Sea bass sperm freezability is influenced by motility variables and membrane lipid composition but not by membrane integrity and lipid peroxidation

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Animal Reproduction Science 131 (2012) 211– 218

Contents lists available at SciVerse ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

ea bass sperm freezability is influenced by motility variables andembrane lipid composition but not by membrane integrity and lipid

eroxidation

. Martínez-Páramoa,∗, P. Diogoa, M.T. Dinisa, M.P. Herráezb, C. Sarasquetec, E. Cabritac

CCMAR-Center for Marine Science, University of Algarve, Campus Gambelas, 8005-139 Faro, PortugalDepartment of Molecular Biology, Area of Cell Biology, University of León, 24071 León, SpainICMAN-Institute of Marine Science of Andalusia, Spanish National Research Council, Av. Republica Saharaui 2, 11510 Puerto Real, Cádiz, Spain

r t i c l e i n f o

rticle history:eceived 19 January 2012eceived in revised form 9 March 2012ccepted 14 March 2012vailable online 29 March 2012

eywords:uropean sea bassperm cryopreservationperm cryo-resistanceperm freezabilityipid peroxidationperm motility

a b s t r a c t

Cryopreserved sperm quality depends on the characteristics of fresh sperm. Thus, it isnecessary to establish a group of variables to predict the cryopreservation potential ofthe fresh samples with the aim of optimizing resources. Motility, viability, lipid peroxida-tion and lipid profile of European sea bass (Dicentrarchus labrax) sperm were determinedbefore and after cryopreservation to establish which variables more accurately predict thesperm cryopreservation potential in this species. Cryopreservation compromised spermquality, expressed as a reduction of motility (46.5 ± 2.0% to 35.3 ± 2.5%; P < 0.01) andviability (91.3 ± 0.7% to 69.9 ± 1.6%; P < 0.01), and produced an increase in lipid peroxi-dation (2.4 ± 0.4 to 4.0 ± 0.4 �moles MDA/mill spz; P < 0.01). Also, significant changes wereobserved in the lipid composition before and after freezing, resulting in a reduction inthe 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) incryopreserved sperm, as well as an increase in lysophosphatidylcholine (4.4 ± 0.3% to4.8 ± 0.3%; P < 0.01) and C24:1n9 fatty acid (0.5 ± 0.1% to 0.6 ± 0.1%; P < 0.05). Motility, veloc-ity, cholesterol/phospholipids ratio, monounsaturated fatty acids and the n3/n6 ratio werepositively correlated (P < 0.05) before and after freezing, whereas, viability and lipid peroxi-dation 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 corre-lated (P < 0.05) with lipid peroxidation. Therefore, the results demonstrated that motilityand plasma membrane lipid composition (CHO/PL) were the most desirable variables deter-mined in fresh samples to predict cryo-resistance in European sea bass sperm, taking intoaccount the effect of both on cryopreserved sperm quality.

. Introduction

In general, cryopreservation has been widely usedor reproductive practices, germplasm conservation andenetic improvement of resources in several species of

∗ Corresponding author. Tel.: +351 289 800 900x7374;ax: +351 289 800 069.

E-mail address: [email protected] (S. Martínez-Páramo).

378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.anireprosci.2012.03.008

© 2012 Elsevier B.V. All rights reserved.

mammals (Watson and Fuller, 2001). However, despite itsapplication to preserve the genetic profile of threatenedspecies (He et al., 2011; Martínez-Páramo et al., 2009)or strains with biotechnological interest (Robles et al.,2009), cryopreserved sperm is scarcely used for routinefertilization practices. Factors such as reduced motility

and fertilization ability, embryo development failure orreduced offspring survival and quality (Cabrita et al., 2010;Pérez-Cerezales et al., 2010, 2011) limit the use of cryop-reserved fish sperm. However, this may be counteracted

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by improving cryopreservation protocols and adding cer-tain compounds that protect cells during freezing–thawing(Cabrita et al., 2001; Martínez-Páramo et al., 2012) orby a more precise selection of samples prior to freezing(Cabrita et al., 2011). There are several reports relatingpost-thaw sperm quality to the initial features of freshsamples. Therefore, during recent years, numerous vari-ables (motility, viability, cell concentration, osmolality, pH,enzymatic activity, membrane composition and antioxi-dant activity) have been used to characterize sperm toestablish which samples can be cryopreserved (Groisonet al., 2010; Lahnsteiner et al., 2010; Rurangwa et al., 2004).This procedure would guarantee the reduction of risksand damage associated with the use of bad quality sam-ples.

