Evaluation of DNA damage as a quality marker for rainbow trout sperm cryopreservation and use of LDL as cryoprotectant

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Theriogenology 74 (2010) 282–289 www.theriojournal.com

Evaluation of DNA damage as a quality marker for rainbow trout sperm cryopreservation and use of LDL as cryoprotectant S. Pérez-Cerezalesa, S. Martínez-Páramob, J. Beirãoa, M.P. Herráeza,* a

Department of Molecular Biology, Area of Cell Biology, University of León, 24071 León, Spain b Center for Marine Science-CCMAR, University of Algarve, 8005-139 Faro, Portugal

Received 3 November 2009; received in revised form 18 December 2009; accepted 5 February 2010

Abstract Defining reliable and objective biomarkers of sperm quality is a complex matter, because it does not rely on a particular characteristic of the milt. Susceptibility to cryopreservation varies between ejaculations and throughout the year, and the evaluation of fresh sperm does not always provide accurate information about their fertilization ability after freezing and thawing. DNA is one of the cell components prone to suffering cryodamage and several studies have pointed out the importance of the maintenance of its integrity during sperm cryostorage. The authors analysed sperm from rainbow trout for four weeks during the natural reproductive season. Viability, DNA integrity, and fertilization ability were evaluated. Furthermore, in order to increase membrane and DNA protection during sperm cryopreservation, the authors optimized the use of LDL fraction from egg yolk as a cryoprotectant during the analysed period. Results revealed that the evaluation of DNA damage in fresh sperm reveals subtle cell damage, not evidenced in fresh sperm by the other parameters. DNA fragmentation increased from 8 to 31% during the reproductive season, indicating pre-freezing differences that render the cells more susceptible to cryodamage. Also, the use of 12% LDL (low density lipoprotein) fraction, instead of the commonly used pure egg yolk, improved sperm quality after freezing. When LDL was used, post-thaw quality remained constant throughout the analysed period, providing around 60% of eyed embryos. In contrast, when egg yolk was used, post-thaw quality decreased significantly at the end of the season and the percentage of eyed embryos dropped from 60% to 27%. Results demonstrated that reduction in DNA integrity takes place during the reproductive season affecting susceptibility to cryodamage and that the protective effect of egg yolk is very much improved when only their LDL fraction is added to the cryopreservation extender. © 2010 Elsevier Inc. All rights reserved. Keywords: Comet assay; DNA damage; Sperm cryopreservation; Trout sperm; LDL; Onchorhynchus mykiss

1. Introduction Sperm cryopreservation is presumed to be a common practice in the future of fish farming, its uses ranging from facilitating genetic management of broodstock, to transporting the gametes of individuals with an interesting genotype between farms or companies. * Corresponding author: Tel.: ⫹34987291912; Fax: ⫹34987291917. E-mail address: [email protected] (M.P. Herráez). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.02.012

Many efforts have been made in the development of different and specific procedures for sperm cryopreservation in species of commercial interest [1] and there have been some experiences in transferring this knowledge to fish farms. One of the bottlenecks of this transfer is considered to be the lack of standardization of procedures, mainly due to the variable results obtained when the same method is applied in different populations, individuals, or even in samples from the same male [2,3]. Many advances have recently been made in

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the study of gamete cryobiology, requiring established protocols to be reviewed and improved, with particular care being taken in the evaluation of samples considered suitable for cryopreservation. These studies revealed new factors that reduce sperm efficiency during cryopreservation that could also affect further embryo development. In this sense, different authors [4 – 8] showed that sperm cryopreservation promotes DNA fragmentation in rainbow trout, seabream, seabass, and loach. It has been reported that fertilization with DNAdamaged sperm could increase the rate of abnormal karyotype in the offspring of carp [9] and increase the percentage of abortions, as well as the risk of cancer and abnormal weight in newborn mice [10]. Hence, the evaluation of this parameter and reduction of DNA injury shall be a priority in the design of new freezingthawing procedures and extenders, in order to increase fertilization rates, and also to ensure good embryo development, the health of the offspring, and the maintenance of the genotypes to be preserved. The mechanisms by which DNA damage is produced are not completely understood but several studies point out the possibility that oxidative and mechanical events could be responsible for DNA fragmentation and base modification [7,11,12]. Recent works developed by our group on trout sperm revealed that DNA fragmentation significantly increases after cryopreservation, and suggested that oxidation of specific bases could occur during freezing-thawing [6,7]. In order to reduce these chromatin modifications two aspects should be taken into account: the evaluation of possible differences in resistance to damage between samples and the use of less damaging freezing methods. Regarding the first question, previous studies performed in rainbow trout by Cabrita and colleagues demonstrated that sperm samples have different susceptibility to suffering membrane damage during cryopreservation, and that the selection of more resistant samples results in higher quality after thawing [13]. Similar differences in susceptibility to DNA injury could be expected and the identification of resistant samples could improve post-thaw quality. One of the factors affecting cryopreservation success is the timing of sperm collection, being generally accepted that milt produced in the middle of the spawning season provides the most appropriate samples for freezing [2,3]. On the other hand, the addition of antioxidants to the freezing extender to reduce oxidative events is common practice, but does not seem to completely avoid chromatin fragmentation. Hu et al. [14] demonstrated that the addition of the LDL fraction of egg yolk to the

