ICMAN Institute of Marine Science of Andalusia

Puerto Real, Spain

ICMAN Institute of Marine Science of Andalusia

Puerto Real, Spain

Time filter

Source Type

Martinez-Paramo S.,University of Algarve | Diogo P.,University of Algarve | Dinis M.T.,University of Algarve | Herraez M.P.,University of León | And 2 more authors.
Animal Reproduction Science | Year: 2012

Cryopreserved sperm quality depends on the characteristics of fresh sperm. Thus, it is necessary to establish a group of variables to predict the cryopreservation potential of the fresh samples with the aim of optimizing resources. Motility, viability, lipid peroxidation and lipid profile of European sea bass (Dicentrarchus labrax) sperm were determined before and after cryopreservation to establish which variables more accurately predict the sperm cryopreservation potential in this species. Cryopreservation compromised sperm quality, expressed as a reduction of motility (46.5 ± 2.0% to 35.3 ± 2.5%; P<0.01) and viability (91.3 ± 0.7% to 69.9 ± 1.6%; P<0.01), and produced an increase in lipid peroxidation (2.4 ± 0.4 to 4.0 ± 0.4μmoles MDA/mill spz; P<0.01). Also, significant changes were observed in the lipid composition before and after freezing, resulting in a reduction in the cholesterol/phospholipids ratio (1.4 ± 0.1 to 1.1 ± 0.0; P<0.01), phosphatidylcholine (47.7 ± 0.8% to 44.2 ± 0.8%; P<0.01) and oleic acid (8.7 ± 0.2% to 8.3 ± 0.2%; P<0.05) in cryopreserved sperm, as well as an increase in lysophosphatidylcholine (4.4 ± 0.3% to 4.8 ± 0.3%; P<0.01) and C24:1n9 fatty acid (0.5 ± 0.1% to 0.6 ± 0.1%; P<0.05). Motility, velocity, cholesterol/phospholipids ratio, monounsaturated fatty acids and the n3/n6 ratio were positively correlated (P<0.05) before and after freezing, whereas, viability and lipid peroxidation were not correlated. Motility and the cholesterol/phospholipids (CHO/PL) ratio were negatively correlated (P<0.05) with each other and the CHO/PL ratio was positively correlated (P<0.05) with lipid peroxidation. Therefore, the results demonstrated that motility and plasma membrane lipid composition (CHO/PL) were the most desirable variables determined in fresh samples to predict cryo-resistance in European sea bass sperm, taking into account the effect of both on cryopreserved sperm quality. © 2012 Elsevier B.V..


Cabrita E.,ICMAN Institute of Marine Science of Andalusia | Ma S.,University of Algarve | Diogo P.,University of Algarve | Martinez-Paramo S.,University of Algarve | And 2 more authors.
Animal Reproduction Science | Year: 2011

During cryopreservation, dilution in the extender media reduces the seminal plasma constituents being cells more vulnerable to oxidative stress. Vitamins (C and E) and the amino acids taurine and hypotaurine are powerful antioxidants naturally present in seminal plasma. Whether their effect may improve sperm quality and reduce sperm DNA damage after cryopreservation in fish sperm still remains unclear. Thus, the aim of the present work was to analyse the effect of extender supplementation with several antioxidant components on post-thawed sperm motility, viability and DNA integrity of two commercial species, gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax). Sperm collected from ten to twelve individuals was cryopreserved in ten different extenders containing: taurine and hypotaurine (1 and 10. mM), ascorbic acid (1 and 10. mM), α-tocoferol (0.1 and 0.5. mM) or 1. ml/l of a commercial cell antioxidant supplement. Cell viability, motility and DNA fragmentation were determined in post-thawed samples. Addition of antioxidants (vitamins and amino acids) to D. labrax and S. aurata extenders did not significantly increase the parameters of motility (TM, PM, VCL, VSL and Lin) or viability, although 1. mM taurine slightly increased the percentage of motile cells (TM) in S. aurata. DNA fragmentation (DNA in tail and Olive tail moment) in D. labrax sperm was higher in treatments containing vitamins than amino acids or control. However in S. aurata sperm, antioxidants especially taurine and hypotaurine, significantly reduced both DNA fragmentation parameters, protecting DNA against strand breaks. These results suggest a species-specific effect depending on the type of antioxidants used. © 2011 Elsevier B.V.


