Keywords

FormalPara Introduction

Cannulation of animals is a needed method when aiming at administrating a long-term treatment or searching how some blood parameters are changing throughout a given period of time, usually for 24 h or some days at the very most. Working with aquatic animals, the objective is of the utmost importance as the method avoids any multiple handling possibly accompanied with deleterious and unexpected consequences on the physiology of the animal.

In the late 1980s and early 1990s, a time where sturgeon investigations were being developed in some western countries, namely, France, new methodologies were promoted, one of them being the cannulation which was achieved by the two authors of the present chapter. The objectives were to document: (a) the stress due to ammonia intoxication (Salin 1992), (b) the stress due to a hypoxia (Nonnotte et al. 1993, Maxime et al. 1995), (c) the impact of spawning procedures on both stress indicators and reproduction potential during the final steps of reproduction (Williot 1997; Williot et al. 2011), (d) the effects of spawning procedures on steroid profiles during the final steps of reproduction (Williot 1997; Chap. 17) and (e) the reference content for all blood parameters (Williot 1997; Williot et al. 2011).

The present cannulation methodology was kindly transmitted to UC Davis (USA) and applied rapidly by Belanger et al. (2001) and further by Lankford et al. (2003).

In the meantime, on the one hand, a similar technique was applied to explore the effects of hypercapnia on blood of the White sturgeon (Acipenser transmontanus) (Crocker and Cech 1998), and on the other hand a cannulation of the dorsal aorta through the roof of the mouth was implanted in Adriatic sturgeon (Acipenser naccarii) (Di Marco et al. 1999).

The objective of the present chapter is to give a detailed description of the process set up by the authors allowing one to apply the methodology in further physiological investigations on sturgeon.

1 Needed Material

1.1 Fish and Holding Tanks

For fish weighing in the range of 7–9 kg (Williot et al. 2011), 2 m diameter tanks (Fig. 24.1) were used to hold one spawner per tank. In fact, the procedure can be applied to a wider range of fish weight. Water depth was maintained around maximum 50 cm to allow checking the fish and blood sampling very easily. Water supply was oxygen-saturated via an aerated-enriched column placed vertically on the periphery of the tank. The outlet is in central position and represented by a grid.

Fig. 24.1
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A 2 m diameter tank for holding the fish

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1.2 Cannulation

A V-shaped table equipped with grown moss and supplying water through a small pipe to be inserted in the mouth of the fish is needed (Fig. 24.2). A disinfectant (usually an iodine solution called betadine) is used to wash the skin area as well as the tools to be inserted (syringe and cannula).

Fig. 24.2
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A V-shaped table with fish with a small pipe in its mouth allows the fish breathing

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The Tuohy syringe (or trocar) (No 15 or 17) (Figs. 24.3, 24.4, 24.5 and 24.6) allows positioning the cannula into the vasculature. The cannula is inserted into the Tuohy syringe. A cannula (n°3404; internal–external diameters are 0.76–1.22 mm) is composed of a 2.5-m-long polyethylene capillary (Biotrol, France) (Fig. 24.7).

Fig. 24.3
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A complete Tuohy syringe

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Fig. 24.4
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The two parts of a Tuohy syringe

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Fig. 24.5
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A magnified view of the lower part of the Tuohy syringe

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Fig. 24.6
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Another magnified view of the lower part of the Tuohy syringe. It gives a good indication on the angle that has to be respected in inserting the Tuohy syringe into the fish

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Fig. 24.7
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A still packed cannula

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Scalpel, needle, current syringe, scissors and suture material complete the needed surgical equipment. The cannula is implanted behind the anal fin, as shown Fig. 24.8.

Fig. 24.8
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Ventral view of a cannulated fish. 1. Upper white arrow, coming out of the cannula; 2. lower red arrows, three subcutaneous bridges

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The needed remaining materials are (a) a physiological solution (Table 24.1), (b) a lighter to close up the external extremity of the cannula, (c) a piece of Styrofoam (as a float) (Fig. 24.9) and (d) an elastic band.

Table 24.1 Physiological solutions used to fill up the cannula
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Fig. 24.9
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View of a cannulated fish with the cannula (white arrow) and Styrofoam float (black arrow) allowing easy, secure and non-stressful blood sample

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2 Chronology of the Process

  1. (a)

    Anaesthetise the fish (for about 5mn in a bath containing clove oil (emulsified in ethanol; 1:10, clove oil/ethanol) added to water at a concentration of 40 ppm) (Williot et al. 2011).

  2. (b)

    Set up the fish ventral side-up on V-shaped table with a continuous supply of oxygenated water in the mouth (Fig. 24.2) according to the method described by Doroshov et al. (1983).

  3. (c)

    Prepare one of the ends of the cannula with a bevelled in order to suit the end of the Tuohy syringe and smooth things over. Drive it into the Tuohy syringe.

  4. (d)

    Fill up the cannula with the physiological solution.

  5. (e)

    Disinfect the small area behind the genital papilla.

  6. (f)

    Place the Tuohy syringe with a correct angle in the vertical symmetry plan of the fish, and then sink it very carefully into the musculature up to the blood which is seen entering the capillary when the aorta is reached.

  7. (g)

    Sink the capillary for about 10 cm into the vasculature and then take off the syringe.

  8. (h)

    Push the physiological solution inside the cannula until blood is no more visible in the cannula with a current syringe.

  9. (i)

    Close the cannula with a flame with a lighter or with a cap.

  10. (j)

    Perform the three subcutaneous bridges with the scalpel and then the needle.

  11. (k)

    Thread the cannula through the three subcutaneous bridges.

  12. (l)

    Make three cross-stitches in between the bridges.

  13. (m)

    Fix the end of cannula onto the Styrofoam piece with an elastic band.

  14. (n)

    The equipped fish is ready to be put back into its tank.

This operation lasted 20–25 min for experienced people.

3 Effectiveness

The present methodology of sturgeon’s cannulation proved to be extremely useful, especially in studying the effects of stressors. The most delicate step is to place the cannula properly into the caudal vasculature. In the majority of the cases, the cannula was in function for up to 3 days. In one experiment, despite the care in attaching the cannula, two females out of 16 lost their cannula, i.e. 12% (Williot et al. 2011). Despite the cannulas, ovulated eggs were currently collected, and fish were again blood sampled afterwards. Further, no one fish died which means that the cannula methodology is safety.

It is worth mentioning that this procedure could be applied for longer time (i.e. 1 week) without neither haemorrhage nor infection. Finally, the method could be also applied to the dorsal vein.