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extender, rather than the whole egg yolk, improves boar sperm quality after cryopreservation in terms of motility, membrane integrity, and reduction of DNA damage. To our knowledge the only reports on the use of LDL fraction for fish sperm cryopreservation were provided by Babiak et al. [15], who extracted this fraction using a different purification method and did not find any improvement in fertilization or hatching rates in pike and rainbow trout sperm frozen with several extenders. With the aim of reducing DNA injury promoted during cryopreservation of rainbow trout sperm, the objectives of this work were to study sperm freezability throughout the reproductive season, paying particular attention to DNA damage and to test the use of LDL fraction extracted from chicken egg yolk in the cryopreservation extender. 2. Materials and methods 2.1. Broodstock and gamete collection Male and female rainbow trout (Oncorhynchus mykiss) were maintained in Las Zayas fish farm (León, northern Spain), under natural photoperiod and gametes were obtained in February, during the natural reproductive period. Sperm was obtained in the fish farm facilities by introducing a catheter in the urogenital pore to avoid urine contamination during the process and performing abdominal massage. After sperm collection, samples were kept in falcon tubes (15 and 50 ml) on ice until arrival at the laboratory. Sperm was processed within 90 min from the collection. Females were anaesthetized with 100 mg/l MS-222 (Sigma, Madrid) and transported to the University facilities where the required oocytes for fertilization trials were collected by stripping. 2.2. Reagents All media components were purchased from Sigma (Sigma-Aldrich Spain, Madrid) or Merck (Merck, Spain, Barcelona) except when otherwise stated. 2.3. Experiment 1: Selection of the optimal LDL concentration for cryopreservation Sperm was collected from 4 males, analysed and pooled in similar amounts from each male (2 ⫻ 1010 cell/ml). The pool was divided into 6 aliquots which were cryopreserved following the protocol described by Herráez et al. [16] with different egg yolk extracts. For freezing, sperm was diluted 1:3 (sperm:extender) in #6

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from Erdhal and Graham (0.7 mM CaCl2 ⫻ 2H2O, 1.08 mM MgCl2 ⫻ 6H2O, 1.49 mM Na2HPO4, 34.30 mM KCl, 100 mM NaCl, 0.52 mM citric acid, 55.5 mM glucose, 20 mL KOH solution 226 mM, 20 mL bicine solution 324 mM, and 323 mOsm/kg, pH 7.4) containing 10% egg yolk or different concentrations of LDL (5, 9, 12, 15, and 20% (v/v)) and 7% Me2SO as permeable cryoprotectant. LDL was obtained using the protocol described by Moussa et al. [17]. The diluted sperm was kept at 4 °C for 10 min to equilibrate, and during this time was loaded into 0.5 ml French straws (10 straws for treatment). The loaded straws were then kept for 10 min in a floating device at 2 cm above liquid nitrogen in a Styrofoam box, for sperm freezing. After this, they were immersed in Liquid nitrogen and stored in a nitrogen container until analysis. Thawing was carried out in a water bath at 25 °C for 30 s and the sperm analyses were immediately performed. DNA damage and sperm viability (membrane integrity) were evaluated for each male in fresh sperm and from all cryopreserved procedures. 2.4. Experiment 1: Evaluation of sperm freezability Sperm from 12 males was collected at the beginning (day 1), middle (day 14), and end (day 28) of February (4 males each time) and was separately cryopreserved using 10% egg yolk or 12% LDL (based on the results of experiment 1). DNA damage and membrane integrity were evaluated in fresh and frozen samples. Fertilization trials were performed in triplicate on day 1 and 28 using eggs pooled from 2 females and fresh and cryopreserved sperm with egg yolk and with 12% LDL. For fertilization, 100 eggs were mixed with 500 ␮l of fresh sperm diluted 1:3 (sperm:extender) in #6 from Erdhal and Graham or one straw (6.6 ⫻ 107 spermatozoa/egg) and 10 ml of the activating solution DIA (94.97 mM NaCl, 49.95 mM Glicine, 19.98 mM Tris) was immediately added. The eggs were incubated in a recirculating water system at 10 °C in the dark, under controlled conditions, until they reached eyed stage. 2.5. Sperm Viability (plasma membrane integrity) Spermatozoa membrane integrity was evaluated using the fluorescent “live/dead Kit” (Molecular probes, Madrid), containing propidium iodide (PI) and SYBR-14 and the flow cytometer FACSort Plus Analyzer (Becton–Dickinson, USA). Each sample was diluted to 1.6 ⫻ 107 cell/ml in SFMM (110 mM NaCl, 28.18 mM KCl, 1.22 mM MgSO4 ⫻ 7H2O, 1.77 mM CaCl2 ⫻ 2H2O, 10.05 mM Bicine, 9.99 mM HEPES) solution at a final volume of 1 ml. SYBR was added to a final concen-