Motility is the most common variable used for deter-mining sperm quality (Cosson et al., 2008). Also, in manycases, a correlation between sperm motility and its abil-ity to fertilize eggs has been established for some species(Dietrich et al., 2005; Ottesen et al., 2009). In general, spermmotility decreases after cryopreservation, and fish spermcontains different spermatozoa subpopulations accordingto motility pattern, which may be differentially affected bythe cryopreservation protocol (Beirão et al., 2011). Plasmamembrane composition and integrity are key factors affect-ing sperm functionality (Am-in et al., 2011; Argov et al.,2007; Lahnsteiner et al., 2009). During freezing and thaw-ing, the formation of ice crystals together with osmoticshock, promote cell breakages that reduce the percent-age of viability (Asturiano et al., 2007). Moreover, severalauthors have demonstrated that the cryopreservation pro-cess modifies the spermatozoa plasma membrane lipidprofile (Cerolini et al., 2001; Chakrabarty et al., 2007),inducing changes in phospholipids and cholesterol organi-zation, which modify cellular homeostasis, leading to lossesin sperm function.

Different studies have shown that the variables used tocharacterize sperm quality respond differently to the cry-opreservation process depending on the species (Rurangwaet al., 2004). Therefore some variables determined in freshsperm may predict the capacity for sperm cryopreservationin one species but fail to do so in another. Motility predictedsperm cryopreservation potential in several mammalianspecies such as goat and boar (Dorado et al., 2010; Floreset al., 2009) and in some fish species such as Atlantic cod(Butts et al., 2011). In other studies, a positive correla-tion occurred between early changes in sperm membraneintegrity and post-thaw quality of boar spermatozoa (Penaet al., 2007). Furthermore, Zilli et al. (2004) determinedthe �-d-glucuronidase activity and the ATP concentrationin sea bass sperm and considered that fertilization abilityin both fresh and cryopreserved sperm could be reliablypredicted by determining these biochemical parameters.Taking into account that sperm freezability depends on ini-tial sperm quality, it is of paramount importance to definea group of variables that accurately predict which malesare “good” or “bad” sperm donors for cryopreservation or

which samples may have more chances of resisting cryo-damage. Moreover the possibility of defining key qualityvariables that ensure the characterization of sperm qual-ity in frozen samples can contribute to a faster analysis,

ction Science 131 (2012) 211– 218

avoiding time-consuming screening of quality descriptorsin frozen samples.

In the present work, sperm from European sea bass(Dicentrarchus labrax) was characterized before and aftercryopreservation, to determine which of the variablestested were useful for forecasting sperm cryopreservationpotential in this species. Thus, motility, viability, lipid per-oxidation and lipid profile of European sea bass sperm weredetermined with the aim of establishing possible correla-tions between sperm quality before and after freezing.

2. Materials and methods

2.1. Sperm collection, cryopreservation and thawing

European sea bass males, supplied by the Aqualvorfishfarm (Odiaxere, Lagos, Portugal), were used for thisexperiment. Sperm was collected during the natural repro-ductive cycle, from the beginning of November to the endof February to obtain samples of different quality. A totalof 32 pools containing sperm from 10 to 12 males (meanweight 840 ± 132 g) were obtained.

Immediately after extraction, sperm was diluted (1:6,v/v) in a non-activating mineral medium (NAM: 59.8 mMNaCl, 1.5 mM KCl, 12.9 mM MgCl2, 3.5 mM CaCl2, 20 mMNaHCO3, 0.4 mM glucose and 1% (w/v) BSA, pH 7.7) to avoidmotility activation (Fauvel et al., 1998). Diluted sperm wasmaintained at 4 ◦C for further analyses of motility, viability,level of lipid peroxidation and lipid extraction.

