 119.8.0%, and none differed from 100% except Gln [20 µM (85.7%) and 50 µM (87.6%)] and Val [50 µM (119.8%)] using the UND method. The UND method had a coefficient of variation ranging from 0.9% (Val) to 7.8% (His), whereas for the DER method the range was 2.2% (Glu) to 8.8% (Asp). The highest correlation coefficient (>0.90) and CCC (>0.90) were observed for Arg, Ile, Leu, Met, Thr, Trp, Val, and Gln, with the Bland-Altman plot test showing minimal mean bias for these AA. Lowest values were observed for His (r = 0.46; CCC = 0.45), Lys (r = 0.76; CCC = 0.75), Ala (r = 0.83; CCC = 0.73), and Glu (r = 0.65; CCC = 0.42). The UND method showed linearity, precision, and accurate recovery rates for most AA, with most essential AA having comparable values between the 2 methods. However, the DER method had greater 12C AA area signal sensitivity, linearity, and recovery rates.

Detection of bovine inflammatory cytokines IL-1β, IL-6, and TNF-α with a multiplex electrochemiluminescent assay platform

Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare. Our objective was to develop a multiplex assay for the bovine inflammatory cytokines IL-1β, IL-6, and TNF-α using the Meso Scale Discovery U-PLEX platform. "Do-It-Yourself" ELISA kits that contained polyclonal antibodies, both unlabeled and biotinylated, and the specific recombinant bovine cytokine standard, were purchased for each of these three cytokines. The biotinylated antibodies were coupled to linkers that bind to specific locations within each well of the U-PLEX plate. Unique linkers were used for each of the cytokines. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to performing an optimization on the multiplex assay containing reagents for all three cytokines. To calculate cytokine concentrations, standard curves were developed using the recombinant cytokines and were run concurrently on each plate. Standard curves for IL-1β and TNF-α were run at concentrations ranging from 0 to 50,000 pg/mL, and for IL-6 from 0 to 10,000 pg/mL. The average lowest level of detection concentration measured by the standard curves were 5.3 pg/mL, 0.92 pg/mL, and 22.34 pg/mL for IL-1β, IL-6, and TNF-α respectively, as determined by data from seven plates containing bovine plasma samples from a combination of healthy and diseased cattle. The U-PLEX platform was a viable means to develop custom analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents.

Fat substitutes based on bovine blood plasma and flaxseed oil as functional ingredients

Technological and structural properties of edible flaxseed oil oleogels with dehydrated full bovine blood plasma as structuring agent were evaluated, envisioned oleogels as potential sustainable saturated fat replacers in high-fat content culinary preparations. Gelation process was induced by heating o/w emulsions at 60 °C and 80 °C, followed by a freeze-drying step to the removal of water. Structural properties were evaluated by infrared spectroscopy while technological characteristics were inferred by measuring total lipids content, oil binding capacity, textural parameters, lipid oxidative stability, and color development during 30 days of chilled storage. Results suggest that the high-temperature gelation process yields oleogels of higher gel strength and stability, although the lower temperature yields higher oxidative stability oleogels. An apparent correspondence between technological properties and gel network structures was also observed, and even though further studies regarding fatty acids bioavailability and behavior of the oloegels in each food matrix are required, bovine blood plasma-based oleogels seems to be a promising alternative as saturated fat replacers in culinary preparations, also allowing the incorporation of flaxseed oil as a source of linolenic acid for reducing cholesterol and blood sugar levels. In this way, it is sought not only the development of healthier fats substitutes but also the recovery of a waste to generate a more sustainable ingredient.

