FATTY ACID DYNAMICS DURING INDUCED SEXUAL MATURATION IN MALE EUROPEAN EEL. RELATIONSHIP WITH STEROIDS AND SPERM QUALITY PARAMETERS
Introduction
Little is known about the role of fat and fatty acids in European eel (Anguilla anguilla) spermatogenesis. In fish, fatty acid composition greatly affects sperm quality and fertilization outcome, and temperature plays an important role in gonadal development. The aim of this work was to study the changes in the fat content and analyse quantitatively the fatty acid composition of different tissues from males hormonally-induced under three thermal regimes, looking for the relationships between fatty acids, steroids plasma levels and sperm quality parameters.
Material and methods
Male eels (n=300; mean body weight 100±2 g) were subjected to three thermal regimes (T10: 10 °C first 6 weeks, 15 °C next 3 weeks and 20 °C last 6 weeks; T15: 15 °C first 6 weeks and 20 °C last 9 weeks; and T20: constant 20 ºC). Fish were hormonally treated for the induction of maturation with weekly intraperitoneal injections of human chorionic gonadotropin (1.5 IU/g). Eight fish per treatment were sacrificed weekly during the first eight weeks and five animals per treatment were sacrificed during the last five weeks. Samples of testis, liver and muscle were kept at -80 ºC until lipid extraction and fatty acid analysis. The testis development stages (S1-S6) were established by histology. Sperm was collected by abdominal pressure and volume (using a graduate tube) and density (using a Neubauer Improved haemocytometer) were estimated. For motility parameters analysis, sperm was activated with seawater and evaluated by triplicate using a CASA software (ISAS; Proiser R+D, S.L.). Samples from blood plasma were collected to analyse the plasma concentrations of E2, DHP, T and 11KT.
Results
The eels reached spermatogenesis earlier and showed the best sperm production parameters under the treatment T20. All the thermal treatments induced similar results in the liver fatty acids during spermatogenesis, registering an increase in palmitic and eicosapentaenoic (EPA) acids when eels began to produce sperm. Regarding testis fatty acids evolution during spermatogenesis, Figure 1 shows a principal component analysis carried out in testis fatty acids. All the fatty acids located on the right on the component plot (Fig. 1A) were found in higher amounts coinciding with S2-S3 stages as shows the corresponding score plot (Fig. 1B) and decreased when the eels produced sperm (S4-S6). EPA, arachidonic (ARA) and docosahexaenoic (DHA) acids were found on the second component and maintained constant levels throughout the testis development.
In the liver, EPA decreased when the sperm volume increased. Also in the liver, the levels of polyunsaturated fatty acid (PUFA) precursors (α-linolenic and linoleic) decreased when the sperm motility increased. In the testis, ARA levels were negatively correlated with different sperm velocity parameters. Finally, when the eels were producing sperm, EPA and DHA were then positively correlated with T and 11KT.
Discussion and conclusion
The eels reached spermatogenesis earlier and showed the best sperm production parameters under the treatment T20, suggesting that eel spermatogenesis is closely regulated by water temperature. The levels of palmitic acid and EPA increased in the liver when eels began to produce sperm, suggesting de novo biosynthesis of fatty acids (being palmitic the main product, Cook and Mc Master, 2002) and, a probable subsequent mobilization to the testis, due to their important function during spermatogenesis. The maintenance of ARA and EPA levels in testis may have a physiological meaning (as prostaglandins precursors, i.e.; Asturiano et al., 2000), while the maintenance of DHA levels may have a structural one (spermatozoa membranes formation).
The consumption of ARA during the final sperm maturation phases coincided with an increase in the speed of spermatozoa, supporting not only the importance of the role of this fatty acid for fish male testis maturation (Wade et al., 1994), but especially conditioning sperm quality.
DHP, as in other teleost species, seems to be an essential element in the initiation of the early stages of spermatogenesis and our results provide substantial evidence that DHP could modulate the metabolism of lipids in the liver during early spermatogenesis. Finally, similar to results found in mammals, our results show that in the eel there are mechanisms regarding the role of EPA and DHA as modulators of androgens synthesis, particularly during the final phase of the maturation of sperm.
All this information could be useful to develop suitable broodstock diets to improve the sperm quality and subsequently, the larval development in this species.
Acknowledgements
Funded by EU (PRO-EEL, grant agreement n°245257). DSP had a grant from the COST Office (COST Action FA1205: AQUAGAMETE) to make the steroids analyses in Norway, and grant to attend the meeting.
References
Asturiano, J.F., Sorbera, L.A., Zanuy, S., Carrillo, M., 2000. Effects of polyunsaturated fatty acids and gonadotropin on prostaglandin series E production in a primary testis cell culture system for the European sea bass. Journal of Fish Biology 57, 1563-1574.
Cook, H.W., Mc Master, C.R., 2002. Fatty acid desaturation and chain elongation in eukaryotes. In: Vance, D.E, Vance, J.F. (Eds.). Biochemistry of lipids, lipoproteins and membranes: Elsevier, pp. 181-204.
Wade, M.G., Van der Kraak, G., Gerrits, M.F., Ballantyne, J.S., 1994. Release and steroidogenic actions of polyunsaturated fatty acids in the goldfish testis. Biology of Reproduction 51, 131-139.