For cryopreservation, 10% DMSO (final concentration;v/v) was added to the diluted sperm and, following theprotocol described by Martínez-Páramo et al. (2012). Themixture was immediately loaded into 0.5 ml straws (I.M.V.,France) and frozen for 15 min at 6.5 cm above a liquid nitro-gen surface, and then immersed in the nitrogen and storedin a container until used. They were thawed at 35 ◦C for15 s.

2.2. Determination of quality variables

2.2.1. Sperm motilitySperm motility was analyzed in fresh and cryopreserved

sperm using computer-assisted sperm analysis (CASA).Sperm placed in a Makler chamber (0.5 �l of dilutedsperm; 1:6, v/v in NAM) was activated with 20 �l of arti-ficial sea water (513.3 mM NaCl, 10.7 mM KCl, 11.7 mMCaCl2, 54.8 mM MgSO4 and 11.6 mM NaHCO3), and imme-diately, digitalized images obtained using an 10× negativephase contrast objective in a light microscope (NikonE200, Tokyo, Japan) were recorded with a Basler camera(Basler Afc, Ahrensburg, Germany) at 10, 20, 30 and 45 spost-activation. Images were processed with ISAS software(Proiser, Valencia, Spain) to determine total spermatozoamotility (TM, %), progressive motility (PM, %), velocity (VCLand VSL, �m/s) and linearity index (LIN, %).

2.2.2. Cell viability

Cell viability was determined before and after cryop-

reservation using the double fluorescent dye SYBR-greenand propidium iodide (PI) (Invitrogen, Spain). Spermdiluted 1:6 in NAM was re-diluted 1:1000 in the same

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edia to reduce sperm concentration, and SYBR-greenfinal concentration 0.25 �M) and PI (final concentration8 �M) were added to the cell suspension, which was incu-ated for 5 min in the dark at 4 ◦C. Viability was quantifiednder an epifluorescence microscope (Nikon E200, Tokyo,

apan), equipped with triple excitation filter block DAPI-ITC-Texas Red (excitation filter wavelengths: 395–410 nmbandpass, 403 CWL), 490–505 nm (bandpass, 498 CWL),nd 560–580 nm (bandpass, 570 CWL)). To determine theercentage of viable cells (green stained), PI (red) andYBR-green (green) stained cells were counted. At least00 cells per slide and two slides per pool and treatmentere observed.

.2.3. Lipid peroxidationTo determine the lipid peroxidation level, the con-

entration of malondialdehyde (MDA) was quantifiedsing a colorimetric assay (kit BIOXYTECH LPO-586TM,xisResearchTM, USA), following the protocol described byomínguez-Rebolledo et al. (2010), adapted for sea bass

perm by Martínez-Páramo et al. (2012). MDA concentra-ions were calculated from a standard curve and presenteds �moles of MDA per million spermatozoa. Each freshn = 32) or cryopreserved sperm sample (n = 32) was pro-essed in triplicate.

.2.4. Lipid composition: cholesterol/phospholipids ratio,hospholipid classes and fatty acids

Total lipids were extracted from the pooled spermollowing the protocol described by Folch et al. (1957),dapted for sea bass sperm. Fresh or cryopreserved sperm2 ml) was homogenized with 6 ml of chloroform:methanololution (1:2; v/v). After shaking (30 s), 2 ml of chloroformnd 2 ml of acidified 0.9% NaCl solution (one volume of 0.9%aCl + 1/200 volumes of 2 N HCl) were added to the mix-

ure. The tubes were shaken (30 s) and placed on ice (5 min)o be then centrifuged (250 × g, 15 min, 4 ◦C). After cen-rifugation, the upper phase was discarded and 7.6 ml ofolch solution (30 ml CHCl3 + 490 ml MeOH + 480 ml acid-fied 0.9% NaCl solution) was added to the tubes. Afterhaking (30 s), the tubes were centrifuged (250 × g, 15 min,◦C) and the lower phase collected. Chloroform contained

n the phase collected was evaporated under a nitrogentmosphere in a dry bath at 50 ◦C. Lipids remaining in theubes were re-suspended in 300 �l of isopropyl alcohol andtorage in nitrogen atmosphere at −80 ◦C for further anal-sis of cholesterol, phospholipid classes and fatty acids.

