Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

All procedures for working with European eel described in this protocol were approved by the Committee of Ethics of Animal Experimentation at the Universitat Politècnica de València, following the Spanish laws and regulations controlling the experiments and procedures on live animals.

1. Fish Maintenance

1. Bring the European eels to a research facility and put them in a 200 L aquaria with a recirculation system. Use thermostats and coolers to maintain a constant 20 °C temperature.
2. Keep the fish in dark conditions to avoid stress²² and with no food during the experiment.
3. Keep the fish in fresh water during the first 3 days. Then change 1/3 of the water and refill with seawater every other day until reaching a salinity of 37.0 ± 0.3 g/L.

2. Hormonal Treatment

NOTE: The hormonal treatment consists of weekly injections of human chorionic gonadotropin (hCG) throughout the whole duration (nine weeks) of the experiment.

1. Prepare the hCG hormone in advance at a concentration of 1 IU/µL by diluting the hormone in saline solution (0.9% NaCl).
   NOTE: The hormone can be preserved diluted at this concentration for over a week at -18 °C.
2. To anesthetize the fish, prepare a 40 L flexible bucket with 5 L of system water.
   1. In a 250 mL flask, dilute 300 mg of benzocaine in 100 mL of 70% ethanol.
   2. Pour the diluted benzocaine in the bucket (final concentration 0.36 mM) and mix properly. This is for a final benzocaine concentration of 60 mg/L.
   3. Transfer the fish, individually, into the water with benzocaine and wait a few seconds until the benzocaine takes effect and the fish is properly anesthetized.
      NOTE: To confirm that the fish is anesthetized, place the fish in a supine position and check that it stays still in that position.
3. To administrate the hormone, weigh the fish and prepare the hCG hormone at a dose of 1.5 IU/g of fish, in a 1.5 mL plastic tube.
   1. Fill a 1 mL syringe with the hCG hormone from the plastic tube.
   2. Place the anesthetized fish in supine position and with the assistance or the syringe, inject the hormone carefully in the intraperitoneal area.
4. Return the fish to the aquaria and monitor it until fully recovered.
   NOTE: The hormonal administration has to be conducted weekly throughout the experiment. Normally, European eels start spermating after 6-7 weeks of hormonal treatment.

3. Sperm Sampling

1. To obtain the best quality samples, extract sperm 24 h after the hormonal administration^6,23.
2. Anesthetize the fish with benzocaine as described in the step 2.2.
3. Place the anesthetized fish in supine position, clean the genital area with a squirt of distilled water and dry carefully with paper, to avoid feces contamination present in the genital area or accidental sperm activation by contact with seawater from the aquaria.
4. Placing the fingers on both lateral sides of the fish, massage carefully pressing laterally from the pectoral fins to the genital area to force the sperm out.
   1. Repeat the massage until no more sperm comes out from the genital opening.
5. Collect the sperm into 15 mL plastic tubes using a vacuum pump.
6. Dilute the extracted sperm 1:10 in modified Tanaka´s extender solution²⁴ (137 mM NaCl, 76.2 mM NaHCO₃, in distilled water) at 4 ºC.
   NOTE: The sperm in the extender solution should be maintained at 4 ºC.

4. Sperm Quality Evaluation

1. Prepare artificial seawater¹³ (in mM: NaCl 354.7, MgCl₂ 52.4, CaCl₂ 9.9, Na₂SO₄ 28.2, KCl 9.4, in distilled water) with 2% bovine serum albumin (BSA) (w:v), adjust pH to 8.2, osmolality to 1100 mOsm/kg and maintain it at 4 ºC to avoid bacterial growth.
2. Open the software for computer-assisted sperm analysis (CASA) and select the fish sperm module.
   1. Click on Properties to open the system setup of the software. There, set the capture options at 60 images per second. Select negative phase optics, Makler chamber and 10X scale.
   2. Then on the analysis values, select Fish as species and Particles area bigger than 2 µm² and smaller than 20 µm².
   3. On the velocity parameters, set to slow when cells move between 10 and 45 µm/s, set to medium when moving between 45 and 100 µm/s and set to rapid when moving faster than 100 µm/s.
      NOTE: Spermatozoa with velocity slower than 10 µm/s were considered immotile.
   4. Select the progressive values as 80% of straightness (STR) and save the properties.
   5. Click on Capture Field (a window with the live images from the camera will be opened).
      NOTE: The computer-assisted sperm analysis system is formed by a microscope, a camera and an image analysis software.
3. On the counting chamber, put 4 µL of artificial seawater and add 0.5 µL of sperm solution (sperm diluted in extender solution).
   1. Focus the image with the microscope at 10X magnification in the negative phase. 15 s after activation, click on Capture – from video in the computer-assisted sperm analysis software.
   2. Analyze every sample in triplicate and select samples with a motility higher than 70% for cryopreservation.
      NOTE: It is very important to select only high-quality sperm for the success of this cryopreservation protocol.

