Introduction
The European eel (Anguilla anguilla) is a highly valued species targeted for aquaculture production . However, i t is considered a critically endangered species by CITES , and reproduction in captivity seems to be the only realistic alternative to solve both economic and ecological problems (Asturiano, 2020). I t is possible to obtain gametes for artificial fertilization but it requires hormonal treatment , which is a time- consuming and expensive process (Gallego et al., 2012). Moreover , the variable response to these treatments results in high mortality rates during the early stages of development, which could be caused (at least in part ) by low sperm quality and uncontrolled epigenetic factors (Herráez et al., 2017) . Considering the environmental conditions experienced during eels oceanic migration, the effects of low temperature combined with hormonal treatment have been used to better understand the onset o f eel maturation (Peñaranda et al., 2016). Thus , the present study considers the use of low temperature as a pre-treatment before starting a standard hormonal treatment and evaluates its impact on eel maturation and male reproductive performance.
Material and Methods
Eighty-eight male European eels were maintained for 3-4 days in freshwater at 20 ºC . Then, 6 males were sacrificed , as initial control, and the rest were changed to seawater at 10 ºC , except the control group, which was changed to seawater but maintained at 20 ºC. Low water temperature pre-treatments were applied during 1, 2 or 4 weeks to groups of 20 males. Once finished each pre-treatment, 8 fish per group were sacrificed for sampling. The temperature of the water of the remaining males was increased to 20 ºC in 3-4 days and they started receiving standard hormonal treatment ( weekly administration of recombinant chorionic human gonadotropin , rhCG ; Ovitrelle, Spain, 1.5 IU/g fish) . From sacrificed fish (controls and pre-treated) biometric parameters were measured. Blood and testis samples were used to perform steroid (T, 11- KT and E2) analyses and histology, respectively. Once eels were spermiating , sperm volume , motility and kinetic parameters were evaluated weekly using a CASA system.
Results
The eye , fin and hepatosomatic indexes from 1-week, 2- and 4-weeks pre-treated fish were significantly higher than those of the control group. The gonadosomatic indexes registered in the control group were unusually high and significantly differed with 1-week pre-treatment group but not with the 2- and 4-weeks groups. Androgen levels (T, 11- KT) were significantly higher after 4-week s of pre-treatment in comparison with the control. The E2 levels did not show the same increasing profile. The control and 1-week pre-treated males showed significantly higher percentages of undifferentiated and differentiated type A spermatogonia in comparison with early spermatogonia type B . The males after 2- and 4-weeks of pre-treatment revealed a significantly increased proportion of differentiated spermatogonia type A. After the rhCG treatment, males from the 4-weeks pre-treatment group started to produce sperm earlier than males from the other groups. The s perm volume in the 4-weeks pre-treatment group increased during the first weeks of spermiation and in the control group at the end of the experiment, but no significant differences were found. Sperm motility and kinetics parameters were low in pre-treated groups throughout the whole experiment while in the control group increased significantly at the end of the hormonal treatment.
Discussion
The onset of maturation in the European eel is controlled by environmental conditions found during their migration. The application of pre-treatments can be important to induce maturation in aquaculture eels, which do not encounter these conditions (Palstra and van den Thillart , 2009). Several factors such as temperature, salinity, forced swimming and pressure have been used as part of pre-treatments applied to eels (reviewed by Asturiano et al., 2020). Rozenfeld et al. (2019) evaluated the effects of cold seawater in European eel maturation without hormonal treatment . In eels treated at 10 ºC, the T and 11-KT levels were higher and there was an increase in the proliferation of differentiated type A spermatogonia . Our results also corroborate that low- temperature pre-treatment (4- weeks at 10 °C) induces an early sexual development, thus leading to the release of androgens which promote the proliferation of type A spermatogonia . However, the low sperm quality found in the pre-treated groups revealed that thermal pre-treatment followed by rhCG treatment at high temperatures impairs spermatogenesis. Low-temperature pre-treatments induce testis cell proliferation and synchronization but inhibit further maturation. Peñaranda et al. (2016) suggested that low seawater temperature inhibits the gene expression of enzymes responsible for the synthesis of maturation-inducing steroids (MIS) during eels oceanic migration. Thus, the premature maturation of the testis is prevented before eels arrive to the spawning areas and a temperature threshold is re ached allowing the change of the steroidogenic pathway to change from androgens to MIS . In our experiment, low- temperature pre-treated males had a time-dependent and fast response to rhCG treatment ( in comparison with control males maintained at 20 °C), but their spermiation period was shorter and the ir sperm motility parameters were significantly lower. If this is evidencing a lower rate of gene expression in the testis, and thus limiting the final sperm maturation process, is an open question that requires further research.
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