Testing cryopreserved European eel sperm for hybridization (A. japonica × A. anguilla)

Müller, Tamás; Matsubara, Hideaki; Kubara, Yuki; Horváth, Ákos; Kolics, Balázs; Taller, János; Stéger, Viktor; Kovács, Balázs; Horváth, László; Asturiano, Juan F.; Peñaranda, David S.; Urbányi, Béla

doi:10.1016/j.theriogenology.2018.02.021

Cited by 9 publications

(5 citation statements)

References 45 publications

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“…Although DMSO is the most used cryoprotectant for fish sperm (Gallego & Asturiano, 2018b;Martínez-Páramo et al, 2017), methanol has also been widely used in freshwater species such as sturgeons, salmonids, tench or Eurasian perch within others (reviewed by Asturiano et al, 2017). Furthermore, it has been recently used in cryopreservation protocols for Japanese eel sperm (Koh et al, 2017;Müller et al, 2017Müller et al, , 2018. As cryoprotectant, methanol has been reported to penetrate more rapidly the cells and being less toxic than DMSO (Horváth et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

Comparison of European eel sperm cryopreservation protocols with standardization as a target

et al. 2019

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The critical situation of the European eel (Anguilla anguilla) has urged the development of sperm cryopreservation protocols for reproduction in captivity and cryobanking. In the last years, two research groups have developed their own protocols in Spain and Hungary with positive results, but difficult to compare. Here, a series of experiments were conducted to test the quality of thawed sperm after using both protocols, determining which of them produce the best results and aiming for standardization. The quality of thawed sperm was assessed by studying the motility and kinetic values of thawed sperm from both cryopreservation protocols using a computerassisted sperm analysis (CASA-Mot) system. In addition, a viability analysis was performed using flow cytometry to test if the cryoprotectants or the freezing-thawing process led to a reduction in spermatozoa survival. Furthermore, since during cryopreservation the sperm was treated with methylated cryoprotectants (DMSO or methanol) that may induce epigenetic changes in the sperm DNA (cytosine methylation) and could affect the offspring, we conducted a luminometric methylation assay (LUMA) to study the DNA methylation levels induced by both protocols. In this work, all the above-mentioned parameters were analyzed in fresh and frozen-thawed sperm samples. Our results showed that thawed sperm samples from both protocols presented lower sperm motility and velocity, and lower percentage of live cells than those shown in fresh sperm samples. Furthermore, sperm samples from the methanol based protocol showed significantly higher motility, velocity and percentage of live spermatozoa than the same sperm samples treated with the DMSO based protocol. In addition, the DMSO based protocol induced a hypomethylation of sperm DNA compared to fresh samples whereas the methanol based protocol did not alter sperm DNA methylation level. Our results indicate that the methanol based protocol is a more suitable protocol that preserves better the motility and genetic qualities of the European eel sperm.

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Section: Discussionmentioning

confidence: 99%

Comparison of European eel sperm cryopreservation protocols with standardization as a target

et al. 2019

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“…In European eel, several researchers have developed different sperm cryopreservation protocols (Asturiano et al, 2004(Asturiano et al, , 2003Herranz-Jusdado et al, 2018a;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(Müller et al, , 2018, 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 (2018a) 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.…”

Section: Introductionmentioning

confidence: 99%

European eel sperm storage: Optimization of short-term protocols and cryopreservation of large volumes

et al. 2019

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Maturation in captivity of European eel (Anguilla anguilla) requires long and costly hormonal treatments that often lead to asynchronic maturation between sexes. Therefore, optimization of sperm short-term storage methods and cryopreservation protocols can be a key factor for successful artificial fertilization. Two experiments were carried out to optimize the existing protocols.For the short-term storage experiment, sperm was diluted in P1 extender and then stored at different dilution ratios (1:9 and 1:49). The best outcome was then tested at different temperatures (4 and 20 ºC) and in constant agitation or still. In the cryopreservation experiments, large sperm volumes (cryotubes of 2 and 5 ml), different cooling rates (freezing tubes 1 or 3 cm above liquid nitrogen during 15 and 20 minutes), and different extender compositions (methanol 10% was used as cryoprotectant, and complemented with FBS 20%, BSA 5% or egg yolk 5%) were tested. Sperm kinetic parameters were analyzed with a CASA-Mot system both in fresh and short-or long-term stored samples.In the short-term storage trial, sperm quality did not show significant differences in the first 24 h after sperm collection between the different storage conditions tested. For longer time, 1:49 dilution ratio showed significantly better results than 1:9, and low temperature (4 ºC) was better for sperm preservation after 3 days.Cryopreserved sperm samples showed good motility results when they were frozen in cryotubes of 2 and 5 ml, with no significant differences compared to samples cryopreserved in lower volumes (straws of 0.5 mL). Furthermore, the combination of methanol (10%) and egg yolk (5%) as freezing medium, induced significant higher post-thawing motility values (over 50%) than the control (methanol 10%), whereas the addition of FBS (20%) and BSA (5%) led to a significant reduction of the sperm motility. The establishment of these storage and cryopreservation protocols will be important for the improvement of European eel artificial reproduction programs.

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“…Despite the low percentage of fertilized eggs, lower than that observed with fresh sperm, embryos developed and a few larvae from cryopreserved sperm were obtained at 55 h after fertilization. Similarly, Müller et al [39,56] successfully used cryopreserved European eel sperm in fertilization trials, but in this case, they used Japanese eel eggs and successfully obtained hybrid larvae of A. japonica x A. anguilla. The sperm used in this fertilization trial was cryopreserved following a protocol based on those described by Müller et al [34] and Szabó et al [35], using a modified Tanaka solution [25] as extender and methanol 10% as cryoprotectant (Table 1), obtaining progressive motility results in the thawed sperm samples of 12.3 ± 10.87%.…”

Section: European Eel Sperm Cryopreservationmentioning

confidence: 99%

Eel sperm cryopreservation: An overview

et al. 2019

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The eels are teleost fishes from the order Anguilliformes that includes several species with high commercial value. Due to the high interest for aquaculture production of some eel species and for the need to restore eel species that are endangered, several research groups have directed their research toward developing protocols to cryopreserve the spermatozoa of Japanese eel (Anguilla japonica) and European eel (Anguilla anguilla).In this review, we provide an overview on the different protocols that have been developed so far. The first developed protocols used DMSO as cryoprotectant in both species with good success, obtaining sperm motilities of over 45% in Japanese eel and over 35% in European eel. Moreover, sperm cryopreserved using DMSO was successfully used in fertilization trials, although with low fertilization rates. However, recent studies show that DMSO produce epigenetic changes in eel sperm and therefore, the last developed protocols used methanol as cryoprotectant instead. Cryopreservation protocols using methanol as cryoprotectant, showed improved motility values in both Japanese and European eel. In addition, the latest protocols have been adapted to cryopreserve larger volumes of sperm of up to 5 mL, which is useful for larger scale fertilization trials.The present study introduces the state of the art and future perspectives of the eel sperm cryopreservation to be applied in aquaculture and biological conservation programs.

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Testing cryopreserved European eel sperm for hybridization (A. japonica × A. anguilla)

Cited by 9 publications

References 45 publications

Comparison of European eel sperm cryopreservation protocols with standardization as a target

Comparison of European eel sperm cryopreservation protocols with standardization as a target

European eel sperm storage: Optimization of short-term protocols and cryopreservation of large volumes

Eel sperm cryopreservation: An overview

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