Cholesterol was quantified in the lipid extracts (n = 32)y spectrophotometry using the colorimetric assay CHOD-AP Kit (Biolabo, France), following the manufacturer’snstructions. The cholesterol concentration in the samples

as determined using a cholesterol standard curve. Theamples and standard curve were measured spectropho-ometrically at 490 nm and data expressed as nmoles ofholesterol per million spermatozoa.

The phospholipids were quantified following the pro-

ocol described by Bartlett (1959), adapted for fish sperm.he nmoles of phosphate per million spermatozoa in eachample were quantified by absorbance determination at30 nm using a phosphorous standard curve.

ction Science 131 (2012) 211– 218 213

The phospholipid classes were quantified by HPLC aftercolumn filtration of the lipid extracts using silica gel car-tridges (Waters, Spain), following the protocol described byBeirão et al. (2012). The HPLC (Waters 2695) was equippedwith a UV detector (Waters 996), a Waters Spherisorb col-umn PSS838521 (Waters, Spain) (5 �m, 250 mm × 3.0 mmi.d.) and a mobile phase (acetonitrile/methanol/phosphoricacid, 130/5/1.5, v/v/v) at a constant flow rate of 1.0 ml/minwas used for phospholipid class separation. The peaksobtained were detected at 203 nm and identified by com-parison with standards. The results are presented as apercentage of each phospholipid class in comparison withthe total phospholipids detected.

The fatty acid classes were determined by gas chro-matography following the protocol described by Berry et al.(1965). The gas chromatograph (Perkin Elmer AutosystemXL) was equipped with a flame ionization detector anda fused silica capillary column Omegawax 250 (Supelco,Spain) (30 m × 0.25 mm i.d. × 0.25 �m film thickness). Thechromatography conditions for the analysis were: initialtemperature set at 50 ◦C for 2 min; warming to 200 ◦C ata rate of 7 ◦C/min; 2 min at 200 ◦C; warming to 220 ◦C at arate of 0.5 ◦C and 5 min at 220 ◦C, using helium as the carriergas. Peaks were identified using a fatty acids methyl esterstandards mixture (C4 – C24:1) and methyl nonanoate wasused as internal standard. The results are presented as apercentage of the total fatty acids detected.

2.3. Statistical analysis

SPSS18.0 software was used for statistical analysis.Data were expressed as means ± S.E.M., and normalized bylogarithmic or arcsine transformation when results wereexpressed as percentages. The effect of cryopreservationon sperm motility variables was determined using a gen-eral linear model with the Bonferroni correction (P < 0.05).Significant differences before and after freezing for theremaining variables tested were determined using Stu-dent’s t-test (P < 0.05).

To predict sperm freezability, bivariate correlationsbetween data from fresh and cryopreserved sperm wereanalyzed with Pearson’s correlation coefficient (P < 0.05)for each variable.

Partial correlations between sperm quality parameters,controlling for the effect of cryopreservation, were ana-lyzed with Pearson’s correlation coefficient (P < 0.05).

3. Results

3.1. Sperm motility

All motility variables decreased linearly from 10 to45 s post-activation, and showed a reduction after cry-opreservation (P < 0.01). Fresh sperm motility decreasedlinearly (P < 0.01) from 46.5 ± 2.0% 10 s post-activation to7.4 ± 1.0% 45 s post-activation, and after cryopreserva-tion, the percentage of spermatozoa motility decreased

(P < 0.01) from 35.3 ± 2.5% to 4.0 ± 0.9% at 10 s and 45 spost-activation, respectively (Fig. 1 A). Numbers of pro-gressive spermatozoa were reduced by half (P < 0.01) inthe cryopreserved samples (6.0 ± 0.9%) in comparison with

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near velion. Baroni, P < 0

Fig. 1. Mean values of total motility (A), progressive motility (B), curvilifresh and cryopreserved European sea bass sperm, until 45 s post-activatbetween fresh and cryopreserved samples (general linear model, Bonferr

fresh sperm (10.5 ± 1.0%; Fig. 1B). Sperm velocity (VCLand VSL) showed the same pattern. After cryopreservation,VSL decreased more quickly within time than VCL, whichshowed a stationary phase until 30 s post-activation. Thus,because VCL showed a reduction of around 7 �m/s (Fig. 1C),VSL decreased 15 �m/s between 10 s and 30 s (Fig. 1D). Lin-earity was also reduced after cryopreservation (P < 0.01),with values of 56.5 ± 1.3% and 50.2 ± 2.1% being recordedfor fresh and cryopreserved sperm, respectively, 10 s afteractivation with sea water (Fig. 1E).