New approach to derivatisation for oestradiol esters detection in animal blood plasma using negative chemical ionisation GC-MS

ABSTRACT In 1996, the EU prohibited the use of substances with anabolic action for food-producing animals (EU Directive 96/22/EC). In cases of illegal use of steroid hormones, these substances are usually applied to the animals in the form of esters. The reliable determination of intact steroid esters in animal tissues or body fluids is an unequivocal proof of illegal treatment of animals with EU prohibited anabolic substances. Previously our laboratory developed a sensitive method for determination of oestradiol benzoate and other steroid esters in blood plasma using LC-MS/MS, validated according to Commission Decision 2002/657/EC. This study describes a GC-MS method which has been developed for five oestradiol esters in blood plasma. The sample preparation procedure consisted of protein precipitation, phospholipids removal and cleaning on an alumina column. Oestradiol esters were derivatised with 2, 3, 4, 5, 6-pentafluorobenzoyl chloride (PFBCl) and pyridine in dichloromethane. The measurement of oestradiol esters was carried out by GC-MS/NCI with Cool On-Column injection. Methane was used as a negative chemical ionisation reagent gas. The method for determination of oestradiol esters in blood plasma has been validated according to Commission Decision 2002/657/EC. Decision limits for all analytes were observed below 0.05 ng mL−1. The method is robust for bovine and porcine plasma analyses and can be applied both for screening and confirmatory determination in routine residue monitoring.

Effects of spray-dried bovine plasma protein in milk replacers fed at a high plane of nutrition on performance, intestinal permeability, and morbidity of Holstein calves

Spray-dried plasma protein (SDP) has been shown to improve growth and intestinal function in young calves when included in milk replacers (MR) fed at conventional rates. Use of an SDP and wheat protein blend to replace a portion of whey protein has been shown to perform similarly to using an all-whey protein control MR. However, a trend in the dairy industry is to feed calves for greater rates of growth during the preweaning period. The purpose of this study was to determine the effects of increasing amounts of SDP inclusion in MR on growth and health of calves fed at a high plane of nutrition. Young (<7 d) Holstein calves were offered starter and assigned to 1 of 5 MR treatments: an all-milk protein (whey) control MR (0SDP, n = 26) or MR containing 5% SDP (5SDP, n = 20), 7.5% SDP (7.5SDP, n = 14), 10% SDP (10SDP, n = 20), or 12% of an approximate 1:1 SDP plus wheat protein blend (PW, n = 17). All MR were formulated to contain 26% CP and 16% fat and were fed at a maximum rate of 1 kg of powder (as fed) from d 8 to 36. Amounts of MR powder were decreased by 25%/wk from d 37 to weaning at d 57. Thereafter, calves were provided only starter feed until the end of the study at d 63. On d 4, 15, 36, and 57, intestinal permeability was assessed via oral administration of lactulose and d-mannitol followed by analysis of lactulose and mannitol in blood at 60 min after administration. Increasing SDP led to a small linear decrease in MR consumed. There was a tendency for a positive linear relationship between increasing SDP and average daily gain of body weight, and SDP had mixed effects on body frame variables. Increasing SDP tended to increase fecal scores and increased the amount of fluid therapy given. Diet had no effect on intestinal permeability. Increasing SDP led to an increase in serum total cholesterol and serum urea N and tended to have a quadratic effect on serum glucose concentration on d 36. Calves fed PW tended to have increased withers height, increased intestinal permeability on d 36, and an increased likelihood of being medicated for any reason or being medicated for respiratory illness, but growth and health were not different from the control diet otherwise. Feeding PW led to an increase in serum total cholesterol and tended to lead to increased serum glucose concentration on d 36. Results of this study indicate that SDP can be included at up to 10% as fed in the MR of calves fed at a high plane of nutrition (1 kg/d of MR powder, as fed) with improvements in average daily gain. Additionally, a 1:1 SDP plus wheat protein blend can be used at 12% inclusion with no difference in most health and growth parameters.

