Effect of MS222 on Hemostasis in Zebrafish

Deebani, Afnan; Iyer, Neha; Raman, Revathi; Jagadeeswaran, Pudur

doi:10.30802/aalas-jaalas-18-000069

Cited by 19 publications

(17 citation statements)

References 19 publications

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“…By contrast, gilthead sea bream ( Sparus aurata ) showed significantly increased cortisol levels following the exposure to even 25 mg/L MS-222 or 0.075 mg/L 2-phenoxyethanol ( 48 ). In zebrafish, 15 mg/L of buffered MS-222 altered various hematological characters, including hematocrit, coagulation, and the amount of blood collected ( 49 ). The influence of a given anesthetic on the biochemical profile of a sample obviously depends on the dose and on the treated species.…”

Section: Anesthesia and Blood Sampling Proceduresmentioning

confidence: 99%

Blood Will Tell: What Hematological Analyses Can Reveal About Fish Welfare

2021

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Blood analyses provide substantial information about the physiological aspects of animal welfare assessment, including the activation status of the neuroendocrine and immune system, acute and long-term impacts due to adverse husbandry conditions, potential diseases, and genetic predispositions. However, fish blood is still not routinely analyzed in research or aquaculture for the assessment of health and/or welfare. Over the years, the investigative techniques have evolved from antibody-based or PCR-based single-parameter analyses to now include transcriptomic, metabolomic, and proteomic approaches and from hematological observations to fluorescence-activated blood cell sorting in high-throughput modes. The range of testing techniques established for blood is now broader than for any other biogenic test material. Evaluation of the particular characteristics of fish blood, such as its cell composition, the nucleation of distinct blood cells, or the multiple isoforms of certain immune factors, requires adapted protocols and careful attention to the experimental designs and interpretation of the data. Analyses of fish blood can provide an integrated picture of the endocrine, immunological, reproductive, and genetic functions under defined environmental conditions and treatments. Therefore, the scarcity of high-throughput approaches using fish blood as a test material for fish physiology studies is surprising. This review summarizes the wide range of techniques that allow monitoring of informative fish blood parameters that are modulated by different stressors, conditions, and/or treatments. We provide a compact overview of several simple plasma tests and of multiparametric analyses of fish blood, and we discuss their potential use in the assessment of fish welfare and pathologies.

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Section: Anesthesia and Blood Sampling Proceduresmentioning

confidence: 99%

Blood Will Tell: What Hematological Analyses Can Reveal About Fish Welfare

2021

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“…The top 1 μl of clear citrated plasma was pipetted out without disturbing the red cell layer and used for kinetic coagulation assays [ 7 ]. After blood collection, the fish was immediately euthanized by MS222 overdose [ 8 ].…”

Section: Methodsmentioning

confidence: 99%

Microkinetic coagulation assays for human and zebrafish plasma

Iyer

Jagadeeswaran²

2021

Blood Coagulation &Amp; Fibrinolysis

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Coagulation assays, prothrombin time (PT), and partial thromboplastin time (PTT) are tests to measure the clotting ability of plasma and used in evaluating patients suffering from bleeding disorders. These assays require 100 μl of human plasma. In zebrafish, dilute plasma with exogenously added human fibrinogen was used. Our objective is to create a microkinetic coagulation assay for human and zebrafish plasmas using 1 μl plasma under conditions similar to PT and PTTs. Here, we developed an assay using the Take3 plate with wells holding up to 6 μl, which can be loaded in a microplate reader for measuring the absorbance of fibrin formation. In this assay, we used 1 μl of citrated zebrafish or human plasma followed by the addition of either thromboplastin or Dade ACTIN or factor X activator from Russell viper venom as an activating agent and CaCl 2 . We found 4 or 3 μl of the final volume of reaction was optimal. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established curves using dilute plasma. This kinetic coagulation was inhibited by heparin and was reduced significantly in coagulation factor deficient plasmas. These results validated our microkinetic coagulation assays. Moreover, we derived clotting times from these kinetic curves, which were identical to human PT, PTT, and Russel's viper venom time. In conclusion, we established a microkinetic assay that could measure blood coagulation activity in models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.

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“…On a clean paper towel, zebrafish was placed on its lateral side, head covered with a wet Kimwipe, and the skin surface was gently wiped with Kimwipe. After making a lateral incision between the ventral and the dorsal fins and in between second and fourth black stripes from the dorsal end, blood was collected by using a pipette tip and was used in further experiments 25 , 26 . The above procedure was approved by the Institutional Animal Care and Use Committee of the University of North Texas, and animal experiments were performed in compliance with the institutional guidelines.…”

Section: Methodsmentioning

confidence: 99%

RNaseH-mediated simultaneous piggyback knockdown of multiple genes in adult zebrafish

Raman

Ryon

Jagadeeswaran

2020

Sci Rep

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We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders.

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Effect of MS222 on Hemostasis in Zebrafish

Cited by 19 publications

References 19 publications

Blood Will Tell: What Hematological Analyses Can Reveal About Fish Welfare

Blood Will Tell: What Hematological Analyses Can Reveal About Fish Welfare

Microkinetic coagulation assays for human and zebrafish plasma

RNaseH-mediated simultaneous piggyback knockdown of multiple genes in adult zebrafish

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