tration of 100 nM and left to incubate in the dark for 10 min. Then, PI was added to a final concentration of 12 ␮M and kept in the dark for 10 min. After this time, analysis by flow cytometry was performed. The red fluorescence emitted by PI was detected using a 610 nm filter and the green fluorescence emitted by SYBR-14 with a 516 nm filter. The flow cytometer was equipped with standard optics and an Argon ion laser (Innova 90, Coherent, USA), with CellQuest software for Macintosh (version 3.0, Becton–Dickinson, USA) for acquisition and analysis, and with FacComp software (version 1.0) for daily setup and quality control. The evaluation was carried out in triplicate in all samples. The results were reported as means ⫾ SEM. 2.6. DNA damage DNA fragmentation was analysed using the Comet assay (SCGE), which enables chromatin fragmentation to be evaluated in each individual cell, as previously described [7]. Fresh and cryopreserved sperm was diluted in SFMM to a final concentration of 8 –10 ⫻ 106 mL⫺1. Frozen sperm was prepared immediately after thawing to prevent sperm degradation. Slides for the sperm smears were prepared in advance as described by Cabrita et al. [6]. All buffers were prepared fresh. After spreading the sperm, the slides were immersed in cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% Triton-X, 1% Lauroyl sarcosine sodium salt, 4 mM lithium diiodosalicylate, pH 8) and incubated for 1 h at 4 °C. Then they were placed in an electrophoresis cube (Bio-Rad, Spain) containing electrophoresis buffer (0.3 M NaOH, 1 mM Na2–EDTA, pH 12) for 20 min at 4 °C. Electrophoresis was performed for 10 min at 25 V and 300 mA at 4 °C. The slides were then neutralised with 0.4 M Tris, pH 7.5 for 5min at 4 °C. This operation was repeated three times to ensure the elimination of all alkali and detergents. The slides were fixed by immersion in a pure methanol solution for 3 min and were then left to drain in the air and stored protected from light and dust. For comet visualization, 40 ␮L of ethidium bromide 0.5 ␮g/mL were pipetted onto the slides which were then covered with a coverslip. Samples were observed with an epifluorescence microscope (Nikon Eclipse E800) fitted with a 510 –560 nm excitation filter and a 590 nm barrier filter. Images were acquired from the microscope with a Nikon DXM1200F digital camera, acquiring approximately 50 cells from each slide using Nikon ACT-1 software (v. 2.62, Nikon). All the images were analysed with the Komet imaging system software (version 5, Kinetic Imaging, UK). Two slides were

S. Pérez-Cerezales et al. / Theriogenology 74 (2010) 282–289 Table 1 Cell membrane integrity and percentage of Tail DNA from fresh sperm and sperm cryopreserved with different extenders containing different concentrations of LDL fraction of egg yolk or 10% pure egg yolk.