3.2. Cell viability and lipid peroxidation

Cell viability was reduced by cryopreservation (P < 0.01),the percentage of viable cells decreasing from 91.3 ± 0.74%in fresh sperm to 69.9 ± 1.6% after freezing and thawing(Fig. 2A).

Lipid peroxidation, quantified as �moles of malon-dialdehyde, was greater in the cryopreserved sperm(4.0 ± 0.4 �moles MDA/106 spz) than in the fresh samples(2.4 ± 0.4 �moles MDA/106 spz; P < 0.01; Fig. 2B).

3.3. Lipid composition: cholesterol/phospholipids ratio,phospholipid classes and fatty acids

The cholesterol/phospholipids ratio decreased (P < 0.01)from 1.4 ± 0.1 in fresh sperm to 1.1 ± 0.0 after cryopreser-vation (Table 1). The main phospholipid classes identified

ocity (VCL) (C), straight line velocity (VSL) (D) and linearity index (E) ins indicate S.E.M. Different lowercase letters show significant differences.05).

were: phosphatidylserine (PS), phosphatidylethanolamine(PE), phosphatidylcholine (PC) and lysophosphatidyl-choline (LPC). The amount of PS and PE did not showsignificant differences before and after freezing, represent-ing values of around 8% and 40%, respectively (Table 1).However, the proportion of PC and LPC was altered by cry-opreservation, showing an opposite reaction. Thus, becausePC decreased after cryopreservation, the percentage of LPCincreased in cryopreserved samples (Table 1).

Most of the fatty acids identified in the membranes werenot affected by the cryopreservation protocol. Only oleicacid (C18:1n9) and the fatty acid C24:1n9 showed differ-ences, decreasing and increasing respectively (P < 0.05), incryopreserved samples in comparison with fresh sperm(Table 1).

3.4. Correlations

The analyses of correlations between fresh and cryop-reserved samples for the different variables determinedshowed a positive correlation for the following variables:total (P < 0.01) and progressive motility (P < 0.05), straightline velocity (VSL; P < 0.05), cholesterol/phospholipids ratio(P < 0.01) and fatty acid composition (monounsaturated

fatty acids and C24:1n9 with P < 0.05 and C18:0-stearicacid, C18:1n9-oleic acid, C20:1n9 and n3/n6 ratio withP < 0.01) (Table 2). The remaining variables were not corre-lated before and after freezing.

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Table 1European sea bass sperm lipid composition: cholesterol/phospholipidsratio, phospholipids (%) and fatty acid classes (%), before and after cryop-reservation, expressed as mean values ± SEM. Different superscripts in thesame row show differences between fresh and cryopreserved sperm sam-ples (Student’s t-test, P < 0.05). CHO/PL ratio: cholesterol/phospholipidsratio; FA: fatty acids, EPA: eicosapentaenoic acid; DHA: docosahexaenoicacid; AA: arachidonic acid.

FRESH CRYOPRESERVED

CHO/PL ratio 1.4 ± 0.1a 1.1 ± 0.0b

Phospholipids (%)Phosphatidylserine (PS) 7.9 ± 0.6 9.7 ± 0.8Phosphatidylethanolemine (PE) 39.7 ± 0.6 42.0 ± 0.7Phosphatidylcholine (PC) 47.7 ± 0.8a 44.2 ± 0.8b

Lysophosphatidylcholine (LPC) 4.4 ± 0.3b 4.8 ± 0.3a

Fatty acids (%)Saturated FA

C16:0 (palmitic acid) 22.1 ± 0.4 21.5 ± 0.4C18:0 (stearic acid) 6.6 ± 0.2 6.4 ± 0.1Total 31.6 ± 1.0 31.7 ± 0.9

Monounsaturated FAC16:1n9 (Palmitoleic acid) 1.0 ± 0.0 1.0 ± 1.0C18:1n9 (Oleic acid) 8.7 ± 0.2a 8.3 ± 0.2b