NEONATAL INTENSIVE CARE OF 10 HOSPITALIZED GIRAFFE CALVES (GIRAFFA CAMELOPARDALIS) REQUIRING HAND-REARING

This retrospective case series describes the clinicopathologic findings, diagnoses, treatment, and outcomes of 10 hand-reared newborn giraffe (Giraffa camelopardalis) calves admitted to a university teaching hospital for intensive care. Ten calves (five males, five females; nine reticulated giraffes [Giraffa camelopardalis reticulata], one Masai giraffe [G. c. tippelskirchi]), were admitted under 2 days of age. Inadequate transfer of passive immunity was suspected in 5 of 10 calves based on assessment of serum total solids and globulin values. These calves were treated with oral frozen bovine colostrum and/or intravenous hyperimmune bovine plasma. Diarrhea occurred in 6 of 10 calves and was managed with supportive care, fecal microbiota transplantation, and limiting milk intake (offering 10% body weight [BW] in milk per day, while feeding <2 L per meal at 2- to 4-hr intervals). Less common diagnoses included pneumonia (n = 3) and mycoplasma-associated septic arthritis (n = 1). Eight calves received systemic antimicrobial therapy. Hyperlactatemia (lactate > 5 mmol/L; n = 8) and hypercreatininemia (creatinine > 2.0 mg/dl, n = 7) were the most common presenting laboratory abnormalities, which resolved with intravenous fluid therapy. All neonatal giraffes survived to discharge after a median hospitalization of 9.5 days (range, 5-37 days) and were successfully hand-reared at their place of birth. In conclusion, neonatal giraffe calves can be intensively managed in a hospital environment. Diarrhea was a common clinical problem and can be related to feeding regimens. Intravenous hyperimmune bovine plasma infusion was well tolerated to manage failure of transfer of passive immunity in calves with inadequate colostrum administration. The current study supports that compromised neonatal giraffe calves may carry an excellent prognosis after early, intensive intervention.

An efficient microfluidic device based on electromembrane extraction for the simultaneous extraction of acidic and basic drugs

The simultaneous extraction of acidic and basic compounds is considered a great challenge. In this work, an efficient and fast microfluidic device is described for the simultaneous determination of acidic and basic drugs by two electromembrane extraction, offering extraction efficiencies over 98% for all analytes in human urine samples and solving the difficulties encountered to date. The sample is submitted into the device and the collected acceptor phase is directly analyzed by diode array detector and high-pressure liquid chromatography (HPLC). The device consisted of three poly(methylmethacrylate) layers and four electrodes to perform EME in two steps in a single device. Two acidic analytes (ketoprofen and naproxen) and two basic analytes (amitriptyline and loperamide) were selected as model analytes. The device proposed works under stable electric field conditions, low current intensities that confers great stability to the supported liquid membrane. After a comprehensive study of the SLM, 1:1 2-nitrophenyl octhyl ether:dodecanol was selected as optimal. This device has also been successfully applied in 1:2 diluted bovine plasma samples with recoveries over 84% and a relative standard deviation below 6%. This microfluidic device needs small sample volumes (lower than 50 μL) and offers short extraction times (10 min) and excellent clean-up. Furthermore, it has proven to be a robust and reproducible device after more than 30 consecutive extractions, and thanks to the low potential required (5 V), it allows its compatibility with a single battery.

ITRAQ-based proteomic analysis of bovine pre-ovulatory plasma and follicular fluid

Bovine follicular fluid (FF) creates a unique microenvironment in follicles necessary for follicle growth, oocyte maturation, and estradiol (E2) production. The objective of this study was to analyze changes in proteins in FF and plasma (PL) from animals with high E2 (HE2) or low E2 (LE2) during the preovulatory period. Beef cows were synchronized, and follicular dynamics and ovulatory response were monitored using transrectal ultrasonography. Nine cows were selected and slaughtered, blood samples were collected at slaughter and FF was aspirated from dominant follicles (DF; >10 mm). Abundant proteins (albumin, IgG, IgA, and alpha-1-antitrypsin) were depleted from both PL and FF. Peptides were labeled with iTRAQ reagents and quantified using 2-dimentional liquid chromatography ESI-based mass spectrometry. Estradiol was associated with protein changes in PL and FF. Protein expression changes between FF HE2 and FF LE2 were greater than between PL HE2 and PL LE2. There were 15 up-regulated proteins and 10 down-regulated proteins in FF HE2 compared to FF LE2, and 7 proteins up-regulated and 9 proteins down-regulated in PL HE2 compared to PL LE2. Several of the differentially expressed proteins function in follicle development and were mainly categorized under cellular process and metabolic process. Pathway analysis identified the up- and down-regulated proteins were predominantly associated with the complement and coagulation cascades. The data demonstrate E2 regulates a wide range of reproductive associated proteins in bovine PL and FF and can provide the basis for further investigation of specific processes involved in such regulation.

