Blood sampling is a regular practice that is applied for experimental research as well as for diagnostic purposes. In line with the 3R principles, the obvious advantage of this non-lethal approach highlights the importance of developing methods based on small quantities of blood which can be obtained repetitively, making it possible to follow up individual fish.

Although the procedure can be performed quickly the fish should be properly anesthetised for the procedure (Fig. 1). To prevent damaging the skin surface and to facilitate handling a V shaped stand covered with a plastic bag suffice to hold the fish in the right position is useful for caudal bleeding (Fig.  2).

Blood can be also sampled from the ventricle, the dorsal aorta or approaching the caudal vein from the side just below the lateral line. When fish are to be terminally sampled any approach could be valid, however, for non lethal sampling and particularly for repeat bleeding, our experience has shown that damage is minimal when a sample is taken from the caudal vessel at the peduncle region and approached ventrally (Figs. 3 and 4). The internal anatomy of the muscle blocks in the area reduces the post-sampling bleeding, a very desirable feature if fish are to be put back into the water. Additionally, the healing process does not involve scaring the fillet portion of the skeletal muscle, something  very important for farmed fish at harvest.

Sampling for histological examination

The main focus of this manual is the necropsy technique, although when additional information is included this is to assist with a better understanding of functional morphology and identification of abnormality. We did not intend to cover the sampling procedures for different specific or complementary analysis. However, during the description of the technique we have provided tips highlighting where particular sampling procedures should be performed and admit our bias toward “protecting” the tissues for histological examination. This is because of their relative vulnerability as a sample compared to those required for molecular or virology diagnostics for instance. If the post processing of the sample requires maceration, then a bit of “roughness” at sampling might not make a difference and the main objective will be to prevent contamination (virology, bacteriology or molecular diagnostics). For histological assessment, the integrity of organs and tissue structure itself is a paramount factor, similar to the asepsis and sterility would be for bacteriology for example.

We have selected some images to illustrate a general sampling procedure when histology samples are taken. The order of sample collection reflects that applied during experimental infections, where usually some tissues and organs need to be shared for different purposes (histology, molecular and virology, for example).

Always perform the sampling as quick after death as possible. Independent of the fish size, tissue samples should not exceed a max thickness of 1cm (although it can be a long strip of 1cm) to allow the fixative to penetrate adequately within the first few hours. There are several options for fixatives but we are assuming a 10% neutral buffered formalin. Maintain a ratio of 10 (up to 50) parts of fixative to 1 part of tissue in a wide mouthed and leak proof container. The later prevents compressing the tissues and the high ratio prevents tissues from being squashed, guaranteeing good fixation. When sampling a lesion do not take the affected portion only, but make a cut to include a margin of (normal) surrounding tissue. Always include a sample of non-affected tissue.