C20:1n9 2.6 ± 0.2 2.7 ± 0.2C22:1n9 0.2 ± 0.0 0.2 ± 0.0C24:1n9 0.5 ± 0.1b 0.6 ± 0.1a

Total MUFAs 13.0 ± 0.4 12.8 ± 0.3Polyunsaturated n-3 FA

C20:5n3 (EPA) 9.3 ± 0.2 9.0 ± 0.2C22:5n3 1.9 ± 0.1 2.0 ± 0.1C22:6n3 (DHA) 37.3 ± 0.8 37.8 ± 0.7Total n3 52.1 ± 0.9 52.4 ± 0.9

Polyunsaturated n-6 FAC18:2n6 2.2 ± 0.1 2.0 ± 0.0C20:4n6 (AA) 0.2 ± 0.0 0.2 ± 0.0Total n6 3.2 ± 0.1 3.2 ± 0.1

Total PUFAs 55.3 ± 0.9 55.6 ± 0.9n3/n6 ratio 16.9 ± 0.7 17.3 ± 0.8

twtc

Table 2Correlations between the sperm quality variables analyzed in freshand cryopreserved sperm. Correlations with P < 0.05 are signed * andP < 0.01 are denoted **; VSL: straight line velocity; CHO/PL ratio: choles-terol/phospholipids ratio; MUFAs: monounsaturated fatty acids.

Pearson correlation (r)

Total motility 0.465**

Progressive 0.361*

VSL 0.373*

CHO/PL ratio 0.704**

C18:0 (stearic acid) 0.528**

C18:1n9 (oleic acid) 0.508**

C20:1n9 0.580**

C24:1n9 0.429*

results of the present study revealed a reduction in motil-

FS

AA/EPA ratio 0.02 ± 0.00 0.03 ± 0.00DHA/EPA ratio 4.0 ± 0.1 4.2 ± 0.1

Correlations between variables showed strong posi-ive correlation (P < 0.01) between the motility variables

ith each other and cell viability (Table 3). On the con-

rary, the cholesterol/phospholipids ratio was negativelyorrelated (P < 0.05) with total motility and progressive

ig. 2. Mean values of European sea bass sperm viability (A) and lipid peroxidatio.E.M. Different lowercase letters show significant differences between fresh and

MUFAs 0.410*

n3/n6 ratio 0.536**

spermatozoa, and positively correlated (P < 0.05) with lipidperoxidation (Table 3). Regarding fatty acid composition,total saturated fatty acids and stearic acid (C18:0) showed apositive correlation (P < 0.05) with total motility and linear-ity, respectively, whereas stearic acid, EPA (C20:5n3) andC24:1n9 were negatively correlated (P < 0.05) with lipidperoxidation (Table 3).

4. Discussion

The characterization of the European sea bass spermdescribed in the present research demonstrated that cry-opreservation compromises sperm quality, as has beenwidely demonstrated in different species (Suquet et al.,2000; Tiersch et al., 2007; Watson, 2000). The furtherapproach in the present study by trying to understandwhich of the variables used to characterize fresh spermquality are able to accurately predict cryo-resistance inEuropean sea bass sperm samples, allowing the selectionof donors to optimize resources in artificial fertilizationpractices, besides the improvement of cryopreservationprotocols attending to objective parameters. In general,

ity variables after freezing. The cryopreserved sperm hada lesser percentage of motile and progressive cells, whichwere slower and had less linear trajectories than fresh

n expressed as �moles of MDA per million spermatozoa (B). Bars indicate cryopreserved samples (Student’s t-test, P < 0.05).

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Table 3Correlations between the sperm quality variables analyzed. Correlations with P < 0.05 are denoted * and P < 0.01 **; TM: total motility; PM: progressivemotility; VCL: curvilinear velocity; VSL: straight line velocity; LIN: linearity index; MDA: lipid peroxidation; CHO/PL ratio: cholesterol/phospholipids ratio;FA: fatty acids; EPA: eicosapentaenoic acid.