A new procedure to prepare transparent, colourless and low-water-soluble edible films using blood plasma from slaughterhouses

Some attempts to produce films using blood plasma proteins can be found in the literature; however, due to their high solubility in water, it is usually necessary to use crosslinkers, which may entail some disadvantages. In this work, a procedure to prepare water-insoluble edible films from bovine and porcine blood plasma without using crosslinkers is described for the first time, with the objective of producing sustainable packaging materials from livestock blood. For this purpose, the blood plasma fraction was acidified and treated with ethanol to precipitate the proteins, which were solubilised in water and mixed with glycerol in order to produce a film-forming solution. The generated films were investigated to determine light absorbance, transparency, microstructure, mechanical properties and solubility at several pHs and compared with a control film prepared with untreated plasma. The new films presented in this work were completely transparent and colourless on visual inspection, in contrast to the yellowish-orange colour of the control films. Furthermore, the microstructure of these new films was more homogeneous, and therefore they showed better mechanical properties than the control film. Finally, these films were found to be highly insoluble in buffer solutions of close to neutral pH, whereas the control film was almost completely solubilised in the same buffers.

Design and Optimization of Quinazoline Derivatives: New Non-nucleoside Inhibitors of Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus (Flaviviridae). In spite of the availability of vaccines, the virus is still causing substantial financial losses to the livestock industry. In this context, the use of antiviral agents could be an alternative strategy to control and reduce viral infections. The viral RNA-dependent RNA polymerase (RdRp) is essential for the replication of the viral genome and constitutes an attractive target for the identification of antiviral compounds. In a previous work, we have identified potential molecules that dock into an allosteric binding pocket of BVDV RdRp via a structure-based virtual screening approach. One of them, N-(2-morpholinoethyl)-2-phenylquinazolin-4-amine [1, 50% effective concentration (EC50) = 9.7 ± 0.5 μM], was selected to perform different chemical modifications. Among 24 derivatives synthesized, eight of them showed considerable antiviral activity. Molecular modeling of the most active compounds showed that they bind to a pocket located in the fingers and thumb domains in BVDV RdRp, which is different from that identified for other non-nucleoside inhibitors (NNIs) such as thiosemicarbazone (TSC). We selected compound 2-[4-(2-phenylquinazolin-4-yl)piperazin-1-yl]ethanol (1.9; EC50 = 1.7 ± 0.4 μM) for further analysis. Compound 1.9 was found to inhibit the in vitro replication of TSC-resistant BVDV variants, which carry the N264D mutation in the RdRp. In addition, 1.9 presented adequate solubility in different media and a high-stability profile in murine and bovine plasma.

In Vitro Immunological Cross-Reactivity of Thai Polyvalent and Monovalent Antivenoms with Asian Viper Venoms.

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV; The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T. albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims.

PSI-7 Differing planes of nutrition affecting the cytokines of bovine seminal plasma in beef cattle

Abstract Bull fertility is highly influential to profits of cow-calf production; however, bulls often experience various planes of nutrition throughout the year. These changes may influence the composition of the ejaculate and their ability to get females pregnant. We hypothesized that differing nutritional planes would affect the cytokines concentration in the seminal plasma of bulls. Mature angus bulls (n = 12) were individually housed and randomly assigned to one of two treatments: 1) over-fed (n = 6) or 2) restricted (n = 6). Bulls were fed the same ration (35% ground hay, 35% cracked corn, 20% distillers’ grain and 10% soybean meal) at differing volumes to achieve desired nutritional planes. Body weight and BCS were taken every two weeks to monitor nutritional planes. Blood and ejaculate were collected once a month to determine cytokine profiles within seminal plasma. Statistical analyses were conducted with GLIMMIX procedure of SAS with bull within treatment as a random effect and time as a repeated measure to determine if nutrition level, time and the interaction influenced seminal plasma cytokine concentration. There were no significant time or treatment×time interactions for any cytokine concentrations. Interferon-γ tended to be increased (P &amp;lt; 0.10) in restricted bulls (3.21±0.95 pg/mL) compared with over-fed bulls (0.64±0.95 pg/mL). The concentrations of IL-8 also tended to be increased (P &amp;lt; 0.10) in restricted (318.89±66.42 pg/mL) compared with over-fed bulls (158.02±66.42 pg/mL). Interleukin-1β in over-fed bulls (24.77±17.51 pg/mL) tended to be decreased (P &amp;lt; 0.10) compared with restricted bulls (65.8±17.51 pg/mL). Interleukin-10 tended to be greater (P &amp;lt; 0.10) in restricted (17.95±2.29 pg/mL) than in the over-fed bulls (12.41±2.29 pg/mL). The concentration of VEGF-A was greater (P &amp;lt; 0.05) in restricted bulls (3,551.28±224.49 pg/mL) than over-fed bulls (2,715.72±224.49 pg/mL). In conclusion, cytokine concentrations were elevated by restricted nutritional levels which could impact the ability of the ejaculate to stimulate uterine gene expression and establish pregnancy.

