Development of a method to assess binding of astaxanthin to Atlantic salmon Salmo salar L. muscle proteins

Several methods were examined to characterize the binding between astaxanthin and salmon muscle protein(s) in order to provide tools for evaluation of the role of muscle proteins on astaxanthin retention in Atlantic salmon Salmo salar L. flesh. The methods included gel filtration chromatography, displacement of a hydrophobic probe and ultrafiltration. With gel filtration chromatography, aggregation of astaxanthin under the experimental conditions was a major problem for the separation of bound astaxanthin from free astaxanthin because the apparent molecular weight of aggregated astaxanthin or astaxanthin micelles was in the range of protein–astaxanthin complexes. Displacement of the fluorescent probe 8‐anilino‐1‐naphthalenesulphonate (ANS) was not effective as astaxanthin quenched the fluorophore so that displacement could not be observed. An ultrafiltration method was developed using 200‐mM sodium cholate for dispersion of astaxanthin aggregates. This allowed unbound astaxanthin to be separated from bound astaxanthin using a 30‐kDa filter. After salmon muscle proteins were solubilized in different fractions by sequential extraction using low ionic strength solutions, the astaxanthin binding of different fractions was assessed using the ultrafiltration method. The significant difference (P<0.05) observed in the astaxanthin binding of the various fractions suggests an application of this assay to detect differences in affinity of proteins for astaxanthin. The results also suggest that proteins other than actomyosin or actin can bind astaxanthin in Atlantic salmon flesh. This method can be used for the identification of astaxanthin‐binding proteins in salmon flesh and other tissues.     

A cannulation technique for urine collection from haddock, Melanogrammus aeglefinus L.

Urine collection from fish is an integral part of metabolic studies designed to measure the excretion of various biochemical compounds. The techniques developed for urine collection in salmonids cannot be applied to gadoid fish due to the anatomical differences in their urinary system. The anterior ureter of haddock (Melanogrammus aeglefinus L.) is an elongated duct that originates from the anterior part of the trunk kidney and courses dorsal‐posterior to the region of the uropore. The posterior ureter originates from the middle of the caudal kidney, and eventually fuses with the anterior ureter before forming a small urinary bladder. After the two ureters join together, a short common duct empties into the urinary bladder and terminates at the uropore behind the anus. Just before the end, the terminal posterior ureter takes a sharp U‐shaped turn, which makes cannulation difficult. We investigated the development of a cannulation technique for urine collection in three different‐sized groups of juvenile haddock. In anaesthetized fish, a catheter was inserted in the uropore in a posterior direction to follow the U‐shaped course of the ureter. After insertion of the catheter into the uropore, the externalized segment of tubing was connected to a needle attached to a syringe. Urine was then aspirated from each of the 20 fish at 15 μL min^?1 for 5 min to measure urine volume and urinary phosphate concentration. The results were reproducible and this cannulation technique has potential for use in other gadoid metabolic studies.     

Optimum dietary ratio of digestible protein and energy for juvenile American eel, Anguilla rostrata, fed practical diets

Triplicate groups of juvenile American eel, Anguilla rostrata, initial weight 8.2 ± 0.24 g, were fed to satiation herring meal based diets formulated with digestible protein/digestible energy (DP/DE) ratios of 19, 20, 21, 22 and 23 g DP MJ DE^?1 (as‐fed basis) for 84 days. Data were collected to determine the effect of dietary DP/DE ratio on feed intake (FI), mean weight (MW), specific growth rate (SGR), feed conversion ratio (FCR), apparent digestibility (AD) of major nutrients, rate of phosphate excretion (RPE) and nutrient retention efficiency (RE). Highest MW, SGR and lowest FCR (P < 0.05) were achieved by feeding 22 g DP MJ DE^?1 with values (mean ± SE) of 22.9 ± 0.07 g fish^?1, 1.23 ± 0.033% day^?1 and 0.91 ± 0.075 g feed g gain^?1, respectively. With exception of lipid, digestibility of all nutrients were the same (P > 0.05) with mean AD coefficients for organic matter, protein, energy and phosphorous of 86.3, 94.1, 89.2 and 34.7%, respectively. Lipid AD was significantly higher (P < 0.05) when DP/DE ratio was 21, 22 or 23 g DP MJ DE^?1 at 92.3% as opposed to when DP/DE ratio was 19 or 20 g DP MJ DE^?1 at 90.3%. The DP/DE ratio had no significant effect (P > 0.05) on RPE and it averaged 0.05 ± 0.002 g phosphate kg fish^?1 day^?1. Nitrogen retention efficiency (NRE) significantly (P < 0.05) increased as DP/DE ratio increased to 21 g DP MJ DE^?1 and was similar thereafter (P > 0.05) at an average of 31.6 ± 0.67%. Energy retention efficiency (ERE) significantly (P < 0.05) increased to 42.9 ± 1.24% as DP/DE ratio increased to 22 g DP MJ DE^?1 and thereafter significantly (P < 0.05) decreased. Lipid retention efficiency (LRE) increased significantly (P < 0.05) to 75.7 ± 0.85% as dietary DP/DE ratio increased to 23 g DP MJ DE^?1. Non‐linear quadratic regression of ERE against dietary DP/DE ratio yielded an estimated optimum DP/DE ratio for juvenile American eel of 22.1 g DP MJ DE^?1.     

