Elsa Cabrita

Cryopreservation of rainbow trout sperm in large volume straws: application to large scale fertilization

Abstract Traditional 0.25- and 0.5-ml straws have been successfully applied to the cryopreservation of rainbow trout sperm. In order to facilitate the fertilization of large egg batches, and with the purpose of transferring this technique to commercial hatcheries, the suitability of 1.8 and 5 ml straws was studied. Sperm was diluted at a 1:3 ratio in #6 Erdahl and Graham extender with 7% DMSO, 10% egg yolk and 7.5 mg/ml Promine. Freezing was performed at different levels above the surface of liquid nitrogen using a styrofoam box and thawing was carried out in a water bath at different temperatures. Freezing and thawing rates inside the straws were registered with a thermocouple. The success of sperm cryopreservation was checked in vitro using several sperm quality parameters: motility, viability and membrane functionality. Fertility tests were also performed on small batches using a ratio of 200 eggs/ml of diluted sperm. Freezing and thawing rates were similar in 1.8- and 0.5-ml straws. However, lower freezing and thawing rates were achieved with 5-ml straws even at lower freezing and higher thawing temperatures. Motility analysis gave similar results in sperm cryopreserved in all the tested straws (0.5, 1.8 and 5 ml). However, cell viability (61.9%) and fertility (73.2%) were slightly lower in large volumes than in 0.5-ml straws (77.4% and 84%, respectively). Fertilization of larger egg batches (1600–2000 eggs) with sperm frozen in 5-ml straws provided similar fertilisation rates (73%), demonstrating that the slight loss in fertility is compensated by the benefits of 5-ml straws. Our results showed that cryopreservation of large sperm volumes could be useful for hatchery purposes.

Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleosts.

Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, but their use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activated using 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and 60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clustering analysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear). We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulation proportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3 and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted, classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples. Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from these techniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterize males according to their reproductive potential.

The influence of certain aminoacids and vitamins on post-thaw fish sperm motility, viability and DNA fragmentation.

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 10mM), ascorbic acid (1 and 10mM), 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 1mM 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.