PSIII-6 Effects of repeated freeze and thaw cycles on serum and plasma metabolite concentrations in beef cattle

Abstract The objective of this study was to determine whether repeated freeze and thaw cycles alter bovine circulating metabolite concentrations, which serve as indicators of nutrient utilization. In research settings, it is common to thaw and refreeze samples when confirming results or performing further analyses; thus, it is important to determine if these actions alter metabolites. Jugular blood samples were collected from beef cows (n = 14) and fall-born calves (n = 14) at weaning. Samples were processed into multiple aliquots and stored at -20°C until analysis. Samples were thawed at 4°C for 8 to 10 h on each day of analysis and refrozen for at least 48 h between cycles. Concentrations of serum non-esterified fatty acids (NEFA), serum urea N, plasma triglycerides, and plasma glucose were analyzed using commercially available kits on a UV-visible light plate reader. Data were analyzed with sample type (cow vs. calf), thaw (cycles 1-4), and their interaction in a mixed model. Additionally, R2 were determined among freeze-thaw cycles within each metabolite. Thaw cycle impacted glucose concentrations (P = 0.03); other metabolites were unaffected (P &amp;gt; 0.25). Thaw cycle and its interaction with sample type did not affect metabolite concentrations (P &amp;gt; 0.33). Sample type did not affect triglyceride and BUN concentrations (P &amp;gt; 0.34). There was a type effect (P &amp;lt; 0.0001) for NEFA and glucose; NEFA were greater in cows and glucose was greater in calves. There was a strong relationship between urea N thaw cycles 1 and 2 (R2 = 0.88), which was also apparent between all other urea N, triglycerides, NEFA, and glucose cycles (R2 ≥ 0.79, 0.96, 0.91, and 0.61 respectively). These data indicate that repetitive freezing and thawing of bovine serum and plasma samples has little effect on non-esterified fatty acids, triglycerides, and urea N, but may impact glucose concentrations.

172 Increasing inclusion of fermentable fiber decreases nutrient digestibility in grower pigs

Abstract Fermentable fiber may increase endogenous losses of P and AA, thereby reducing apparent nutrient digestibility. Acacia gum fiber with medium-to-high fermentability and low viscosity was used to investigate its effect on apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of nutrients, and standardized total tract digestibility (STTD) of P in grower pigs. A P-free basal diet (49% corn starch; 18% bovine plasma protein) was formulated to measure basal endogenous P losses (EPL). Three diets were formulated to include 2.5, 5.0, or 7.5% acacia gum fiber at the expense of corn starch in the P-free basal diet. Diets contained 16.1–17.4% CP and 0.31–0.33% total P (DM-basis). The 4 diets were fed to 8 ileal-cannulated barrows (initial BW, 54.6 kg) for four 9-d periods in a double 4 × 4 Latin square. Apparent hindgut fermentation (AHF) was ATTD minus AID. Increasing inclusion of acacia gum quadratically decreased (P &amp;lt; 0.01) AID of DM (∆ 11.1%), linearly decreased (P &amp;lt; 0.05) ATTD of DM (∆ 1.7%) CP (∆ 1.2%), and quadratically increased (P &amp;lt; 0.05) AHF of DM (∆ 9.4%). Basal EPL were 391 and 377 mg/kg DM intake (DMI) for ileum and total tract, respectively. Increasing inclusion of fiber linearly increased (P &amp;lt; 0.05) ileal EPL (∆ 184 mg/kg DMI), and tended to linearly increase (P &amp;lt; 0.10) EPL for total tract (∆ 243 mg/kg DMI). Dietary inclusion of acacia gum tended to linearly decrease (P &amp;lt; 0.10) AID of P, but did not affect (P &amp;gt; 0.10) ATTD, or STTD of P. In conclusion, increasing inclusion of fermentable, low viscous acacia gum fiber decreased diet digestibility of DM and ATTD of CP, but did not affect total tract P digestibility, indicating that increasing fermentable fiber did not increase specific endogenous losses of P in the total tract.