Martinez-Paramo S.,University of Algarve | Diogo P.,University of Algarve | Dinis M.T.,University of Algarve | Herraez M.P.,University of León | And 2 more authors.
Theriogenology | Year: 2012

Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 m. m α-tocopherol or 0.1 m. m ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ± 3.0 μm/sec, 18.3 ± 1.7 μm/sec and 46.9 ± 2.0% in extender containing 0.1 m. m α-tocopherol or 30.6 ± 3.9 μm/sec, 19.5 ± 1.6 μm/sec and 47.9 ± 2.2% in extender containing 1 m. m ascorbic acid respect to the extender control (20.7 ± 3.3 μm/sec, 13.8 ± 1.7 μm/sec and 37.3 ± 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of α-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation. © 2012 Elsevier Inc.


Martinez-Paramo S.,University of Algarve | Diogo P.,University of Algarve | Beirao J.,University of León | Dinis M.T.,University of Algarve | Cabrita E.,ICMAN Institute of Marine Science of Andalusia
Aquaculture | Year: 2012

Precocious maturation is a common feature in European sea bass males which occurs just before reaching the marketable size, reducing growth and feed conversion and representing an important problem that reduces the commercial value of fish. The sperm produced by precocious sea bass males could be useful for reproductive purposes since these males could be selected for breeders and used earlier as a new broodstock. However the characterization of sperm quality in these males during the reproductive season is fundamental in determining its possible use in reproduction or cryopreservation. Thus, in the present work, sperm from precocious European sea bass (Dicentrarchus labrax) males was characterized to determine the effect of seasonality on parameters influencing quality throughout the reproductive period. Several characteristics associated with sperm quality were determined: motility, cell concentration, sperm pH and osmolality, cell viability, lipid peroxidation (malondialdehyde-MDA), phospholipid, cholesterol and fatty acid composition of the plasma membrane. The results demonstrated seasonal variation in some of the parameters tested, the quality of samples being relatively poor at the beginning of the spawning season, corresponding with the highest content of MDA and the lowest motility parameters. Cell viability, phosphatidylcholine and phosphatidylethanolamine, polyunsaturated fatty acids n-3 and saturated fatty acids did not show significant differences during the reproductive period. However, monounsaturated fatty acids and polyunsaturated fatty acids n-6 showed a reduction at the end of the reproductive period. Parameter correlations showed that motility was negatively correlated with lipid peroxidation (-. 0.394, p. <. 0.05), cholesterol/phospholipid ratio (-. 0.449, p. <. 0.05) and osmolality (-. 0.478, p. <. 0.01). Our results demonstrated that lipid peroxidation could explain the seasonal variation in sperm quality, since the highest malondialdehyde value quantified in November is the only parameter associated with the lowest motility recorded at the beginning of the season. Thus, approaches to reduce lipid peroxidation as a consequence of oxidative stress could be useful for improving sperm quality in precocious sea bass males. The parameters quantified are consistent with the interpretation that January is the optimal month in the spawning season to collect sperm from precocious sea bass males for further reproductive purposes. © 2012 Elsevier B.V.


Cabrita E.,ICMAN Institute of Marine Science of Andalusia | Soares F.,IPIMAR | Beirao J.,University of León | Garcia-Lopez A.,ICMAN Institute of Marine Science of Andalusia | And 2 more authors.
Theriogenology | Year: 2011

Improving fertilization success in captive Senegalese sole broodstocks has been a challenge in the last years. Recent reports suggest that low sperm volume and quality could be one of the reasons leading to poor fertilization rates, although further studies are needed to reach a conclusive explanation. Here, we report on several experiments focused on this issue. Seasonal profiles of plasma androgen levels (testosterone and 11-ketotestosterone) and sperm production and quality parameters were assessed, although no statistical correlations among them were identified. The response of males to female presence/absence was also analyzed. Long-term isolation from females decreased male androgen levels at the peak of the reproductive period, suggesting some kind of disrupting effects on the endocrine system. On the other hand, short-term exposure of previously isolated males to ripe females decreased androgen levels, possibly reflecting a rapid steroidogenic shift promoting final maturation of spermatozoa, and increased sperm viability, motility and velocity, thus, supporting the concept of positive effects of female contact on male sole performance. Further evidence sustaining the relevant female-to-male communication in sole reproduction was obtained after treating the females with progestagen 17α,20β-dihydroxy-4-pregnen-3-one (regarded as pre-ovulatory pheromone in fish) and registering a significant increase in sperm viability, velocity and motility in surrounding males. Finally, we found that a single administration of a 20 μg/kg GnRH analogue in males was effective in stimulating androgen release and sperm quality, although the effects were transient and thus, the use of sustained hormone delivery methods were suggested for improving efficiency. Our results point to velocity, viability, and motility as the most sensitive parameters in sole sperm, although further studies will have to evaluate whether these parameters have any relation with fertilization success in captive broodstocks of this important aquaculture species. © 2011 Elsevier Inc.


