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Abstract: The use of high quality gametes from both males and females during in vitro fertilization (IVF) trials is an essential step in order to achieve high fertilization and hatching rates. Although aquaculture hatcheries have focused more on egg rather than spermatozoa quality, some studies have demonstrated that sperm quantity and quality have a great influence both on fertilization/hatching success and the subsequent development of the embryo and larvae. In this study we have demonstrated that sperm/egg ratio and sperm quality are factors strongly related to each other in the pufferfish (Takifugu niphobles). Our results suggest that both factors should be taken into account as unique interrelated elements, making possible to obtain high fertilization rates using a successful combination of small amount of high quality sperm or high amount of low quality sperm. In addition, coefficients of correlation and determination among all the sperm motion parameters provided by a CASA system and fertilization/hatching rates were estimated for the first time in a marine species. Positive significant correlations were found in some parameters such as total and progressive motility (0.68 and 0.7 respectively). However, curvilinear velocity (VCL), straight line velocity (VSL) and average velocity (VAP) showed the highest coefficients of correlation (0.82, 0.8, and 0.81, respectively). In this respect, spermatozoa velocity appears to be a key factor in the fertilization process, especially when the number of spermatozoa per egg is limited in the aqueous environment. [Copyright &y& Elsevier]

Abstract: The first goal of this study was the development of a short-term storage method for pufferfish (Takifugu niphobles) sperm. In this respect, the best results were obtained by diluting the sperm in a seminal-like solution and keeping it in a Petri dish in chilled storage (4°C). This method was successful in preserving sperm quality parameters without resulting in differences in fresh sperm for a relatively long-term period (7days), for use in aquaculture matters. The addition of bovine serum albumin (BSA) to the medium did not improve the results. On the other hand, both the osmolality and the ion composition of the media are essential factors which can modulate the sperm motility parameters. The osmolality of the activating medium was the most important factor in triggering pufferfish sperm motility, and osmolalities of 750–825mOsm/kg were necessary to initiate this process, demonstrating that it appears to be a dose-independent mechanism. Regarding the ion composition of the activation media, this study has shown that despite the spermatozoa being able to initiate movement without any ion in the activation medium, the absence of ions can negatively affect the kinetic parameters of the spermatozoa. Finally, in natural conditions (seawater), the activation of sperm motility generates intracellular increases in Ca2+ and K+, suggesting that these ions play an essential role in the activation mechanism of pufferfish sperm. [Copyright &y& Elsevier]

Abstract In the past three decades the European eel Anguilla anguilla experienced up to 99% decline in recruitment in some parts of its distribution range, thus breeding in captivity is nowadays considered key in order to save this species. With this in mind, obtaining high quality gametes is fundamental, as is the ongoing study of new hormonal treatments in order to improve current methods. Therefore, the aim of this research study was i) to assess the effect of two hormonal treatments (OVI, a recombinant α-choriogonadotropin; and VET, a human chorionic gonadotropin purified from female urine) on the reproductive performance of European eel males, and, after choosing the best hormone, ii) to compare the effects of three doses in order to cut the costs of artificial maturation. Our results indicated that the type of hormone used (recombinant vs purified gonadotropins) significantly affected the progression of spermiation in European eel males, and that the recombinant hormone (OVI) produced better results in terms of sperm quantity and quality in most of the weeks of the treatment, remaining thus an effective treatment to induce spermiation in this species. On the other hand, in terms of the doses experiment, our results showed that from the lowest to the highest dose (0.25 to 1.5 IU/g fish) all the treatments were able to induce the whole spermiation process. However, a weekly dose of 1.5 IU/g fish of recombinant hormone (OVI) was necessary in order to provide a notable amount (volume and density) of high quality (motility and velocity) samples throughout the treatment. Finally, the economic analysis demonstrated that the recombinant hormone (OVI, 1.5 IU/g fish) had a greater profitability than the other treatments, making it possible to obtain high-quality sperm for a lower price. In this context, and considering the fact that in the first few weeks of any hormonal treatment there is no high-quality sperm production, long-term hormonal therapies are necessary in order to lessen the cost of high-quality European eel sperm. Highlights • The type of hormone used (recombinant vs purified) significantly affected the spermiation process in European eel males. • The recombinant hormone produced the best results both in sperm quantity and quality, and on the economic performance. • Recombinant hormones are recommended for inducing the spermiation process in this species for aquaculture purposes. [ABSTRACT FROM AUTHOR]

Abstract: Since 1960, the European eel (Anguilla anguilla) has suffered a dramatic reduction in natural stocks. Breeding in captivity is considered an alternative, but obtaining high quality sperm seems basic on this regard. The main objective of this study was to assess the effects of three thermal regimes (two of them variable: T10 and T15; and one of them constant: T20) and three hormonal treatments with different hormones (hCG, hCGrec and PSMG) on the induction of maturation in European eel males. In the case of the thermal regimes, our results demonstrated that the onset and progression of spermiation are strongly influenced, and perhaps closely regulated, by water temperature. T20 demonstrated the best results in all the sperm parameters (volume, density, motility, kinetic features, etc.) throughout most weeks of treatment, becoming a reliable and productive method for inducing spermiation in this species. In the case of hormonal treatments, the onset and progression of spermiation in European eel males were influenced by the hormone used. In this respect, hCGrec produced the best results in all the sperm parameters including volume, density, motility, kinetic features, etc., throughout most weeks of treatment, thus becoming an effective alternative treatment to the standard hCG treatment used to induce spermiation in eel species. Moreover, hCGrec gave rise to the best economical profitability, making it possible to obtain good quality sperm samples at a lower price than by using the other two hormonal treatments. [Copyright &y& Elsevier]

Abstract: The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to characterise European eel spermatozoa morphometrically, as a basis for future studies on the morphological effects of methods for sperm cryopreservation and sperm quality. This characterisation was carried out measuring several spermatozoa morphology parameters (head length, width, area and perimeter) by scanning electron microscopy (SEM), in comparison with measurements developed in European eel spermatozoa with computer-assisted morphology analysis (ASMA). Spermatozoa head morphology showed differences in width (1.15±0.01μm versus 1.12±0.01μm), perimeter (14.68±0.13μm versus 13.72±0.19μm) and area (5.36±0.06μm2 versus 1.12±0.01μm2) for ASMA and SEM, respectively. When head length was evaluated, significant differences were found, being higher for SEM methodology (5.09±0.04μm versus 4.29±0.03μm). The curved and elongated spermatozoa head in eels means a problem for the ASMA system (Sperm Class Analyser®, Morfo Version 1.1, Imagesp, Barcelona, Spain), causing an error in the length measurements. However, similar results were obtained by both techniques when spermatozoa head length was considered as the greater length between two points within the object (4.29±0.03μm versus 4.31±0.04μm for ASMA and SEM, respectively). In conclusion, this is one of the first applications of ASMA in fish and the first in this species, and confirms this system as a useful tool with wide applications in future fish spermatozoa studies. Width, perimeter and area could be used as parameters for the spermatozoa morphology evaluation, whereas the length requires a new programming of the Imagesp software. [Copyright &y& Elsevier]