Bovine seminal plasma osteopontin: Structural modelling, recombinant expression and its relationship with semen quality

Osteopontin (OPN) is a multifunctional phosphoprotein that has been linked to fertility in bulls. However, the exact mechanism by which OPN contributes to fertilisation is yet unknown. The biotechnological use of OPN in bovine reproduction is promising but some gaps remain unfilled. The present work aimed: (a) to verify whether the seminal plasma OPN is associated with seminal traits and a standard breeding soundness exam; (b) to predict OPN interactions with integrins, CD44 and glycosaminoglycans through molecular docking; and (c) to develop a protocol for recombinant expression of OPN from vesicular gland cDNA. Ejaculates from top ranked bulls had higher amounts of seminal plasma OPN in comparison with bulls classified as questionable (p < .01). The structural modelling and molecular docking predictions indicated that bovine OPN binds to heparin disaccharide, hyaluronic acid and hyaluronan. In addition, docking studies described the binding complexes of OPN with CD44 and the integrin heterodimers α5β1 and αVβ3. Finally, expression of rOPN-6His was successfully obtained after 3hr of induction with 0.5mM IPTG at 37°C and a denaturing purification protocol resulted in efficiently purified recombinant OPN. The present results contribute to the development of biotechnological uses of OPN as a biomarker in bovine reproduction.

Improving the accuracy of unbound fraction measurement of drug-protein binding by preconditioning the RED membrane inserts.

Aim: The objective of this study was to evaluate the rapid equilibrium dialysis (RED) device in protein binding assays in diluted protein matrices and to improve the accuracy of the unbound fraction (fu) measurement. Methodology: Protein binding assays of drug compounds in bovine serum albumin solutions and human plasma with different folds of dilution were performed using theRED device with and without preconditioning of thedialysismembrane inserts, and the results were compared with those using other approaches in this study. Results & conclusion: Preconditioning of the RED membrane insertsimproved the fu data accuracyofdrug-protein binding assay in matriceswith relatively lowprotein contents and such impact could be compound dependent.

Relative Deficiency in Albumin Methionine Content is Associated With Decreased Antioxidant Capacity of Equine Plasma

Relative to other species, horses seem particularly susceptible to oxidative stress. Plasma albumin plays an important role in preventing oxidative damage, in part due to its methionine (MET) content. Equine albumin is highly unusual in that it contains no MET residues. Whether or not this causes deficient antioxidant capacity in equine plasma relative to that of other species has not yet been explored. The objective of this study was to compare the redox status of equine (no MET) to that of bovine (moderate amount of MET) plasma. Plasma was collected from healthy, nonpregnant Quarter Horse mares (n = 10) and adult, healthy, nonpregnant, dried Holstein cows (n = 15). Measures of total antioxidant capacity and oxidative stress were assessed for each plasma sample using multiple commercially available assays: total antioxidant capacity, thiol detection, thiobarbituric acid reactive substances, and advanced oxidation protein products. Plasma from horses had significantly (P < .05) lower thiol content and thiobarbituric and reactive substances and higher advanced oxidation protein products than plasma from cattle. A difference in total antioxidant capacity was not observed; however, our study was underpowered to establish a meaningful comparison. Based on these findings, the lack of MET in equine albumin appears to translate to a lower antioxidant capacity of equine plasma. Our findings are consistent with previous reports in other species that identify MET as having an important role in the antioxidant capacity of albumin. Our results also highlight the complex system of antioxidant defenses in plasma that counteract the harmful effects of oxidants.