Fresh 5 % LDL 9 % LDL 12 % LDL 15 % LDL 20 % LDL Egg Yolk

Cell viability (%)

% Tail DNA

98.7 ⫾ 0.39a 16.1 ⫾ 1.07f 30.8 ⫾ 1.37e 37.6 ⫾ 1.35c 34.4 ⫾ 2.30d 41.6 ⫾ 1.46b 13.7 ⫾ 0.77g

7.9 ⫾ 0.71f 58.2 ⫾ 3.66a 36.6 ⫾ 2.71d 32.1 ⫾ 2.29e 36.2 ⫾ 1.88d 39.8 ⫾ 3.01c 43.1 ⫾ 4.58b

Values are expressed as mean ⫾ SEM. Different letters in the same column means significant differences (P ⬍ 0.05).

analysed per male and treatment. For each analyzed spermatozoon, the pixels observed in the tail of the comet represent DNA fragments (damaged DNA) and the nucleus represents the head of the comet in which the undamaged DNA is located. From the several parameters analyzed by the Komet software, the percentage of tail DNA (% DNAt) was used to quantify the DNA damage. Data were expressed as mean of percentage of tail DNA. 2.7. Statistical analysis Statistical analysis was carried out using the computerized package generated by SPSS 14.0 software for Windows. The results were expressed as means ⫾ SEM. Mean values of percentage of tail DNA were obtained from 400 cells (100 from each male) and mean values of viability were obtained from 106 cells from each sample. Means were compared and analysed by one-way ANOVA. Membrane integrity and DNA damage throughout the study period were also analysed using a Linear Mixed-Model Analysis.

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damage, LDL promoted a decrease in fragmentation (% Tail DNA) reaching the best results when 12% was added to the extender (32.0 ⫾ 2.29). Concentrations of LDL higher than 12% resulted in increased DNA fragmentation. 3.2. Experiment 2 The analysis of fresh sperm revealed that during February no changes were observed in membrane integrity (range 97.6 –98.9%) (Fig. 1). Nevertheless, DNA fragmentation increased during the reproductive season (Fig. 2), showing an initial Tail DNA of 7.9% ⫾ 0.5, which increased significantly to 32.1% ⫾ 1.2 at the end of the analysed period. After freezing with 12% LDL in cryopreservation extender, viability remained constant at over 40% without significant variations between dates. On the other hand, the use of egg yolk promoted a significant decrease in membrane integrity when compared with LDL as well as during the sampling period, the lowest value being obtained on 28 (3.2% ⫾ 0.51) (Fig. 1). With regard to DNA fragmentation, the percentage of Tail DNA increased throughout time with the two extenders, as indicated by the linear mixed model analysis. Values increased from 46% on the first sampling day to 56.9% ⫾ 1 on the last when 10% of egg yolk was used, and from 27.7% to 49.2% ⫾ 1.14 in samples frozen with 12% LDL. Evaluation of embryos at the eyed stage revealed that 99.3% ⫾ 2.5 and 96.2% ⫾ 3.2 of eggs were fertilized with fresh sperm and reached this stage in

3. Results 3.1. Experiment 1 As shown in Table 1, fresh sperm showed high membrane integrity (98.7 ⫾ 0.39) and low DNA damage (7.9 ⫾ 0.71 tail DNA). Sperm cryopreserved with 10% egg yolk suffered significant injury in plasma membrane functionality after freezing/thawing, showing the lowest percentage of viable cells (13.7% ⫾ 0.77) and a high level of fragmented DNA (43.1 ⫾ 4.58 Tail DNA). When LDL was used instead of egg-yolk, an increase in cell membrane integrity was observed whatever the concentration, reaching 41.6 % ⫾ 1.46 of viable cells when 20% was used. In terms of DNA

Fig. 1. Cell membrane integrity of fresh sperm and sperm cryopreserved with 12% LDL or 10% egg yolk, throughout the analysed period. Bars indicate ⫾ SEM. Different letters indicate significant differences between treatments as showed by linear mixed model (P ⬍ 0.05).

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4. Discussion

not be used for cryopreservation [2,3]. The analysed period comprised the 4 central weeks of the natural reproductive season. During that time, the analysis of fresh sperm indicated that cell viability and fertility rates remained constant at almost 100% indicating the good, and apparently constant, quality of the samples used, in the range of the quality recommended by different authors for cryopreservation [16,19]. These data did not reflect any variation in quality throughout time and could be considered an index of homogeneity, but the evaluation of chromatin fragmentation did reveal very significant differences between samples, showing that fragmentation increased during the analysed period. Increased DNA damage could be related to the ageing of spermatozoa stored in the ducts and the presence of immature, apoptotic, or necrotic cells [20]. The increase in DNA fragmentation did not affected fertilization rates, probably because an excess of spermatozoa per oocyte facilitated fertilization by the noninjured spermatozoa and because part of the damaged DNA of spermatozoa could be repaired by the oocyte during the first cleavage [21,22]. Samples cryopreserved with the egg yolk containing extender suffered very different degrees of damage, viability, DNA integrity, and fertilization capacity decreasing throughout time and giving very poor results on day 28. These data reflect that near the end of the reproductive season the spermatozoa are more prone to damage by freezing-thawing and that egg yolk did not provide enough protection to either membrane stability or DNA structure. The decrease in the percentage of eyed embryos in comparison with that obtained with