Beirao J.,University of León | Zilli L.,University of Salento | Vilella S.,University of Salento | Cabrita E.,ICMAN Institute of Marine Science of Andalusia | And 3 more authors.
Journal of Applied Ichthyology | Year: 2012

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. © 2012 Blackwell Verlag GmbH.


Beirao J.,University of León | Zilli L.,University of Salento | Vilella S.,University of Salento | Cabrita E.,ICMAN Institute of Marine Science of Andalusia | And 3 more authors.
Biology of Reproduction | Year: 2012

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 plasma membrane (HM) and flagellar membrane (FM), after cryopreservation with an extender containing 5% dimethyl sulfoxide (DMSO) either alone or with AFPI or AFPIII (1 μg/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 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 polyunsaturated) 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. © 2012 by the Society for the Study of Reproduction, Inc.


Pacchiarini T.,ICMAN Institute of Marine Science of Andalusia | Olague E.,ICMAN Institute of Marine Science of Andalusia | Sarasquete C.,ICMAN Institute of Marine Science of Andalusia | Cabrita E.,ICMAN Institute of Marine Science of Andalusia
Histology and Histopathology | Year: 2014

Busulfan, a cytotoxic alkylating agent used for treatment of chronic myeloid leukemia has effects in mammalian germ cells. In fish species, the use of this compound is of special interest in intra and interspecies germ cell transplants. To determine the effects of busulfan in fish a previous range finding experiment was designed. Survival and growth rate of 150-days after hatching (150DAH) Senegalese sole (Solea senegalensis) juveniles was determined. In a second experiment, the effects of a sublethal busulfan dose in fish germ cell depletion and in somatic tissues were analysed. Sublethal effects of several busulfan treatments (B10-10 days after injection, B20-20 days after injection, B20+-20 days after injection with double injection) were determined in somatic and gonadal tissues. Alterations were registered through histopathological techniques, TUNEL (cell apoptosis) and quantified at molecular level (Q-PCR analyses) using the vasa mRNAs (Ssvasa1-2 and Ssvasa3-4 mRNAs) as molecular markers for germinal cells in Senegalese sole juveniles. Several sublethal effects were observed with 40 mg kg-1 busulfan, a non-lethal dose, such as: pyknosis in liver, increase of melanomacrophage centres and blood stagnation in spleen and interruption of gonadal development. Females were more affected by busulfan treatments than males in terms of germ cell disruption, since a significant decrease in the expression of both Ssvasa1-2 and Ssvasa3-4 markers was found in the gonad of treated females rather than males. At 10 days post-treatment (B10), females already presented a decrease in germ cell proliferation, as confirmed by Q-PCR. Ssvasa expression proved to be a reliable tool for the direct evaluation of the effects of busulfan on Senegalese sole gonadal development, proving that busulfan can be a suitable treatment for causing transient sterility in recipient gonads for germ cell transplantation.


PubMed | ICMAN Institute of Marine Science of Andalusia
Type: Journal Article | Journal: Histology and histopathology | Year: 2014

Busulfan, a cytotoxic alkylating agent used for treatment of chronic myeloid leukemia has effects in mammalian germ cells. In fish species, the use of this compound is of special interest in intra and interspecies germ cell transplants. To determine the effects of busulfan in fish a previous range finding experiment was designed. Survival and growth rate of 150-days after hatching (150DAH) Senegalese sole (Solea senegalensis) juveniles was determined. In a second experiment, the effects of a sublethal busulfan dose in fish germ cell depletion and in somatic tissues were analysed. Sublethal effects of several busulfan treatments (B10-10 days after injection, B20-20 days after injection, B20-20 days after injection with double injection) were determined in somatic and gonadal tissues. Alterations were registered through histopathological techniques, TUNEL (cell apoptosis) and quantified at molecular level (Q-PCR analyses) using the vasa mRNAs (Ssvasa1-2 and Ssvasa3-4 mRNAs) as molecular markers for germinal cells in Senegalese sole juveniles. Several sublethal effects were observed with 40 mg kg busulfan, a non-lethal dose, such as: pyknosis in liver, increase of melanomacrophage centres and blood stagnation in spleen and interruption of gonadal development. Females were more affected by busulfan treatments than males in terms of germ cell disruption, since a significant decrease in the expression of both Ssvasa1-2 and Ssvasa3-4 markers was found in the gonad of treated females rather than males. At 10 days post-treatment (B10), females already presented a decrease in germ cell proliferation, as confirmed by Q-PCR. Ssvasa expression proved to be a reliable tool for the direct evaluation of the effects of busulfan on Senegalese sole gonadal development, proving that busulfan can be a suitable treatment for causing transient sterility in recipient gonads for germ cell transplantation.

Loading ICMAN Institute of Marine Science of Andalusia collaborators
Loading ICMAN Institute of Marine Science of Andalusia collaborators