You are likely to get less inter-animal variation if your rats were fasted.

The reason we use fasting is to eliminate some of the variability between different animals. When you leave rodents with unlimited access to food, not all of them eat at the same time or even the same amounts. If you have multiple animals in a single cage, you will often notice some differences in their weights. All of this plays into what you may see in serum levels of various measurements, especially parameters that are known to be effected by feeding status, such as the ones you are measuring.

If your experiment requires levels from animals that fed ad libitum, that's a different matter.

Dear Ozlem, yes of course, in my experience, if you do not consider fasting for the animal, you will have big difference in blood biochemical and hormonal results. The animal may have different weight, different appetite, different eating time  and so on. So if you measure feeding affecting analytes e.g. Glucose, Triglyceride, ....you will obtain a very different result in comparison to fasting state. For measuring lipid soluble analytes, e.g. determination thyroxine (T4), Tse (testosterone),...these analytes prefer chylomicrons as solvent and leave water phase to lipid phase, and you will encounter to false negative results. Finally in some exception you can blood collection without fasting but generally it is recommended blood sampling in fasting sate (without food and free access to water).

best

mehdi 

Dear Tausif Alam and Dear Mehdi Hedayati,

Thank you very much for your kind interest. Actually, rats were at individual cages during my experiment and I noted the amount of food for every day but still I know, I cannot be sure which one eats more and which one eats less before blood sampling, just I know all of them eat at least 2 hours. When all conditions were same, like this situation, for every animal still could I trust my data or not? I have done this experiment and there is no correction chance for me. In your opinion, should I repeat all the experiment or is there any other possibility?

Dear Ozlem, as you know total cholesterol does not affected by fasting or feeding state.

But Triglyceride severely depends on feeding state.

Leptin in chyl serum/plasma (turbid serum/plasma due to presence of chylomicron) will has false negative in competitive immunoassay and false positive in sandwich type immunoassay.

Finally, if the serum/plasma did not turbid, you have not to repeat the experiment for TC, HDL-C, LDL-C and Leptin.

Only TG may affected, and you can ignore its results only.

best

mehdi

Ozlem, 

You do have to consider one important thing in making your decision to repeating the experiment.  Depending on how you data are going to be used (publication, presentation in a meeting etc), would the target audience or evaluators be sufficiently convinced! Because there are routinely established set of methods, they are easily and automatically understood points of references. Values that are known to be not altered by feeding state of animals, will be considered questionable by anyone. But others that are known to change by feeding, are very likely to be questioned by reviewers, if they are meant to be compared to values reported by others in customary fasting state. Mehdi is correct that triglycerides will show the biggest variation in fasted versus fed, ad libitum, animals.

Yes, the shorted the fasting period, the higher the variability you will obtain in the measurement of metabolic analytes

Starvation time is a important criterion while measuring blood parameters. Literature review states that blood parameters are found to vary in a fed state and fasted state. Therefore, depending on the objective of your research, one can decide whether the animals need to fasted or fed. For starvation studies,usually a 6h starvation time period is best recommended. When this may not be possible, an overnight starvation is recommended. Again, care needs to be taken when many animals have to handled as there will be time difference between the first and last animal to be handled and this causes a huge variance in the data.

For wistar rats how long is the best fasting time to measure Triglycerides in serum?

The attached link to the article may clarify your question.

Article Fasting of mice: A review