Name: handling and treatment of male european eels (anguilla anguilla) for hormonal maturation and sperm cryopreservation
Uploaded: 2018-01-28T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation at JoVE.com

This publication was funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), the COST Office (COST Action FA 1205, AQUAGAMETE), and the Research Centre of Excellence - 1476-4/2016/FEKUT.

All procedures for working with European eel described in this protocol were approved by the Committee of Ethics of Animal Experimentation at the Universitat Polit&#232;cnica de Val&#232;ncia, following the Spanish laws and regulations controlling the experiments and procedures on live animals.
1. Fish Maintenance
<ol>
	<li>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 &#176;C temperature.</li>
	<li>Keep the fish in dark conditions to avoid stress22 and with no food during the experiment.</li>
	<li>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 &#177; 0.3 g/L.</li>
</ol>
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.
<ol>
	<li>Prepare the hCG hormone in advance at a concentration of 1 IU/&#181;L 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 &#176;C.</li>
	<li>To anesthetize the fish, prepare a 40 L flexible bucket with 5 L of system water.
	<ol>
		<li>In a 250 mL flask, dilute 300 mg of benzocaine in 100 mL of 70% ethanol.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Fill a 1 mL syringe with the hCG hormone from the plastic tube.</li>
		<li>Place the anesthetized fish in supine position and with the assistance or the syringe, inject the hormone carefully in the intraperitoneal area.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3. Sperm Sampling
<ol>
	<li>To obtain the best quality samples, extract sperm 24 h after the hormonal administration6,23.</li>
	<li>Anesthetize the fish with benzocaine as described in the step 2.2.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Repeat the massage until no more sperm comes out from the genital opening.</li>
	</ol>
	</li>
	<li>Collect the sperm into 15 mL plastic tubes using a vacuum pump.</li>
	<li>Dilute the extracted sperm 1:10 in modified Tanaka&#180;s extender solution24 (137 mM NaCl, 76.2 mM NaHCO3, in distilled water) at 4 &#186;C. 
	NOTE: The sperm in the extender solution should be maintained at 4 &#186;C.</li>
</ol>
4. Sperm Quality Evaluation
<ol>
	<li>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 &#186;C to avoid bacterial growth.</li>
	<li>Open the software for computer-assisted sperm analysis (CASA) and select the fish sperm module.
	<ol>
		<li>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.</li>
		<li>Then on the analysis values, select Fish as species and Particles area bigger than 2 &#181;m2 and smaller than 20 &#181;m2.</li>
		<li>On the velocity parameters, set to slow when cells move between 10 and 45 &#181;m/s, set to medium when moving between 45 and 100 &#181;m/s and set to rapid when moving faster than 100 &#181;m/s. 
		NOTE: Spermatozoa with velocity slower than 10 &#181;m/s were considered immotile.</li>
		<li>Select the progressive values as 80% of straightness (STR) and save the properties.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>On the counting chamber, put 4 &#181;L of artificial seawater and add 0.5 &#181;L of sperm solution (sperm diluted in extender solution).
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Sperm Freezing Method
<ol>
	<li>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.
	<ol>
		<li>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).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56835/56835fig1.jpg" /> 
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. <a href="//cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56835/56835fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>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.</li>
	<li>With the help of a pipette, add 480 &#181;L of the cryopreservation solution into the 500 &#181;L straws and if necessary, mark them by closing one end with modelling clay.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Thawing Method
<ol>
	<li>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 &#186;C.</li>
	<li>Transfer the straws with frozen sperm from the storage tank into the Styrofoam box with liquid nitrogen using long forceps.
	<ol>
		<li>Put each straw into a water bath at 40 &#186;C for 13 s.</li>
		<li>Pour the sperm into a 1.5 mL plastic tube by cutting the closed ends of the straw with scissors.</li>
	</ol>
	</li>
	<li>Analyze sperm motility using computer-assisted sperm analysis system as explained in the step 4.1.</li>
	<li>Keep 100 &#181;L of sperm to analyze the viability of spermatozoa with a flow cytometer.</li>
</ol>
7. Flow Cytometry
<ol>
	<li>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.
	<ol>
		<li>Prepare the green fluorescent staining solution by diluting it from the stock solution (1 mM) 1:10 in Tanaka&#180;s medium to a working solution of 100 &#181;M.</li>
		<li>Do not dilute the PI solution. The stock solution is at 2.4 mM.</li>
	</ol>
	</li>
	<li>For each sample, take 50 &#181;L of fresh or thawed sperm and add 0.5 &#181;L of green fluorescent staining working solution (final concentration 1 &#181;M) and 2 &#181;L of PI solution (final concentration 100 &#181;M).</li>
	<li>Incubate the samples containing the dyes (PI and green fluorescent staining) in the dark for 5 min.
	<ol>
		<li>Dilute the samples in 500 &#181;L of Tanaka&#180;s extender solution and analyze with the flow cytometer.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Adjust the voltages of the different lasers: SS= 199; FS= 199; FL1= 377; FL3= 372</li>
		<li>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.</li>
		<li>Select the reading mode Single tube fixed position mode.</li>
		<li>Put the sample in the right number of the carousel</li>
		<li>Read the sample (pressing F9) and save the data collected to an excel file (pressing F7) for further analysis.</li>
	</ol>
	</li>
</ol>