Cardiovascular effects of a continuous rate infusion of lidocaine in calves anesthetized with xylazine,midazolam, ketamine and isoflurane

ObjectiveTo assess the cardiovascular changes of a continuous rate infusion of lidocaine in calves anesthetized with xylazine, midazolam, ketamine and isoflurane during mechanical ventilation.Study designProspective, randomized, cross-over, experimental trial.AnimalsA total of eight, healthy, male Holstein calves, aged 10 ± 1 months and weighing 114 ± 11 kg were included in the study.MethodsCalves were administered xylazine followed by ketamine and midazolam, orotracheal intubation and maintenance on isoflurane (1.3%) using mechanical ventilation. Forty minutes after induction, lidocaine (2 mg kg^?1 bolus) or an equivalent volume of saline (0.9%) was administered IV followed by a continuous rate infusion (100 μg kg^?1 minute^?1) of lidocaine (treatment L) or saline (treatment C). Heart rate (HR), systolic, diastolic and mean arterial pressures (SAP, DAP and MAP), central venous pressure (CVP), mean pulmonary arterial pressure (mPAP), pulmonary arterial occlusion pressure (PAOP), cardiac output, end-tidal carbon dioxide (Pe’CO₂) and core temperature (CT) were recorded before lidocaine or saline administration (Baseline) and at 20-minute intervals (T20-T80). Plasma concentrations of lidocaine were measured in treatment L.ResultsThe HR was significantly lower in treatment L compared with treatment C. There was no difference between the treatments with regards to SAP, DAP, MAP and SVRI. CI was significantly lower at T60 in treatment L when compared with treatment C. PAOP and CVP increased significantly at all times compared with Baseline in treatment L. There was no significant difference between times within each treatment and between treatments with regards to other measured variables. Plasma concentrations of lidocaine ranged from 1.85 to 2.06 μg mL^?1 during the CRI.Conclusion and clinical relevanceAt the studied rate, lidocaine causes a decrease in heart rate which is unlikely to be of clinical significance in healthy animals, but could be a concern in compromised animals.     

Effect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro Maturation

In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.     

Short communication: Progressive motility of frozen–thawed canine semen is highest five minutes after thawing

Progressive motility is usually estimated by visual inspection using a light contrast microscope at X 100 immediately after semen collection or immediately after thawing frozen semen. Standard operating procedures have never been established for this test. The objective of this experiment was to examine time‐dependent changes of motility after thawing cryopreserved canine semen. Semen of 35 dogs was collected, and volume, concentration, progressive motility, morphology, membrane integrity and HOS test were evaluated. For cryopreservation, CaniPRO^® Freeze A&B was used. Semen was thawed and diluted using CaniPRO^® culture medium. After thawing, semen was evaluated as before. In addition, every sample was evaluated for progressively motile sperm cells 0, 5, 20 and 60 min after thawing. Progressive semen motility was significantly highest five minutes after thawing.     

RESEARCH PAPER: Comparison between two methods for cardiac output measurement in propofol‐anesthetized dogs: thermodilution and Doppler

ObjectiveTo compare cardiac output (CO) measured by Doppler echocardiography and thermodilution techniques in spontaneously breathing dogs during continuous infusion of propofol. To do so, CO was obtained using the thermodilution method (CO_TD) and Doppler evaluation of pulmonary flow (CO_DP) and aortic flow (CO_DA).Study designProspective cohort study.AnimalsEight adult dogs weighing 8.3 ± 2.0 kg.MethodsPropofol was used for induction (7.5 ± 1.9 mg kg^?1 IV) followed by a continuous rate infusion at 0.7 mg kg^?1 minute^?1. The animals were positioned in left lateral recumbency on an echocardiography table that allowed for positioning of the transducer at the 3rd and 5th intercostal spaces of the left hemithorax for Doppler evaluation of pulmonary and aortic valves, respectively. CO_DP and CO_DA were calculated from pulmonary and aortic velocity spectra, respectively. A pulmonary artery catheter was inserted via the jugular vein and positioned inside the lumen of the pulmonary artery in order to evaluate CO_TD. The first measurement of CO_TD, CO_DP and CO_DA was performed 30 minutes after beginning continuous infusion (T0) and then at 15‐minute intervals (T15, T30, T45 and T60). Numeric data were submitted to two‐way anova for repeated measurements, Pearson’s correlation coefficient and Bland &; Altman analysis. Data are presented as mean ± SD.ResultsAt T0, CO_TD was lower than CO_DA. CO_DA was higher than CO_TD and CO_DP at T30, T45 and T60. The difference between the CO_TD and CO_DP, when all data were included, was ?0.04 ± 0.22 L minute^?1 and Pearson’s correlation coefficient (r) was 0.86. The difference between the CO_TD and CO_DA was ?0.87 ± 0.54 L minute^?1 and r = 0.69. For CO_TD and CO_DP, the difference was ?0.82 ± 0.59 L minute^?1 and r = 0.61.ConclusionDoppler evaluation of pulmonary flow was a clinically acceptable method for assessing the CO in propofol‐anesthetized dogs.