Great question! Looking forward to other answers. Here is my opinion based on my experience: the most critical part in not "messing" with any parameter is speed: the shorter the time from killing to organ collection and snap freezing your samples or preparing your primary cells, the greater is the chance that parameters are as "in vivo" as possible. So killing the mouse by breaking the neck is the fastest and if performed well, will convey least stress (of course if you wrestle with your mouse for 5 min before you manage to break the neck, stress hormones will do their job on messing up some parameters you could measure). One draw back is, that blood collection post mortem isn´t really advised. So if you need blood, you need to put the mouse to sleep. CO2 messes up the blood pH, fiddles with hypoxia response, etc. Isoflurane puts the mouse to sleep, surely has some impact on some parameter, and I´d recommend that to collect blood by cardiac puncture followed by breaking the neck and organ collection.

So there is no "easy" straight answer what the best method is. It all depends on what parameters you are interested in and if they are likely to change quickly over time (signalling) or a bit more slowly (mRNA expression/protein expression) or quite slow (in my case organ iron levels).

But indeed, it´s very important to think about how to kill your "animal" as this applies to all other lab animals we use... .

Hi Christoph,

Thanks for your valuable answers.

We used to collect the blood sample through cardiac puncture. We usually estimate the parameters related to inflammation.

We usually follow the high doses of the anaesthetic agent to kill the animals after completion of experiment and before collecting the samples.

So is this procedure affects the parameters?

Christoph’s answer is almost perfect.

I would only suggest not calling the procedure “euthanasia”, since I believe that it is NOT euthanasia. You can say “life was terminated”, “life termination was achieved by”, “exitus was induced”, or similar. The expression “euthanasia” should be reserved for the acting or not acting to assure actualization of the interests of a being that will die. Our experiment and killing the animal in the end could not be interpreted as to be primarily in the interests of the animal.

I developed the entire argument which free PDF could be found here:

http://www.omicsonline.org/2155-9627/2155-9627-2-105.php

Hej Ravinder,

I´ll not be able to answer your question with the little detail you gave and even with more detail (anaesthetic, what parameters exactly, duration of your treatment and the time it takes until you have your sample in a stable way (e.g. snap frozen)) I´d only be able to give you a rough estimate. If you are indeed assuming that a parameter could be affected by your way of killing the animal, you should try a different method that is not using the same mode of killing and the fastest way to get from live-animal to stable sample. As written above, breaking the neck - if done right - is the quickest way and you´ll not have any drug-sideeffects. Before setting up such a validation experiment, you could also check literature about your anaesthetic to see which side effects it has and if anything is known about that drug and your parameters...but I guess you'll find very little :-/

I think that if you can, overdose of anesthetics should be avoided. Anesthetic may be quickly distributed in blood and tissue. The effect may be independent of whether animal still has circulation since the agent is already distributed and is acting locally.

Numbers of anesthetic agents have some effects on inflammatory response. Some responses may be quite fast (leukocyte activation, permeability of blood vessel wall, local vasodilatation, leaking vessels). See our papers, many of them are available in PDF format and free.

Yet, to examine microcirculation by intravital microscopy and blood or tissue levels of various mediators of inflammation we used the rat model of sepsis and seldom mouse. At the end of the experiment, since intravenous line was available, we used to inject about 1ml saturated KCl solution i.v. that produces immediate cardiac arrest. You probably do not have i.v. line in mice. In mice we produced i.v. catheters by heating with the lighter 100microL pipette tip and quickly pulling it apart. This method produces up to 100 micrometer diameter catheter which you can use to cannulate mouse vessels (artery). If you want a simple method, Christoph is right, neck dislocation is probably the fastest method.