Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurora spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqpla and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl(2), the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl(2)(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified. (C) 2011 Elsevier Inc. All rights reserved.

In the present study, sexual gonadal differentiation and first sexual maturation of Meagre (Argyrosomus regius) was studied, based upon the annual changes in gonadosomatic index (GSI), gonadal histology, and the plasma steroid hormones, testosterone (T), 11-ketotestosterone (11-KT), and estradiol (E2). In addition, spermatozoa characteristics were evaluated by measuring sperm motility and morphology. Results demonstrated that Meagre completes sex differentiation at 10 to 12 mo of age, and are group-synchronous spawners, which reach puberty at 2 (mean length 26.8 0.7 cm, mean weight 920 75 g; N 10) and 3 (mean length 35.8 0.8 cm, mean weight 1610 89 g; N 10) years of age for males and females, respectively. In males, during the sex differentiation period, T levels were significantly higher with respect to those of 11-KT; this suggests that T has a key role in the early phases of the sex differentiation. During the spawning season an increase in plasma concentrations of all hormones was observed with 11-KT levels being significantly higher that those of T. In females, during the sex differentiation period, there was an increase in E2 plasma levels, while during the first spawning season, a significant increase of T and E2 levels were measured. Regarding sperm characteristics, the measured curvilinear velocity (VCL) and straight-linear velocity (VSL), resulted in the same order of magnitude with respect to those measured in other marine fish, while the average path velocity (VAP) was similar to that measured in the European Eel. The head of Meagre spermatozoa presents as oval shaped with a surface area of approximately 3.66 m2 and a perimeter of approximately 6.65 m. All these findings represent an important basis for further investigation on the reproductive biology of this specie and may assist the farmers to improve seed production in aquaculture.

Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) toreduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to the procedures of fish sperm cryopreservation

The long-term goal of this research project is to set up efficient protocol that can be used to develop a standardized approach for vitrification of marine fish spermatozoa. In particular, the aim of the present study was to develop a vitrification protocol for sea bream (Sparus aurata) spermatozoa. To draw up the protocol, we tested two different dilution media (1% NaCl and Mounib medium), three different vitrification devices (loops, drops and cut straws), different cryoprotectants (CPs) and three different equilibration times (30, 60 and 120 s). The effect of the different vitrification procedures on spermatozoa quality was checked by measuring spermatozoa motility rate and viability, mitochondrial membrane potential and the fertilizing ability of both fresh and post-thawed gametes. The best result was obtained by dropping directly into liquid nitrogen 20 ml of spermatozoa suspension (drop-wise method) diluted with Mounib buffer containing 10% Me2SO þ 10% glycerol. The addition of a mixture of anti-freezing proteins, AFPI and AFPIII, to Mounib buffer significantly increases the spermatozoa quality following vitrification so confirming the usefulness of AFPs in improving the quality of gametes subjected to the vitrification process. The present study proves that vitrification offers an alternative to conventional sperm cryopreservation also in this species.