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Int. 16 (6), 561-572 (2008).</li><li>M&#252;ller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Japanese+eel+(Anguilla+japonica+Temminck+&amp;+Schlegel,+1846)+propagation+using+cryopreserved+sperm+samples.">Japanese eel (Anguilla japonica Temminck &amp; Schlegel, 1846) propagation using cryopreserved sperm samples.</a> J. Appl. Ichthyol. 33 (3), 550-552 (2017).</li></ol>

The authors declare that they have no competing financial interests.

This protocol describes the complete process for European eel maturation, handling and sperm cryopreservation. The husbandry conditions described here are optimal for fast maturation and production of high volumes of high-quality sperm in this species 6,7,25. The success of this cryopreservation protocol and its potential use for fertilization after thawing depend greatly on the quality of fresh sperm 26. Therefore, the selection of high-quality sperm is of great importance. Note that subjective sperm quality evaluation depends on the skills, perception and training of the researcher who evaluates the samples 5,27,28 and can lead to very different quality estimations depending on the researcher 29. Therefore, the use of computer-assisted sperm analysis is highly recommended to select the best quality samples for cryopreservation.
Results of the post-thawing sperm quality presented here showed a reduction in the parameters of motility, velocity, and cell survival. This is consistent with the available bibliography, even though there exist great variation between species 26,30. For instance, in a study with Atlantic salmon (Salmo salar), fresh sperm with a motility of 70 - 95% was frozen using different cryoprotectants (DMSO and methanol). The sperm motility after thawing was significantly lower than in fresh samples, with motility values in the best protocol of 8.2%, yet fertilization rate using thawed sperm was as high as 42.8%, which represented 95% of the control (fresh sperm) 31. In a different study with the sperm of Atlantic halibut (Hippoglossus hippoglossus), no significant reduction in sperm motility was found after cryopreservation and fertilization rate using thawed sperm was over 95% 32.
In European eel, previous studies also showed a reduction in sperm motility after cryopreservation independently of the cryoprotectant used 16,18. In addition, cryopreserved sperm from European eel of similar quality has been used successfully for fertilization 8. In that study, Asturiano et al. used DMSO as the cryoprotectant. The use of DMSO has some manipulation disadvantages since this cryoprotectant activates the sperm and therefore needs to be frozen immediately upon addition of DMSO. Also, insemination with thawed sperm needs to be conducted immediately after thawing. The decrease of the sperm pH can partially solve this problem 19, but the protocol is more delicate than the protocol presented here with methanol as cryoprotectant.
Several studies have shown positive results using methanol as the cryoprotectant. For instance, in a study conducted with Japanese eel (Anguilla japonica) using a very similar protocol to the one presented here, with 10% methanol as cryoprotectant, the authors successfully fertilized eggs using fresh and cryopreserved sperm. In this study, they obtained fertilization rates of 17% with no significant differences between fresh and cryopreserved sperm 33.
The protocol presented here has proved to preserve sufficient sperm quality after thawing, and show similar sperm characteristics after thawing than protocols using different extenders and cryoprotectants, but with the advantages of easy handling pre- and post-cryopreservation. It is very important to follow accurately the cooling and thawing times described here as well as using high-quality sperm. In future studies, fertilization trials will be tested using this protocol.

Over the last 25 years, the number of European eels (Anguilla anguilla) arriving at the European coast have decreased steadily by 90% 1,2,3. There are several factors that explain this drastic drop including pollution, infections, overfishing and habitat destruction. All of this has had a profound effect on this species, leading to the inclusion of the European eel on the International Union for Conservation of Nature (IUCN) list as critical endangered 4. Consequently, the development of techniques and protocols for reproduction in captivity are necessary.
The maturation of the European eel in captivity is acheived by hormonal treatment 5,6,7 but the production of gametes in both sexes is difficult to synchronize 8. Even though the development of new androgen implants has shown to accelerate oogenesis in eels 9,10, the timing of final maturation in females is still highly variable and difficult to control 11. Therefore, short-term storage of sperm 12,13,14 and cryopreservation techniques are necessary for reproduction management, making gamete synchronization unnecessary 8.
Cryopreservation of European eel sperm has been developed since 2003 15,16. Several researchers designed successful protocols using either dimethyl sulfoxide (DMSO) or methanol as cryoprotectants 16,17,18,19,20. Although both protocols have been successfully used, the obtained cell viability of thawed sperm cryopreserved with DMSO is lower than with methanol 20,21. Moreover, eel sperm is activated on contact with DMSO and requires more tedious sperm manipulation 19, therefore methanol is a more suitable cryoprotectant for European eel sperm than DMSO.
Here, the protocol for optimal handling and hormonal treatment of the European eel will be described below. In addition to this, the best European eel sperm cryopreservation protocol using methanol as a cryoprotectant and a protocol for the assessment of sperm quality in this species will also be described.

