Technical FOCUS : use of fluorescent markers to evaluate restocking efficiency

Fluorescent dyes

The most widely used chemical markers for fish are fluorochromes, such as tetracycline hydrochloride, oxytetracycline, calcein or alizarin (red S or complexone). These vital markers link specifically onto alkaline-earth metals such as calcium, and create a fluorescent compound which emits in a specific wavelength (calcein 530 nm ; tetracycline 560 nm ; alizarin red S 580 nm) when excited and examined through a fluorescence microscope. These compounds are metabolized in bony tissues, which in fish are found in the internal skeleton, fins and scales. SCIMABIO offers experimental designs using these different markers in projects dealing with the evaluation of restocking efficiency. We use mainly alizarin red S for otolith markings and calcein when fish cannot be sacrificed, as fluorescence is visible externally in fins.

Alizarin marking

This marking protocol was described in 2006 by Caudron and Champigneulle (see article). The fish are bathed in an alizarin red S solution for several hours, which ensures a permanent mark visible in the otoliths. Detection takes place under a fluorescence microscope equipped with an alizarin filter.


Marking can be realized at different stages in the life cycle in order to induce different marks in the otoliths and thus separate different stocking practices (see articles on the arctic char and trout). Thus marking at the fingerling stage produces a small circular mark, close to the core. Later markings produce a fluorescent ring further from the center (see focus picture). This method can be relied upon since marked specimens have been kept in fisheries for several years and can still show the otolith mark in 100% of cases. Moreover, the samples (heads) can be frozen, and professional as well as recreational fishermen can contribute to the gathering of these samples.

Calcein marking

The major drawback of otolith marking is the necessity to sacrifice the fish. In some situations, this is not a problem (sampling by fishermen), but when monitoring rare or threatened populations, a non-lethal method must be chosen. In these cases, we propose calcein marking.

Calcein is a fluorescent molecule, which combines naturally to the calcium found in the host, on the skeleton, scales and fin rays. It offers the convenience of being detected on live fish.

Calcein marking is a two-step process:

  • A preliminary step, called ‘osmotic shock’, consisting in a saline solution (NaCl) bath.This step, innocuous for trout, induces an osmotic potential, and heightens absorption of the marking solution.
  • The next step is a concentrated calcein bath (n° CAS 1461-15-0).

Detection takes place in the dark with a US patented portable device (SE-MARK detector ; Western Chemical Company). The head and fin rays are the most fluorescent tissues.

PROCEDURE FOR CALCEIN MARKING ON TROUT FINGERLINGSmarquage calcéine de truites et observation de la fluorescence

This method, developed in the last ten years in U.S. fisheries, has recently been tested in a natural environment. A recent study on trout in Swiss streams has shown the possible use of calcein on monitoring individuals for a year. Beyond that timeframe, the marker disappears with solar radiation, and fluorescence retention varies according to shading rate (see article).

SCIMABIO supports you and your projects

Depending on the management issue, we can assist you on several levels :

  • Protocol conception,
  • Marking operations,
  • Conception of sampling kits,
  • Otolith analysis or direct observation of fin rays,
  • Results analysis.

Don’t wait… Contact us.