Fish sperm cryopreservation is considered as a valuable technique for artificial reproduction and genetic improvement (Chao & Liao, 2001; Kopeika et al., 2007; Rana, 1995; Suquet et al., 2000). Semen quality must be monitored when attempts are made to increase the efficiency of artificial fertilization, to cryopreserve only sperm of high quality, and to evaluate frozen-thawed sperm. Cryopreserved sperm usually shows, with respect to fresh sperm, a lower quality, since the freezing–thawing procedure affects DNA and protein integrity (Labbe et al., 2001; Zilli et al., 2003, 2005), membrane lipids (Maldjian et al., 2005; Müller et al., 2008), sperm motility (Linhart et al., 2000; Ritar, 1999; Rodina et al., 2007; Zilli et al., 2005), fertilization ability (Gwo & Arnold, 1992; Rana, 1995), and also larval survival (Suquet et al., 1998). Spermatozoa genome alteration due to cryopreservation may affect only late embryonic development and larval survival (Kopeika et al., 2003a, 2003b, 2004; Suquet et al., 1998), but not the early events in embryonic development, because these are controlled by maternally inherited information (Braude et al., 1988). On the contrary, defects in sperm proteins (degradation and/or change of the phosphorylation state) may compromise sperm motility, fertilization ability, and the early events after fertilization (Cao et al., 2003; Huang et al., 1999; Lessard et al., 2000). The most common parameters used to evaluate sperm quality are fertilization ability, motility (rate and duration) and cellular (chemical and/or biochemical) parameters. Fertilizing capacity is the most conclusive test of sperm quality but the use of this marker is laborious and requires the availability of eggs (McNiven et al., 1992). Motility is normally evaluated as percentage and duration, but some authors also use velocity, flagellum beat frequency, or other parameters measured by computer-assisted sperm analysis (Ciereszko et al., 1996; Cosson et al., 2000; Rurangwa et al., 2001). Cellular bio markers has been used to evaluate spermatozoa quality of different fish species such as Atlantic salmon (Aas et al., 1991; Hwang & Idler, 1969), rainbow trout (Ciereszko & Dabrowski, 1994; Lahnsteiner et al.,1996a, 1998) and sea bass (Zilli et al., 2004). All these parameters have been also used to evaluate the effect of cryopreservation on spermatozoa quality. Here we reviewed data obtained by our group, on the effect of freezing-thawing procedures on sea bass and sea bream sperm. In particular, data concerning the effect of cryopreservation on bio-chemical parameters, DNA integrity, protein profile and phosphorylation state, are reported.

The successful fertilization achievement is, theoretically, affected by the fatty acids composition of the different sperm membrane domains, which has effects on plasma membrane fluidity, fusogenicity, signal transduction, and other processes. In the present study we analyzed the fatty acids composition of two different membrane domains (head membrane and flagella) of Sparus aurata sperm samples, and its relation to other quality parameters: motility and viability. Results showed that flagella had a higher rate of unsaturated (MUFA and PUFA) fatty acids compared with the head membrane. While flagellar fatty acid composition correlated with motility parameters, suggesting their relation with the need of fluidity for the flagellar beating, head membrane composition better correlated with preservation of sperm membrane integrity.

Changes in the plasma membrane lipid composition have been related to a decrease in sperm quality during cryopreservation. Antifreeze proteins (AFPs) have been tested in different species because of their ability to depress the freezing point and their potential interaction with membranes, but controversial effects were reported. In the present study we analyzed separately the lipid composition of two sperm membrane domains, head (HM) and flagellar (FM) membrane, after cryopreservation with an extender containing 5% DMSO either alone or with AFPI or AFPIII (1μg/ml). We used sperm from a teleost, Sparus aurata, because the lack of acrosome avoids changes of lipid profiles due to capacitation process or acrosomal losses during freezing/thawing. Comparing with the control (cryopreservation with 5% DMSO alone) the addition of AFPIII increased the velocity, linearity of movement and the percentage of viable cells. In addition, freezing with DMSO alone increased the phosphatidyl-serine content, as well as the saturated fatty acids and decreased the unsaturated ones (mainly PUFA) both in HM and FM. These changes in the lipid components were highly avoided with the addition of AFPIII. HM had a higher amount of saturated fatty acids than FM and was more affected by cryopreservation without AFPs. The percentage of viable cells was positively correlated with the amount of unsaturated fatty acids in the HM whereas the motility parameters were positively correlated with both FM and HM amount of unsaturated fatty acids. AFPs, especially AFPIII, seem to have interacted with unsaturated fatty acids stabilizing the plasma membrane organization during cryopreservation and contributing to improve sperm quality after thawing.