<table><tbody><tr><td>AQUACEN BENZOCA&amp;Iacute;NA +D2A2:D24</td><td>AQUACEN</td><td>3394ESP</td><td>Benzocaine</td></tr><tr><td>NaCl</td><td>Avantor&amp;nbsp;</td><td>3624-19</td><td></td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>305501</td><td>Syringe 1 mL</td></tr><tr><td>FC500</td><td>Beckman Coulter</td><td></td><td>Flow cytometer</td></tr><tr><td>Air pump 100</td><td>EHEIM</td><td>4011708370032</td><td>Modified for suction</td></tr><tr><td>Eppendorf 1.5</td><td>Eppendorf</td><td>175508</td><td>Plastic tubes 1.5 mL</td></tr><tr><td>Falcon tubes</td><td>Falcon</td><td>175747</td><td>Plastic tubes 15 mL</td></tr><tr><td>Flexible bucket</td><td>Fiel</td><td>1040101</td><td>40 L, Black color.</td></tr><tr><td>Ethanol</td><td>Guinama SL</td><td>Mg96270</td><td></td></tr><tr><td>Cyopreservation straws&amp;nbsp;</td><td>IMV Technologies</td><td>14550</td><td>500 &amp;micro;L straws</td></tr><tr><td>Live/Dead Sperm Viability Kit</td><td>Invitrogen</td><td>L7011</td><td>Cytometer Kit</td></tr><tr><td>Modelling clay</td><td>JOVI</td><td>Art 30</td><td>4 different colors</td></tr><tr><td>Precision scale PCB</td><td>KERN &amp;amp; Sohn GmbH</td><td>PCB35002</td><td>Digital scale</td></tr><tr><td>Saline solution Vitulia 0.9%</td><td>Laboratorios Ern, S.A.</td><td>C.N. 999790.8</td><td>Saline solution</td></tr><tr><td>Forceps</td><td>Levantina de Lab. SL</td><td>3710025</td><td>30 cm metal forceps</td></tr><tr><td>Scissors</td><td>Levantina de Lab. SL</td><td>3700014</td><td>Scissors 14cm</td></tr><tr><td>Ovitrelle (r-hCG)</td><td>Merck SL</td><td>EMEA/H/C/000320</td><td>Human chorionic gonadotropin</td></tr><tr><td>Nikon Eclipse 80i</td><td>Nikon</td><td></td><td>Microscope</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>2112211210</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>3257091611</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>1416381210</td><td></td></tr><tr><td>782M Camera</td><td>Proiser</td><td>782M</td><td>Camera for CASA</td></tr><tr><td>ISAS v1</td><td>Proiser</td><td>ISAS v1</td><td>CASA software</td></tr><tr><td>SpermTrack-10</td><td>Proiser</td><td>SpermTrack-10</td><td>Countig chamber for CASA</td></tr><tr><td>Beaker Pyrex 3L</td><td>PYREX</td><td>2110668</td><td>Glass beaker 3L</td></tr><tr><td>Flask Pyrex 250 GL-45</td><td>PYREX</td><td>21801365</td><td>Glass flask 250 mL</td></tr><tr><td>KCl</td><td>Scharlab SL</td><td>PO02000500</td><td></td></tr><tr><td>Methanol</td><td>Scharlab SL</td><td>ME03061000</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Scharlab SL</td><td>MA00370500</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>05482</td><td>Bovine serum albumina</td></tr><tr><td>Styrofoam box</td><td></td><td></td><td>34x34x30 cm and 5cm thick</td></tr></tbody></table>

