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.
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´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.
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.
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
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.
3. Sperm Sampling
4. Sperm Quality Evaluation
5. Sperm Freezing Method
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.
6. Thawing Method
7. Flow Cytometry
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 ± 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 ± 3.1% (mean ± S.E.M., n=18) from fresh to frozen-thawed samples (Table 1 and Figure 2).
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 ± S.E.M. of sperm from 18 samples. Asterisks indicate significant differences between thawed and fresh samples (t-test; p<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. Please click here to view a larger version of this figure.
Fast + medium cells1 (%) | 70.7 ± 2.8 | 27.5 ± 2.1* |
Curvilinear velocity (VCL; µm/s) | 118.8 ± 2.8 | 101.5 ± 1.8* |
Straight line velocity (VSL; µm/s) | 53.3 ± 1.9 | 45.6 ± 1.3* |
Average path velocity (VAP; µm/s) | 73.3 ± 2.1 | 62.0 ± 1.3* |
Beating cross frequency (Hz) | 27.9 ± 0.3 | 27.1 ± 0.6 |
Cell viability (% of alive cells) | 97.7 ± 0.3 | 73.8 ± 2.8* |
1Cells showing curvilinear velocity above 40 µm/s. |
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 ± S.E.M. (n=18). Asterisks indicate significant differences between thawed and fresh samples (t-test; p<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 µm/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.
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.
The authors have nothing to disclose.
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.
AQUACEN BENZOCAÍNA +D2A2:D24 | AQUACEN | 3394ESP | Benzocaine |
NaCl | Avantor | 3624-19 | |
Syringe | BD Plastipak | 305501 | Syringe 1 mL |
FC500 | Beckman Coulter | Flow cytometer | |
Air pump 100 | EHEIM | 4011708370032 | Modified for suction |
Eppendorf 1.5 | Eppendorf | 175508 | Plastic tubes 1.5 mL |
Falcon tubes | Falcon | 175747 | Plastic tubes 15 mL |
Flexible bucket | Fiel | 1040101 | 40 L, Black color. |
Ethanol | Guinama SL | Mg96270 | |
Cyopreservation straws | IMV Technologies | 14550 | 500 µL straws |
Live/Dead Sperm Viability Kit | Invitrogen | L7011 | Cytometer Kit |
Modelling clay | JOVI | Art 30 | 4 different colors |
Precision scale PCB | KERN & Sohn GmbH | PCB35002 | Digital scale |
Saline solution Vitulia 0.9% | Laboratorios Ern, S.A. | C.N. 999790.8 | Saline solution |
Forceps | Levantina de Lab. SL | 3710025 | 30 cm metal forceps |
Scissors | Levantina de Lab. SL | 3700014 | Scissors 14cm |
Ovitrelle (r-hCG) | Merck SL | EMEA/H/C/000320 | Human chorionic gonadotropin |
Nikon Eclipse 80i | Nikon | Microscope | |
CaCl2 | Panreac Quimica SAU | 2112211210 | |
Na2SO4 | Panreac Quimica SAU | 3257091611 | |
NaHCO3 | Panreac Quimica SAU | 1416381210 | |
782M Camera | Proiser | 782M | Camera for CASA |
ISAS v1 | Proiser | ISAS v1 | CASA software |
SpermTrack-10 | Proiser | SpermTrack-10 | Countig chamber for CASA |
Beaker Pyrex 3L | PYREX | 2110668 | Glass beaker 3L |
Flask Pyrex 250 GL-45 | PYREX | 21801365 | Glass flask 250 mL |
KCl | Scharlab SL | PO02000500 | |
Methanol | Scharlab SL | ME03061000 | |
MgCl2 | Scharlab SL | MA00370500 | |
BSA | Sigma Aldrich | 05482 | Bovine serum albumina |
Styrofoam box | 34x34x30 cm and 5cm thick |