I think you had better test Sudan black staining for this section by which you can detect lipid accumulation in hepatocytes. Based on images, the pathological changes in this section are likely related to the accumulation of lipid droplets.

I agree with Niloofar.

The changes look like lipid accumulations.

Thank you for your reply. Niloofar Sadeghi

Unfortunately I only have fixed tissue and no fresh tissue to do the frozen sectioning. Any idea whether there is another method to check for lipids or whether this is often seen in Grouper?

You can detect intracellular lipid content even in paraffin-embedded tissues. Maybe this paper can help you with this protocol.

Thank you Niloofar Sadeghi . This is really helpful

Hi Caroline

These images are suggestive of glycogen overload. Usually lipid accumulation is observed as cytoplasmic vacuoles (correspond to fat droplets, removed during histologic technique), and we don't see vacuolar change. However, massive lipid and glycogen overload can lead to microscopic images similar to these (but usually there are always in the slide some small to large vacuoles).

If you already performed a PAS stain and role out glycogen overload (are you sure that the stain was correctly performed?? you used a positive control as intestine???), if you also role out lipids, then you must think that you have excessive accumulation of water in cytoplasm and organelles (deregulation of Na/K pump).

Usually we perform Sudan black stain or Oil red stain in frozen tissue.

An organ with severe lipid accumulation may float in the fixative, and have a yellowish coloration and greasy appearance - did you see the organs macroscopically???( in glycogen and water overload the organ is not greasy and have a paler coloration)

Regarding the granules you see, it seems protein debris, may be protein inclusions. If it was a mammal it could be α1-Antitrypsin deficiency, among other alterations, but your images are not from a mammal...it has nucleated erythrocytes.

Are severe swelling of hepatocytes in normal? - they are not normal

whatever the species observed (avian, reptile or mammal).

Is this liver of wild grouper or from farm?Usually all fish from farms have a fat liver.

Fernanda Seixas Travassos Thank you for you reply.

Yes, we had controls for the PAS stain and the controls indicated PAS positive mucin. We are currently repeating the staining with PAS to again confirm the absence of glycogen. We were also able to confirm the absence of excessive accumulations of lipid droplets with a mix of oil red and sudan black...

I think we are seeing excessive water accumulations as you suggested.

The hepatocyte swelling is a problem we saw throughout all the experimental grouper, with the assumption that they are experiencing excessive glycogen accumulations...

a1-Antitrypsin deficiency appears to be a rather rare genetically determined disorder. The fact that all our fish appeared to have these swelling at different degrees make me assume that it is not the a1-Antitrypsin deficiency but maybe a disorder that might have similar underlying mechanisms. I will look a bit more into this and thank you for your time Fernanda Seixas Travassos

If you have any other suggestions I would gladly hear them!