handling and treatment of male european eels (anguilla anguilla) for hormonal maturation and sperm cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Sperm from 18 eels with a sperm motility of 70% or higher, was selected for this study. The results showed a reduction in all quality parameters after thawing compared to those from fresh sperm (Table 1 and Figure 2). The motility results (mean &#177; S.E.M., n=18) showed a higher total motility and a progressive motility in fresh sperm than the total motility and progressive motility found in the post-thaw sperm samples.
The same pattern was found in the analysis of fast sperm cells, where frozen-thawed samples presented a lower ratio of fast cells than fresh samples. In addition, the sperm cell velocities measured were also reduced in the after thawing samples.
Also, results showed that cell viability after thawing presented a reduction in live sperm cells of 23 &#177; 3.1% (mean &#177; S.E.M., n=18) from fresh to frozen-thawed samples (Table 1 and Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56835/56835fig2.jpg" /> 
Figure 2. Motility, curvilinear velocity and cell viability data of fresh sperm and thawed sperm (after cryopreservation). The sperm was cryopreserved for 24 h before being thawed. The values presented are means &#177; S.E.M. of sperm from 18 samples. Asterisks indicate significant differences between thawed and fresh samples (t-test; p&#60;0.05). The parameter motility indicated the percentage of total motile spermatozoa, curvilinear velocity indicated the average velocity of the spermatozoa along a curvilinear trajectory, and cell viability indicated the percentage of alive spermatozoa. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56835/56835fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fast + medium cells1 (%)</td>
			<td>70.7 &#177; 2.8</td>
			<td>27.5 &#177; 2.1*</td>
		</tr>
		<tr>
			<td>Curvilinear velocity (VCL; &#181;m/s)</td>
			<td>118.8 &#177; 2.8</td>
			<td>101.5 &#177; 1.8*</td>
		</tr>
		<tr>
			<td>Straight line velocity (VSL; &#181;m/s)</td>
			<td>53.3 &#177; 1.9</td>
			<td>45.6 &#177; 1.3*</td>
		</tr>
		<tr>
			<td>Average path velocity (VAP; &#181;m/s)</td>
			<td>73.3 &#177; 2.1&#160;</td>
			<td>62.0 &#177; 1.3*</td>
		</tr>
		<tr>
			<td>Beating cross frequency (Hz)</td>
			<td>27.9 &#177; 0.3</td>
			<td>27.1 &#177; 0.6</td>
		</tr>
		<tr>
			<td>Cell viability (% of alive cells)</td>
			<td>97.7 &#177; 0.3</td>
			<td>73.8 &#177; 2.8*</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">1Cells showing curvilinear velocity above 40 &#181;m/s.</td>
		</tr>
	</tbody>
</table>
Table 1. Summary of results of the different parameters analyzed with computer-assisted sperm analysis system from fresh and thawed samples. Thawed samples were previously cryopreserved for 24 h. All values presented as mean &#177; S.E.M. (n=18). Asterisks indicate significant differences between thawed and fresh samples (t-test; p&#60;0.05). The analyzed parameters were: motile spermatozoa defined as the percentage of total motile cells; progressive motility defined as percentage of spermatozoa that swim forward in an essentially straight line; fast and medium cells defined as percentage of spermatozoa with an average curvilinear velocity above 40 &#181;m/s; curvilinear velocity defined as average velocity of a spermatozoon through its curvilinear trajectory; straight line velocity defined as average velocity of a spermatozoon measured from the first detected position to its last position in a straight line; average path velocity defined as average velocity of a spermatozoon along its spatial average trajectory; beating cross frequency defined as the average rate at which the curvilinear sperm head trajectory crosses its average path trajectory; cell viability defined as percentage of alive spermatozoa.

Watch this Scientific Journal Video about Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation at JoVE.com

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

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

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

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

handling-treatment-male-european-eels-anguilla-anguilla-for-hormonal

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.8K Views.  Universitat Politècnica de València. Protocollen voor Europese aal rijping en sperma cryopreservatie hebben verbeterd in de afgelopen jaar. Dit artikel beschrijft het beste protocol beschikbaar met behulp van humaan choriongonadotrofine (hCG) voor inducerende rijping en methanol als cryoprotectant.

Video: Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Environment

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology has been identified as a way to obtain more detailed information on movement and behavior across a broader spatial scale than is possible with other known technology. The purpose of this method is to provide a detailed step by step instruction on tag implantation for both lampreys and eel. For laboratory studies using actively migrating juvenile Pacific Lamprey (120-160 mm), we determined that the presence of the tag did not alter the swimming ability between tagged and untagged Individuals or have any significant tag loss (&lt;3%). Similar results were determined during laboratory testing of Yellow phase American Eels (113-175 mm). No mortality occurred during a 38-day holding period and there was minimal tag loss (3.8%). The presence of the tag did not have any significant effect on the swimming ability or survival of tagged eels compared to untagged controls and there was minimal tag loss.

This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Biology

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka is also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and, thus, reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including teleost fish. Collecting sperm is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article, therefore, describes two methods of sperm collection in the small model fish, Japanese medaka: testes dissection and abdominal massage. This paper demonstrates that both approaches are feasible for medaka and shows that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. This article also describes a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). These procedures, specified for this useful small teleost model, will greatly enhance understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes

This article describes two quick and efficient methods for collecting sperm from the small model fish medaka (Oryzias latipes), as well as a protocol for reliably assessing sperm quality using computer-assisted sperm analysis (CASA).

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology ...

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