TM PM VCL VSL LIN MDA

PM 0.829** 1VCL 0.626** 0.912** 1VSL 0.557** 0.735** 0.686** 1LIN 0.485** 0.766** 0.871** 0.384** 1Viability 0.331* 0.459** 0.486** 0.451** 0.313*

CHO/PL ratio −0.290* −0.345* 0.349*

C18:0 (stearic acid) 0.342* −0.352*

Saturated FA 0.286*

C18:1n9 (oleic acid) −0.340*

C24:1n9

C20:5n3 (EPA)

Relationships with Pearson parametric correlation (r).

sperm. Previously, Fauvel et al. (1998) described the samereduction on sea bass sperm velocity (VCL and VSL). How-ever, inconsistent with the present results, in a previousstudy it was found that the percentage of total motilityin fresh and cryopreserved sperm were not different, aswas also observed by Zilli et al. (2003). These results couldbe attributed to different cryopreservation protocols used(0.25 �l straws and 1:3 dilution ration in Mounib media)as well as different methods to evaluate motility (a quan-titative method such as CASA was not used). In agreementwith the results obtained by Abascal et al. (2007) andFauvel et al. (1998), sea bass sperm showed short motil-ity duration (around 50 s) that decreased exponentially intime and after cryopreservation. Membrane integrity wasalso affected by cryopreservation. Thus, after freezing, thepercentage of viable cells decreased at the same time aslipid peroxidation increased, although the statistical anal-ysis did not correlate both variables, as also described byOrtega-Ferrusola et al. (2009) in stallion sperm. In mam-mals, lipid peroxidation and membrane breakages werelocated in different parts of the spermatozoa, suggest-ing that they are in part relatively independent processes(Neild et al., 2005). Fish sperm membranes are particularlysusceptible to lipid peroxidation due to greater amountsof polyunsaturated fatty acids. This oxidative process isone of the primary manifestations of oxidative damageinitiated by ROS, and has been linked to altered mem-brane structure and enzyme inactivation (Li et al., 2010).The reduction in viability, observed in frozen sperm fromseveral species, is mainly attributed to osmotic stress dur-ing the cryopreservation process, which is enhanced bythe addition of cryoprotectants, DMSO in this case (Ball,2008; Li et al., 2010). In addition, the cryopreservationprocess modifies the spermatozoa membrane lipid profile,shedding phospholipids, saturated fatty acids and choles-terol, as a mechanism to overcome the stress challengeand enhance survival to cryo-damage (Cerolini et al., 2001;Chakrabarty et al., 2007). Results of the present study indi-cated changes in lipid composition were limited to a fewcomponents. Therefore, besides a reduction in the choles-terol/phospholipids ratio, as was observed by Blesbois et al.

(2005) in bird sperm, only phosphatidylcholine, lysophos-phatidylcholine, oleic acid and the C24:1n9 fatty acid weredifferent between fresh and cryopreserved sperm. Incon-sistent with results in the present study, Chakrabarty et al.

−0.349*

−0.324*

(2007) working with goats, observed an increase in thesperm cholesterol/phospholipids ratio after cryopreserva-tion, explaining this fact as a result of the decrease inphospholipids. However, taking into account the lesserloss of phospholipids in cryopreserved sea bass sperm, thereduction of this ratio was due to a decrease in the propor-tion of cholesterol, as observed by Cerolini et al. (2001) inboar sperm.

The decrease of phosphatidylcholine in the plasmamembrane could be associated with the increasein lysophosphatidylcholine, taking into account thatlysophospholipids are derived from phospholipids by theselective loss of one fatty acyl residue induced by enzymesand/or reactive oxygen species (ROS; Fuchs and Schiller,2009).

Correlations between these previously described vari-ables, provided evidence about the behavior of Europeansea bass sperm during cryopreservation, and providedthe impetus in the present study to determine whichindices could be useful to predict the cryo-resistance ofeach sperm sample. Therefore, results of the present studyshowed that total motility, spermatozoa progressivenessand straight line velocity were positively correlated in thesamples before and after freezing. This fact supports thehypothesis that sperm samples with the greatest motilityand velocity maintain these characteristics after freezing,despite the known reduction promoted by the cryopreser-vation process. In boar sperm, Flores et al. (2009) describeddifferences in several motility variables among sampleswith different resistance to the freezing/thawing cycle. Inaddition, Dorado et al. (2010) demonstrated that CASA-derived motility variables were also good indicators ofgoat semen freezability. Moreover, in results from thepresent study, motility was positively correlated with via-bility. Therefore, although this variable was not correlatedin fresh and cryopreserved sperm, high motility ensuresgreater viability after thawing. Besides motility, onlycholesterol/phospholipids ratio and fatty acid compositionshowed correlation between fresh and cryopreserved sam-ples. The cholesterol/phospholipids ratio showed a strongpositive correlation between fresh and cryopreserved

sperm. The same correlation was observed by severalauthors, although inconsistent results were found depend-ing on the species. In mammals, a greater proportion ofcholesterol improves cryo-survival (Mocé and Graham,

