First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity

Abstract

Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae ('cryolarvae') were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.

Japanese eel (Anguilla japonica Temminck & Schlegel, 1846) propagation using cryopreserved sperm samples

“…() applied modified Tanaka's extender containing 350 mmol/L glucose, 30 mmol/L Tris (pH 8.0) and methanol cryoprotectant for cryopreservation of European eel, Anguilla anguilla, sperm samples that was used for hybridization between Japanese eel and European eel, resulting in hatched hybrid larvae. Asturiano, Sørensen, Pérez, Lauesen, and Tomkiewicz () managed to acquire European eel larvae using 50 mmol/L NaCl, 100 mmol/L NaHCO 3 , 2.5 mmol/L MgCl 2 , 1 mmol/L CaCl 2 , 30 mmol/L KCl, pH 6.5, mOsm/kg ~330 as media and DMSO 10% (v/v) and 25% foetal bovine serum (w/v).…”

“…Cryopreserved sperm containing 76.2 mmol/L NaHCO 3 , 137 mmol/L NaCl, 1.4% soya lecithin and 10% dimethyl sulphoxide (DMSO) was used as the cryoprotectant. Müller et al (2012) Asturiano, Sørensen, Pérez, Lauesen, and Tomkiewicz (2016) managed to acquire European eel larvae using 50 mmol/L NaCl, 100 mmol/L NaHCO 3 , 2.5 mmol/L MgCl 2 , 1 mmol/L CaCl 2 , 30 mmol/L KCl, pH 6.5, mOsm/kg ~330 as media and DMSO 10% (v/v) and 25% foetal bovine serum (w/v).…”

Improvement of techniques for sperm evaluation and cryobanking in European eel.

“…To avoid this drawback, Peñaranda et al (2009) tested different combinations of pH and NaHCO3 concentrations, that resulted in the development of an improved medium based on the P1 medium, but containing 100 mM NaHCO3 and pH 6.5 that partially prevented the activation effect of DMSO. With this protocol, they obtained post-thaw spermatozoa motility values close to 40%, which is well sufficient for fertilization trials, that were conducted by Asturiano et al (2016). In this work, they successfully produced fertilized eggs and larvae.…”

“…However, in the case of the eel both protocols have yielded high post-thaw sperm viability (58 to 63%) and motility values ranging between 18 and 38% (Asturiano et al, 2017). Furthermore, the fertilizing capacity of the Spanish protocol (from now on referred as DMSO protocol) was successfully tested by producing European eel larvae after fertilization with thawed sperm (Asturiano et al, 2016), and following the Hungarian protocol (from now on referred as methanol protocol), hybrid larvae were successfully produced using thawed sperm from European eel and eggs from Japanese eel (Anguilla japonica) (Müller et al, 2012;. In this last study, Müller and collaborators showed that the malformation rate of larvae was higher when using cryopreserved sperm than in the control groups using fresh sperm, suggesting that the cryopreservation methodology needs further refinement.…”

“…In European eel, several researchers have developed different sperm cryopreservation protocols (Asturiano et al, 2003;Herranz-Jusdado et al, 2019a;Müller et al, 2004;Peñaranda et al, 2009;Szabó et al, 2005). Moreover, cryopreserved European eel sperm have been successfully used in fertilization trials (Asturiano et al, 2016) and in hybridization trials with Japanese eel (Anguilla japonica) eggs (Müller et al, 2012;, although an increased rate of larval deformities were observed when fertilizing with cryopreserved sperm (Müller et al, 2018), evidencing the need of refinement of the protocols. Recently, Herranz-Jusdado et al (2019a) have compared the available European eel cryopreservation protocols with the aim of choose the most efficient one and standardize its use, but this protocol still consists of small volumes of 0.5 mL, which is impractical for large-scale fertilization programs needed in e.g.…”