5. Sperm Freezing Method

1. Prepare in advance liquid nitrogen (approximately 2.5 L) in a 34 cm x 34 cm x 30 cm and 5 cm thick Styrofoam box.
   NOTE: Maintain a level of liquid nitrogen of 4-5 cm height at all times.
   1. Build a floating structure to pre-freeze the sperm. Use 2 pieces of polystyrene (20 cm x 5 cm), bind them with 2 plastic tubes, and place the structure on the liquid nitrogen. This structure needs to float over the liquid nitrogen at an approximate height of 3 cm over the surface (Figure 1).

Figure 1. Schematic drawing of the floating structure used for pre-freezing over liquid nitrogen. The structure consists of two pieces of low density Styrofoam of 20 cm x 4 cm x 5 cm connected with plastic tubes of 14 cm. The straws are placed over the plastic tubes at 3 cm over the liquid nitrogen. Please click here to view a larger version of this figure.

2. Prepare the cryopreservation solution by mixing sperm, extender solution and methanol at ratio of 1:8:1 in 1.5 mL plastic tubes and stir gently.
3. With the help of a pipette, add 480 µL of the cryopreservation solution into the 500 µL straws and if necessary, mark them by closing one end with modelling clay.
4. Put the straw on the floating device at a height of 3 cm over the liquid nitrogen for 3 min. Then, place the straw into the liquid nitrogen.
5. After 10-15 min, transfer the straws into a liquid nitrogen storage tank using long forceps and keep them submerged in liquid nitrogen at all times. Here, the sperm can be stored indefinitely.

6. Thawing Method

1. Prepare a Styrofoam box with liquid nitrogen as described in step 5.1, and prepare a water bath using a 3 L beaker with tap water at 40 ºC.
2. Transfer the straws with frozen sperm from the storage tank into the Styrofoam box with liquid nitrogen using long forceps.
   1. Put each straw into a water bath at 40 ºC for 13 s.
   2. Pour the sperm into a 1.5 mL plastic tube by cutting the closed ends of the straw with scissors.
3. Analyze sperm motility using computer-assisted sperm analysis system as explained in the step 4.1.
4. Keep 100 µL of sperm to analyze the viability of spermatozoa with a flow cytometer.

7. Flow Cytometry

1. Use a fluorescent kit containing propidium iodide (PI), which is a red fluorescent compound that stains the nuclei of dead cells, and a membrane-permeant nuclear fluorescent compound, that green stains the nuclei of living cells.
   1. Prepare the green fluorescent staining solution by diluting it from the stock solution (1 mM) 1:10 in Tanaka´s medium to a working solution of 100 µM.
   2. Do not dilute the PI solution. The stock solution is at 2.4 mM.
2. For each sample, take 50 µL of fresh or thawed sperm and add 0.5 µL of green fluorescent staining working solution (final concentration 1 µM) and 2 µL of PI solution (final concentration 100 µM).
3. Incubate the samples containing the dyes (PI and green fluorescent staining) in the dark for 5 min.
   1. Dilute the samples in 500 µL of Tanaka´s extender solution and analyze with the flow cytometer.
4. Turn on the flow cytometer and create a new protocol containing at least 2 plots: SS log vs FS log and FL1 vs FL3.
   NOTE: Both, green fluorescent staining and propidium iodide can be excited with visible-wavelength light. When bound to DNA, the maximum fluorescence emission of these dyes are 516 nm and 617 nm, respectively.
   1. Adjust the voltages of the different lasers: SS= 199; FS= 199; FL1= 377; FL3= 372
   2. Set up the acquisition settings accord to maximum events = 5000 or 15 s (the final concentration of the sample should be around 1 million of cells/mL). Read the sample using a LOW flow.
   3. Select the reading mode Single tube fixed position mode.
   4. Put the sample in the right number of the carousel
   5. Read the sample (pressing F9) and save the data collected to an excel file (pressing F7) for further analysis.