Methods in reproductive aquaculture : marine and freshwater species

GAMETE EXTRACTION TECHNIQUES Reproduction and control of ovulation, spermiation and spawning in cultured fish, E. Mananos, N. Duncan, and C. Mylonas Methods for sperm collection, J-C. Gwo SPERM AND EGG QUALITY EVALUATION Sperm quality assessment, E. Cabrita, V. Robles, and P. Herraez Egg quality determination in teleost fish, F. Lahnsteiner, F. Soares, L. Ribeiro, and M.T. Dinis ARTIFICIAL FERTILIZATION IN AQUACULTURED SPECIES:FROM NORMAL PRACTICE TO CROMOSOME MANIPULATION Artificial fertilization in aquaculture species: from normal practice to chromosome manipulation, B. Urbanyi , A. Horvath, and Z. Bokor SPERM AND EGG CRYOPRESERVATIONChilled storage of sperm and eggs, J. Bobe and C. Labbe Basic principles of fish spermatozoa cryopreservation, J. Cloud and S. Patton Cryopreservation of fish oocytes, T. Zhang and E. Lubzens Embryo cryopreservation: what we know until now, V. Robles, E. Cabrita, J.P. Acker, and P. Herraez Cryobiological material and handling procedures, F. Martinez-Pastor and S.L. Adams PROTOCOLS FOR SPERM CRYOPRESERVATION Cryopreservation of sperm of loach (Misgurnus fossilis), J. Kopeika, E. Kopeika, T. Zhang, and D. Rawson Zebrafish Sperm Cryopreservation with N, N-Dimethylacetamide, J.P. Morris IV, A. Hagen, and J.P. Kanki Sperm cryopreservation of live-bearing fishes of the Genus Xiphophorus, Q. Dong, C. Huang, L. Hazlewood, R.B. Walter, and T.R. Tiersch Use of post-thaw silver carp (Hypophtalmichthys molitrix) spermatozoa to increase hatchery productions, B. Alvarez, A. Arenal, R. Fuentes, R. Pimentel, Z. Abad, and E. Pimentel Cryopreservation of common carp sperm, B. Urbanyi and A. Horvath Sperm cryopreservation from sea perch, Lateolabrax japonicas, S-L. Chen, X.S. Ji, and Y-S. Tian Semen cryopreservation of piracanjuba (Brycon orbignyanus), an endangered Brazilian species, A.T.M. Viveiros and A.N. Maria Cryopreservation of endangered Formosan landlocked salmon (Oncorhynchus masou formosanus) semen, J-C. Gwo, H. Ohta, K. Okuzawa, L-Y. Liao, and Y-F. Lin Protocols for the cryopreservation of Salmonidae semen, Lota lota (Gadidae) and Esox lucius (Esocidae), F. Lahnsteiner and N. Mansour Rainbow trout (Oncorhynchus mykiss) sperm cryopreservation, P. Herraez, V. Robles, and E. Cabrita Sperm cryopreservation of sex-reversed rainbow trout (Oncorhynchus mykiss), V. Robles, E. Cabrita, and P. Herraez Cryopreservation of testicular sperm from European catfish (Silurus glanis), G. Maisse, B. Ogier de Baulny, and C. Labbe Semen Cryopreservation of the African catfish, Clarias gariepinus, A.T.M. Viveiros and J. Komen Cryopreservation of sperm from species of the order Acipenseriformes, A. Horvath, B. Urbanyi, and S.D. Mims Different protocols for the cryopreservation of European eel (Anguilla anguilla) sperm, J.F. Asturiano Cryopreservation of Striped Bass Morone saxatilis Spermatozoa, L.C.Woods III, S. He, and K. Jenkins-Keeran Cryopreservation of semen from striped trumpeter (Latris lineata), A.J. Ritar Cryopreservation of Atlantic halibut sperm, Hippoglossus hippoglossus, I. Babiak Sperm cryopreservation: an actual tool for Seabass (Dicentrarchus labrax) reproduction and breeding control in research and production, C. Fauvel and M. Suquet Notes on Diplodus puntazzo sperm cryopreservation, F. Barbato, S. Canese, F. Moretti, A. Taddei, A. Fausto, L. Abelli, M. Mazzini, and K.J. Rana Sperm Cryopreservation from the Marine Teleost, Sparus aurata, E. Cabrita, V. Robles, C. Sarasquete, and P. Herraez Cryopreservation of seabream semen, J-C. Gwo Cryopreservation of sperm from winter flounder, Pseudopleuronectes americanus, R.M. Rideout, I.A.E. Butts, and M.K. Litvak Cryopreservation of sperm in turbot (Psetta maxima), M. Suquet, O. Chereguini, and C. Fauvel Cryopreservation of grouper semen, J-C. Gwo and H. Ohta Cryopreservation of sperm from Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus), R.M. Rideout and D.L. Berlinsky Cryopreservation of small abalone (Haliotis diversicolor supertexa) semen, J-C. Gwo Cryopreservation of Pacific Oyster Sperm, Q. Dong, C. Huang, B. Eudeline, and T. R. Tiersch A simple method for freezing Pacific oyster (Crassostrea gigas) sperm in quantities suitable for commercial hatcheries, S.L. Adams, J.F. Smith, R.D. Roberts, A.R. Janke, H.F. Kaspar, H.R. Tervit, P.A. Pugh, S.C. Webb, and N.G. King Cryopreservation of Eastern Oyster Sperm, C.G. Paniagua-Chavez and T.R. Tiersch Cryopreservation of sea urchin (Evechinus chloroticus) sperm, S.L. Adams, P.A. Hessian, and P.V. Mladenov Protocol for cryopreservation of Penaeus vannamei sperm cells, M. Salazar, M.Lezcano, and C.Granja Techniques for cryopreservation of spermatophores of the giant freshwater prawn, Macrobrachium rosenbergii (de Man), P. Damrongphol and K. Akarasanon Protocol development for the cryopreservation of spermatozoa and spermatophores of the mud crab, Scylla serrata, S. Bhavanishankar and T. Subramoniam Index

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout.

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage. The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2h (fresh) or 5 days at 4 degrees C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.