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S. Martínez-Páramo et al. / Animal

006; Mocé et al., 2010; Moore et al., 2005). However, inainbow trout spermatozoa, Muller et al. (2008) demon-trated that cholesterol stabilized the plasma membrane ofresh sperm, but this stabilization before cryopreservationrought no improvement to the poor freezing capacity ofhese cells. The cholesterol/phospholipids ratio determineshe physiological properties of the plasma membrane inerms of permeability to water and other molecules, flu-dity and lipid phase transitions (Holt, 2000). Thus, aalance between cholesterol and the other constituentsf the membrane is necessary to ensure the requiredembrane function for successful sperm cryopreserva-

ion (Muller et al., 2008). Partial correlations betweenhe cholesterol/phospholipids ratio and the other vari-bles measured showed that this ratio was positivelyorrelated with lipid peroxidation. This correlation wasreviously demonstrated by several authors (Mazari et al.,010; Tirosh et al., 1997), who reported that the oxidationates of polyunsaturated fatty acids in the lipid bilay-rs were hardly affected by the elevation of cholesteroloncentration. In addition, the cholesterol/phospholipidsatio was negatively correlated with sperm motility androgressiveness, as was also observed by Zalata et al.2010) in human sperm. The authors attributed this cor-elation to alterations in the signaling mechanism thatontrols sperm capacitation. Thus, in the case of sea bassperm, where no capacitation process occurs, the negativeffect of a greater cholesterol/phospholipids ratio on spermotility could be attributed to a reduction in membrane

uidity compromising osmotic regulation and the ionicxchange responsible for motility activation. Therefore,he cholesterol/phospholipids ratio could be considerednother factor to predict freezability, taking into accounthat those fresh samples with a high ratio will presentow motility after freezing/thawing. This fact is in agree-

ent with results obtained by Blesbois et al. (2005), whostablished that greater sperm membrane fluidity, deter-ined by the cholesterol and saturated fatty acids content,

ould be used as an indicator of sperm freezability inhickens, turkeys and guinea fowl. Moreover, in Euro-ean sea bass sperm, the monounsaturated fatty acidsontent was also positively correlated in fresh and cryopre-erved samples, whereas stearic acid (C18:0), C24:1n9 andPA (C20:5n3) showed a negative correlation with lipideroxidation. These correlations confirmed that in Euro-ean sea bass, the sperm lipid composition influenced theryo-resistance of the sample and that specific modula-ion of sperm using these fatty acids could reduce lipideroxidation, one of the principal damages occurring dur-

ng cryopreservation (Ball, 2008; Martínez-Páramo et al.,012).

In conclusion, results of the present study showed thaturopean sea bass sperm freezing capacity could be pre-icted by the analysis of motility and lipid composition.owever, taking into account that the analysis of lipidomposition is a very time-consuming technique, whichmplies the use of qualified technicians, the analysis of

perm motility could be considered the most desirableariable for predicting sperm cryo-resistance, with thedditional advantage that it can be measured within min-tes of collecting the fresh semen.

ction Science 131 (2012) 211– 218 217

Acknowledgments

This research was supported by the CRYOSPERM project(PTDC/MAR/64533/2006) founded by FCT national fundingand AGL2011-28810 project (MICINN); S. Martínez-Páramo was supported by a FCT postdoctoral fellow-ship (SFRH/BPD/48520/2008) co-founded by POPH-QREN,Tipologia 4.1 (FEDER and MCTES) and E. Cabrita wassupported by a Ramón and Cajal research contract (RYC-2007-01650). The authors thank the Aqualvor (Alvor,Portugal) fishfarm for supplying animals.

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