Effects of feeding frequency and protein source in milk replacer for Holstein calves

Whey proteins are a primary component of milk replacers (MR) and are considered the gold standard for calves. Alternative protein sources may decrease MR cost if calf performance is similar. A blend of bovine plasma protein and modified wheat protein might be a good partial replacement for whey protein. Usually MR is fed twice daily, but feeding 3 times daily might increase efficiency of nutrient use, especially with MR containing alternate proteins. Therefore, our objective was to determine the effects of 2 MR, containing either entirely whey protein (CON) or a combination of whey protein, bovine plasma protein, and modified wheat protein (WBP), when fed in either 2 or 3 meals daily on calf growth and health. Female and male Holstein calves (n = 103) housed in individual hutches were studied for the first 63 d of life, with additional measurements obtained at wk 12 of life in group housing. The MR contained 25% CP, 17% fat, and a Lys:Met ratio of 3.1:1. Individual treatments arose from the 2 × 2 factorial arrangement of MR formulation and frequency of feeding. After colostrum, calves were fed MR (12.5% solids) at daily rates of dry matter dependent on age. Amounts were as follows: d 3 to 10 = 0.52 kg/d (2× = 0.259 kg, 3× = 0.173 kg per feeding); d 11 to 20 = 0.68 kg/d (2× = 0.341 kg, 3× = 0.227 kg per feeding); d 21 to 42 = 0.84 kg/d (2× = 0.42 kg, 3× = 0.28 kg per feeding); d 43 to 46, 47, 49, and 51 = 0.42 kg/d, with both 2× and 3× changed to 1 feeding daily and skip days (d 48 and 50) between where calves were not given MR; and d 52 = calves weaned. Starter was fed and intake was measured from d 1 until d 63. Intakes, health scores, attitude scores, and fecal scores were measured daily. Body weight (BW) and growth measurements were obtained weekly until wk 8 and again at wk 12. Blood samples were obtained at 0, 24, and 48 h and then on d 5, 14, 28, and 42 of age. Starter intake was greater for calves fed WBP versus CON during wk 7 to 9. Final BW and hip width at wk 12 were greater for calves fed WBP than for calves fed CON. Calves fed 2× had greater mean and final BW to wk 6 and greater feed efficiency (gain:feed ratio) than calves fed 3×. Blood variables supported the generally similar growth outcomes. Health outcomes did not differ between diets or feeding frequencies. Overall, calves fed WBP had increased starter intake and greater BW gains during wk 7 to 12 than calves fed CON, and calves fed 2× had increased growth and feed efficiency compared with those fed 3×.

Novel Dietary Proteins Selectively Affect Intestinal Health In Vitro after Clostridium difficile-Secreted Toxin A Exposure.

Bacterial gastroenteritis forms a burden on a global scale, both socially and economically. The Gram-positive bacterium Clostridium difficile is an inducer of gastrointestinal bacterial infections, often triggered following disruption of the microbiota by broad-spectrum antibiotics to treat other conditions. The clinical manifestatiaons, e.g., diarrhea, are driven by its toxins secretion, toxin A (TcdA) and toxin B (TcdB). Current therapies are focused on discontinuing patient medication, including antibiotics. However, relapse rates upon therapy are high (20–25%). Here, eighteen dietary proteins were evaluated for their capacity to restore gut health upon C. difficile-derived TcdA exposure. We used bioengineered intestinal tubules to assess proteins for their beneficial effects by examining the epithelial barrier, cell viability, brush-border enzyme activity, IL-6 secretion, IL-8 secretion and nitric oxide (NO) levels upon TcdA challenge. TcdA effectively disrupted the epithelial barrier, increased mitochondrial activity, but did not affect alkaline phosphatase activity, IL-6, IL-8 and NO levels. Intervention with dietary proteins did not show a protective effect on epithelial barrier integrity or mitochondrial activity. However, bovine plasma and potato protein increased alkaline phosphatase activity, egg-white protein increased IL-6 and IL-8 release and wheat, lesser mealworm and yeast protein increased NO levels after TcdA exposure. Hence, dietary proteins can influence parameters involved in intestinal physiology and immune activation suggesting that supplementation with specific dietary proteins may be of benefit during C. difficile infections.