It is well known that one of the bottlenecks in the design of standard procedures for fish sperm cryopreservation is the difficulty to provide homogeneous sperm quality post-thaw. According to many reports, freezability of samples seems to be difficult to predict and very variable results are obtained, even when samples with the same apparent quality are cryopreserved [18]. In order to transfer cryopreservation methods to the industry, and to standardize the freezing protocols, it is very important to identify the parameters whose evaluation is required to guarantee post-thaw quality, as well as to develop more appropriate procedures able to provide a global protection to the spermatozoa, buffering the subtle differences in fresh sperm quality. In our study the use of two different extenders for rainbow trout sperm was analyzed with this purpose. As in other species the quality of rainbow trout sperm fluctuates during the reproductive season and all researchers recommend that early and late milt should

Fig. 3. Percentage of embryos that reached eyed stage from eggs fertilized with fresh sperm and sperm cryopreserved with 12% LDL or 10% egg yolk. Bars indicate ⫾ SEM. Different letters indicate significant differences between values (P ⬍ 0.05).

Fig. 2. Percentage of Tail DNA of fresh sperm and sperm cryopreserved with 12% LDL or 10% egg yolk, throughout the analysed period. Bars indicate ⫾ SEM. Different letters indicate significant differences between treatments as showed by the linear mixed model (P ⬍ 0.05).

both the first sampling (day 1) and the last (day 28), respectively (Fig. 3). From the eggs fertilized with frozen sperm on day 1, 60% ⫾ 4.4 and 65% ⫾ 3.3 reached the eyed stage with both extender additives, LDL, and egg yolk, respectively. However, on day 28, sperm cryopreserved with 12% LDL produced a similar percentage of eyed embryos, whereas success decreased to 27% ⫾ 6.8 when fertilization was with sperm protected with egg yolk.

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fresh sperm is related to the high percentage of fragmented DNA and low cell membrane integrity, especially at the end of the experimental period: a small percentage of cells can fertilize and those spermatozoa carrying fragmented DNA but preserving the capacity to fertilize, could negatively affect early development and increase the rate of abortions. An increase in the rate of abortions during early development with the use of frozen sperm has already been noticed by different authors [5,8,23], and reduction on the fertility rates reported by many researchers are probably related to this fact. None of the evaluated parameters in fresh sperm, except DNA fragmentation, showed any loss of quality, indicating that it should be taken into account in procedures for selecting samples to be preserved. The mean tail DNA in a particular sample is not expected to directly correlate with the fertilizing capacity, which is related to other parameters such as membrane integrity, maintenance of sperm/egg recognition mechanisms, etc [24,25]. Further developmental success is much more dependent on the quality of sperm DNA than fertilization itself. Membrane and DNA integrity are affected in different ways and extent by cryopreservation [7], and are essential for the final success of embryo development. The decrease in the percentage of viable cells, and the increase in the DNA fragmentation, observed on day 28 after freezing with egg yolk, suggest not only a decrease in the fertilization capacity of the sperm, but also an increased chance of being fertilized by a DNA damaged spermatozoa, which could promote a higher rate of developmental failures. Cryopreservation with egg yolk extender is thus a suboptimal method, providing non homogeneous postthaw results. The analysis of pre-freezing DNA fragmentation levels should be required to discard the samples unsuitable for cryopreservation with this protocol, but could be difficult to apply as a routine procedure in fish farming. Recommendation of a threshold level of tail DNA would require an evaluation of milt samples with different levels of damage in the range of 8 –32% (fragmentation on day 1 and 28 respectively). The use of LDL instead of pure egg yolk improved cell protection in terms of membrane integrity and DNA protection at all the tested concentrations. In experiment 1, the addition of LDL was optimized and 12% LDL was considered the most convenient for the second experiment, due to the increase in DNA damage observed at higher concentrations. A similar effect was noticed by other authors [17], who demonstrated that higher concentration of LDL resulted in a decrease in osmotic pressure, because of salt precipitation or the