Effect of cryopreservation on fish sperm subpopulations

The evaluation of the motility data obtained with a CASA system, applying a Two-Step Cluster analysis, identified in seabream sperm 3 different sperm subpopulations that correlated differently with embryo hatching rates. Hence, we designed an experiment to understand the effect of the application of different cryopreservation protocols in these sperm motility-based subpopulations. We analyzed Sparus aurata frozen/thawed semen motility 15, 30, 45 and 60s after activation, using CASA software. Different protocols were applied for cryopreservation: three different cryoprotectants (Dimethyl Sulfoxide (Me(2)SO), Ethylene Glycol (EG) and Propylene Glycol (PG)) each at two different concentrations and two packaging volumes (0.5ml straws, and 1.8ml cryovials) were tested. Different freezing rates were evaluated corresponding to 1, 2, 3, 4 and 8cm above the liquid nitrogen surface for the straws and 1, 2 and 4cm for the cryovials. Motility parameters rendered by CASA were treated with a Two-Step Cluster analysis. Three different subpopulations were obtained: SP1 - slow non-linear spermatozoa, SP2 - slow linear spermatozoa and SP3 - fast linear spermatozoa. We considered SP3 as the subpopulation of interest and focused further analyses on it. Generally, SP3 was the best represented subpopulation 15s after activation and was also the one showing a greater decrease in time, being the least represented after 60s. According to the applied univariate general linear model, samples frozen in straws with 5% Me(2)SO and in cryovials with 10% Me(2)SO at 2 and 1cm from the LN(2,) respectively, produced the best results (closer to the control). Clustering analysis allowed the detection of fish sperm subpopulations according to their motility pattern and showed that sperm composition in terms of subpopulations was differentially affected by the cryopreservation protocol depending on the cryoprotectant used, freezing rates and packaging systems.

Improving Sperm Cryopreservation with Antifreeze Proteins: Effect on Gilthead Seabream (Sparus aurata) Plasma Membrane Lipids

ABSTRACT 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.

Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm

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 mm α-tocopherol or 0.1 mm 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 mm α-tocopherol or 30.6 ± 3.9 μm/sec, 19.5 ± 1.6 μm/sec and 47.9 ± 2.2% in extender containing 1 mm 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.

Vitrification of turbot embryos: preliminary assays

Successful fish embryo cryopreservation is still far from being achieved. Vitrification is considered the most promising option. Many factors are involved in the success of the process. The choice of a proper vitrification solution, the enzymatic permeabilization of embryos to increase cryoprotectant permeability, the adequate container for embryo loading, and the temperature for thawing, were the parameters considered at different developmental stages in the present study. After vitrification, embryo morphology was evaluated under stereoscopic microscopy, establishing the percentage of intact embryos. Two of the studied parameters yielded differences in this percentage, the volume of straw used for embryo loading (1 ml straws were significantly better than 0.5 ml straws, with regard to post-thawed embryo morphologies), and the thawing temperature, achieving 49% of embryos with intact morphology after thawing at 0 degrees C. After thawing, the intact embryos were incubated and periodically observed to detect morphological changes. Changes in the perivitelline space, shrinkage of the yolk and chorion ruptures as well as a progressive whitening of the embryo and yolk were observed. After 8 h all embryos showed clear signs of degradation and during this incubation period no embryo showed any developmental ability.

Sperm cryopreservation of sex-reversed rainbow trout (Oncorhynchus mykiss): parameters that affect its ability for freezing

Abstract Sex control profits the aquaculture industry allowing the obtaining of “all female” rainbow trout populations. Female production is highly profitable since they become sexually mature 1 year later than males, reaching their marketable size before maturation. Sex-reversed rainbow trouts have similar external morphology to normal males but lack sperm ducts, meaning that the animals must be sacrificed to obtain the milt. The peculiarities of the sperm, obtained directly from the testicle, make necessary the development of a specific cryopreservation protocol. In this study, several factors that could affect the freezability of these spermatozoa have been studied: the season of sperm extraction, the method of sperm extraction, and the activation with motility stimulators have been considered. Our results showed that seasonality clearly affects the success of the cryopreservation process, which should always be carried out with sperm obtained in winter season, the natural breeding period. The development of a clean sperm extraction method improved significantly the fertility rates obtained with cryopreserved sperm. The addition of methylxanthines as motility stimulators usually increased motility and fertility rates, but they did not provide significant improvements.