raymond - i stand corrected then. the method I use is CO2 overdose. I have mistakenly called it asphyxiation. but what is the difference? what is the protocol for asphyxiation if it is not a gradual increase in CO2 levels leading to death??

I would imagine that there probably are differences but anaesthesia is probably going to affect blood biochemistry too..in some way or another. However, as you say, you have internal controls. What about taking blood from a live unanaesthetised mouse via a small nick in the tail vein? contact me if you would like more details of how we do this. It depends on how much you need but I have taken 100-200ul this way.

Best way to take blood without any changes in blood biochemistry is swift decapitation

Death by CO2 inhalation will alter the pH of the blood. Additionally, stress hormones will be increased (ACTH, Cort, AVP). However, stress hormones can also increase following anesthesia. Thus, in the end it really depends on your outcome measure if the method of sacrifice makes a difference or not.

I have taken 500ul-1,5ml via brachial artery. The way I do that is after skin, muscle and brachial artery dissection, I cut it and immediatelly take the blood using a micropipette. It's clean, easier and faster to avoid coagulation, that's what I would be worry about.

Anaesthesia can take effects for sure on blood parameters, but the using of a concurrent untreated control solves this problem. Try pentobarbital anaesthesia (Euthasol, Release etc.) intraperitoneally followed by cardiac puncture and there is no need of a special anaesthetic equipment.

Another mixture of anesthesia could be Ketamine/Xilazine mixture administered intraperitoneally at a concentration of 100 mg/10 mg per Kg of body weight. Anesthesia would allow you to euthanaize the mouse by exanguination through the via you like (cardiac puncture/jugular vein/descendant aorta/ etc... Of course anesthesia would affect parameters in the blood (as other responders pointed out) but you would need to think if you could live with such modifications.

However you could also use other points of blood withdrawn as submaxilar puncture, tail vein puncture or even retroorbital extraction, although the later would be advisable to take it under anesthesia.

To avoid anesthesia you should consider the thecnique published by Golde WT added here.

Article A rapid, simple, and humane method for submandibular bleedin...

We routinely take blood by cardiac puncture after CO2 asphyxia and can get about 1 ml by that route. Peripheral venepuncture (eg tail) of concious mice generally gives a much smaller sample of around 100-200 uL, so in part this depends on what exactly you want to measure and the volumes you need. There is lots of speculation about what effects different bleeding or killing methods have, but very little objective data. I think it is hard to judge what stress responses there might be to the different procedures without such objective measures. I would also be interested to know if there are any measured differences in blood chemistry or hormone levels, or indeed any effects on tissue gene expression levels, after the CO2 asphyxia method, compared with sampling via anaesthesia or decapitation. So if you collect data on these issues then please do share it. It would be useful to lots of people I think. We have found that if you kill by CO2 asphyxia then the liver mitochondria are poisoned and completely impossible to use in any mitochondrial assays compared with death by anaesthetic overdose.

Thanks for your comments

Our only two choices really are cardiac puncture during isoflurane anaesthesia, or after CO2 asphyxiation as these are all we can do as dictated by our current IACUC approved protocol. We would like to do the experiments in the near future - probably sooner than our protocol changes could be approved!

Basically we want to do a blanket screen of every possible blood marker as we do not know what may change in our treated/untreated mice. Therefore the max amount of volume obtainable is required.

As I said, we would have internal controls, so the method of collection may not be a problem.

CO2 asphyxiation has one problem that the heart is not pumping and blood is not 'flowing". If you wait too long, cardiac puncture itself will be difficult., and you can end up with coagulated blood. An anesthesized mouse is still alive and blood is free flowing with full cardiac capacity. A better choice, in my opinion and experience.

Also, asphyxiating one mouse at a time is time consuming. If you asphyx 5 mice together, mouse 1 blood is fresher than mouse 5 because cardiac function is absent. Anesthesia is quick, keeps cardiac function, and much more humane.... ANd can be done on 5 mice together, and blood drawn one mouse at a time.

Anaesthetics or CO2 will affect blood biochemistry and haematology for sure. Using negative control and statistical analysis can solve this problem. In your case you can’t use CO2 cause cardiac arrest will take place most probably within few seconds and you can’t take blood by heart puncture method. Best way is Not to use any anaesthetics, just apply skin antiseptic as povidone/iodine near the base of the tail, allow to dray for about 30 seconds. Then make small lateral incision near the base of the tail, that will open the tail vein and venous blood will flow, which can be collected using clean sterile micropipette. Usually you can collect about 0.2 ml blood that harvest about 0.1 ml serum. I think this amount will be enough for assaying the required blood parameters. Best luck.

waleed, with respect one thing that is certainly incorrect in your answer is that it is impossible to use cardiac puncture after CO2 asphyxiation. We routinely use this approach and can get up to 1 ml of blood (0.5 to 0.6 mls of serum).

You can kill rodents by gradual increase in carbon dioxide levels. This results in build up of CO2, anesthesia then death by overdose. It is much less stressful to the animals than asphyxiation and unlike that method, avoids clotting of the blood. CO2 overdose is an acceptable method of euthanasia in the UK while asphyxiation is illegal.

I think it would be helpful to know what blood-parameters you are interested in and if you aim to get values which reflect fully awake and normal mice. My experience (mainly from rats though) is that values for blood glucose, anti-diuretic hormone and of course PCO2, pH and PO2 are highly influenced by the anesthesia-protocol employed.

raymond - i stand corrected then. the method I use is CO2 overdose. I have mistakenly called it asphyxiation. but what is the difference? what is the protocol for asphyxiation if it is not a gradual increase in CO2 levels leading to death??

Thanks John for the useful input, I am glad that you were able to get enough blood even after CO2/euthanasia.  

Are there any restrictions on the maximum blood volume that should be collected under terminal bleeding in mouse by the any of the animal welfare guidelines. Please specify especially with respect to retro orbital bleeding.