In many marine fish species, the spermatozoa are immotile in the testis and seminal plasma, and motility is induced when they are released in the aqueous environment. It is well known that the extracellular factors (hyperosmolality or sperm-activating peptides), controlling sperm motility in marine fish, act on the axonemal apparatus through signal transduction across the plasma membrane. To better understand the molecular mechanism regulating axoneme activation in marine fish, the present review examines the existing literature, with particular emphasis on protein phosphorylation/dephosphorylation process. The present review suggests that: (1) there is no single model that can explain the molecular activation and regulation of sperm motility of the marine fish; (2) only in some species (puffer fish, tilapia, gilthead sea bream, and striped sea bream) protein phosphorylation/dephosphorylation has been shown to be involved in flagellar motility regulation; (3) only a few proteins were identified, which show a change in their state of phosphorylation following sperm activation. A model of molecular mechanism controlling the activation of sperm motility in gilthead sea bream is being proposed here, which could be a useful model to clarify the sperm motility activation process in other species.

The aim of this work was to evaluate the efficiency of a natural antioxidant substance in gilthead sea bream (Sparus aurata) feeds. An olive oil by-product, olive mill vegetation water (VW), contains polyphenols, which have a strong antioxidant activity. A 147 days long growth trial was conducted (monofactorial balanced; 4x3) with the diet as the experimental factor. Two diets (isonitrogenous CP= 40% and isoenergetic GE = 18Mj*kg-1 on DM) were formulated with 1% and 5% of VW inclusion level (VW1, VW5), against a control diet without VW. 600 juvenile gilthead sea breams (mean body weight 114.1±5.7g) were utilised. At the end of the growth trial the productive parameters and somatic indexes were calculated. Antioxidant activity in fish fillets was investigated using TBARS and DPPH assays. Some hematological parameters and digestive enzyme patterns were measured in fish in the middle and at the end of the experiment. The TBARS values showed slight delays in the development of oxidation in the fillet of fish fed with VW. No statistical differences appeared between the fish fed with the experimental diet and the control group, except for maltase activity which increased as the VW inclusion level was increased. The use of VW in a gilthead sea bream diet did not show any detrimental effects in gilthead sea bream production and physiological parameters and slightly improved the conservation of the fish fillets.

In many fish species, the spermatozoa are immotile in the testes and seminal plasma, and motility is induced when they are released in the aqueous environment. Extracellular factors control the activation of the axoneme through signal transduction across the plasma membrane. The present review examines the existing literature concerning the axoneme activation in fish, with particular emphasis on the role played by the protein phosphorylation/ dephosphorylation process, since post-transciptional modifications are involved in the mechanisms of sperm motility activation in many animals with external fertilization (starfish, sea urchins, sea cucumber, fish). We report the current understanding of the role played by the changes of protein phosphorylation state in sperm motility activation of teleost fish and provide additional tools for evaluating the gamete quality before and after cryopreservation procedure and, therefore, improve fish farm management. Statement of relevance: The present review examines the existing literature concerning the signaling pathways involved in the axoneme activation in fish, in order to better understand the molecular mechanism regulating spermmotility initiation and to show how the proteins that change their phosphorylation status after spawning can be used as biomarkers for sperm quality and cryodamage.

The aim of the present study was to evaluate the effect of sea cucumber meal on gilthead sea bream growth. Two diets were used: a fishmeal based diet (control) and a diet containing 18% of sea cucumber meal inclusion (HM). A 100-days growth trial was carried out (120 fish, initial mean body weight of 35.28±9.31 g). The experimental plan used was monofactorial, balanced with three replicates for two experimental treatments (fish diet). The diets were isolipidic (CL 15.80 ±0.5%), isonitrogenous (CP 49.70±1.0%) and isoenergetic (GE 20.00±1.0 MJ kg-1) and feeding rate was 1.5% of biomass. At the end of the trial, growth performances parameters, digestive enzymes (liver and intestinal), haematological parameters, Na+/K+ ATPase activity were measured. Oressigenic and anoressigenic brain neuropeptides were also analyzed in order to investigate other effects of food intake in fish. Productive parameters, blood plasma parameters, enzyme activities and neuropeptides expression were not affected by the introduction of sea cucumber meal in fish feed thus showing that HM can be used as a partial substitute of fish meal in sea bream nutrition. © Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey in cooperation with Japan International Cooperation Agency (JICA), Japan.