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LDL aggregation effect. When this extender was used in our second experiment, viability post-thaw remained constant throughout the analysed period, showing that LDL had a better protective effect than egg yolk, especially on day 28, when the quality of fresh sperm seem to be compromised. DNA damage increased through time, indicating that viability and DNA damage are affected in different ways. Nevertheless, no differences in fertilization rates were noticed between the use of LDL and egg yolk in the first sampling, probably because promoted damages were compensated for by the use of an excess of spermatozoa per egg. At the end of the reproductive season, differences in thawed sperm quality increased significantly, and a higher percentage of eyed embryos was obtained with 12% LDL, which buffered the original differences in fresh sperm quality much better than egg yolk. Egg yolk is a very well known plasma membrane protector and the effect of its low density lipoproteins (LDL) as membrane stabilizers has been widely reported [14,26 –28]. Previous works in mammals have shown a more beneficial effect of the use of the LDL fraction instead of pure egg yolk, not only as a membrane protector but also in motility or in the reduction of DNA damage [14,17]. The fact that isolated LDL provided better results (also observed in our results) could be related to the presence in the egg yolk of other molecules or particles that could produce an antagonistic effect on the cryoprotective role of LDL [29]. Moussa et al. [17] developed a new extraction procedure for LDL from chicken egg yolk, and tested it in bull sperm cryopreservation at concentrations between 2.5% and 20%, showing that the best motility was achieved at between 5 and 10%, higher than when 20% egg yolk was used. Hu et al. [14] compared the use of 9% LDL and 20% egg yolk in boar sperm and obtained significant improvements in motility, DNA damage reduction, and membrane and acrosome integrity with the LDL extract. Despite these promising results, no similar studies have been performed in fish. Babiak et al. [15,30] tested the LDL fraction from chicken egg yolk extracted by the protocol designed by Demianowicz and Strzezek [29] and analysed fertilization rates and embryo survival during development, but no improvements in comparison with pure egg yolk were obtained in rainbow trout or pike sperm after freezing/thawing. Their purification procedure was different from that reported by Moussa [17] and that used in the present study. In the Moussa procedure, new steps such as removing livetins were included to increase the quality of the LDL extract. The results of our study revealed

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that the substitution of commonly used egg yolk by their LDL fraction clearly improves sperm viability and reduces DNA damage in cryopreserved rainbow trout sperm. Membrane protection is shown by the fact that the increased concentration of LDL results in a linear increase in viability. It was suggested that this protective effect was related to the gelation capacity of LDL microspheres and the possibility that the contained phospholipids and triglycerides could protect against ice formation by their inclusion in the membrane or by forming a film around it [31–34]. With regard to DNA, a direct effect of these protectors has not been described and is not expected. According to recent studies there is no correlation between cell viability and DNA damage [35,36]. Our results also support the hypothesis that there is no direct correlation between membrane integrity and DNA damage: the addition of LDL reduced DNA fragmentation, reaching a minimum 12% LDL. However, when higher percentages were used, increased DNA fragmentation was observed whereas membrane integrity remained the same. DNA fragmentation is thought to be caused during freezing/thawing mainly by oxidative processes, but some studies have suggested that other factors such as mechanical and osmotic stress could also contribute to injuries [5,7,37]. The used extender contains citric acid as an antioxidant, but its effect is clearly insufficient to prevent chromatin fragmentation. The protection conferred by the additives to DNA should be considered as an indirect effect of the protection of cell structure: on the one hand, their direct effect on the membrane and seminal plasma proteins could minimize the mechanical stress promoted by changes in the cell shape during ice formation and cryoprotectants/water exchanges and, on the other, the reduction in the rate of cell lysis should diminish the production of reactive oxygen species (ROS) after thawing, thus reducing oxidative stress. These hypotheses should be confirmed in further studies. 5. Conclusions In conclusion and according to our results, the use of 12% LDL instead of pure egg yolk improves the quality of rainbow trout sperm post freezing/thawing and maintains sperm freezability in terms of membrane integrity, DNA damage, and fertilization ability, throughout the analysed period. The evaluation of DNA damage in fresh sperm is a useful tool for pointing out pre-freezing differences in quality that render cells more susceptible to cryodamage, and should be considered in the development of cryopreservation procedures.

Acknowledgments This work was supported by grants LE007A06 from the Junta de Castilla y León (Spain). S. Pérez-Cerezales was supported by a PhD grant from the University of León.

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