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Investigating sperm cryopreservation in a model tunicate,Ciona intestinalis sp. A q

Gerarda Sorrenti a, Anna Bagnoli b, Valentina Miraglia a, Fabio Crocetta a, Valentina Vitiello b,c,Filomena Ristoratore a, Paola Cirino a, Giovanni Sansone b,c, Paolo Sordino a,d,⁄a Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Naples, Italyb Laboratory of Cryobiology, Department of Biological Sciences, University of Naples ‘‘Federico II’’, Via Mezzocannone 16, I-80134 Naples, Italyc CRIAcq, Interdepartmental Research Center for Acquaculture, University of Naples ‘‘Federico II’’, Via Università 100, Portici, I-80055 Naples, Italyd CNR ISAFOM – Institute for Agricultural and Forest Systems in the Mediterranean, National Research Council, Str. le Lancia, Blocco Palma I, Zona Industriale, I-95121 Catania, Italy

a r t i c l e i n f o

Article history:Received 18 July 2013Accepted 12 November 2013Available online 20 November 2013

Keywords:Ciona intestinalisSpermatozoonCryopreservationDimethyl sulfoxideFertilization success

a b s t r a c t

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes issensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advance-ment of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for thestorage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex ofcryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biol-ogy. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semencryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we esti-mated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, �1 �C/minchilling rate (18 �C to 5 �C) and �13 �C/min freezing rate (5 �C to �80 �C), followed by immersion in liquidnitrogen.

� 2013 Elsevier Inc. All rights reserved.

Introduction

Ciona intestinalis sp. A (Tunicata: Ascidiacea) is a benthic her-maphrodite that inhabits temperate coastal environments, whereit usually forms dense clusters in euryhaline areas with nutrientenrichment [8,34]. This species is a model organism that satisfiesseveral requirements in molecular biology of development, com-parative genomics and population biology, due to the phylogeneticposition relative to Vertebrata, genome sequencing and highpotential of colonization [7,8,12,13,44,48,61]. Knockout andtransgenic methodologies have prompted interest in successfulprotocols for culturing, storage and distribution of geneticresources [45–52]. Not only long-term cryopreservation in liquidnitrogen allows low requirements for space/labor, but it also pro-vides out of season supplies for the genetic studies in C. intestinalissp. A.

In aquatic organisms, cryopreservation of eggs and larvae hasbeen investigated with varying degrees of success [4,23,35,41], sothat sperm freezing still represents the major preservation methodfor the maintenance of biological resources [5,21]. Semen cryo-preservation was originally confined to species of patrimonial

importance (threatened or commercially valuable) [20,32,33,53,54], and has now become central to research in genetics [24,30].

Ascidian spermatozoa possess an asymmetric head that con-tains a single mitochondrion and a poorly developed acrosome[6,18,19,38]. Sperm motility starts upon dilution in seawater, andbecomes more active following release of factors by unfertilizedeggs [55]. In C. intestinalis sp. A, sperm motility and viability lastsat least 12 hours after activation [5]. In the process of C. intestinalisfertilization, acrosomal reaction occurs when the male gametecrosses the vitelline coat, a layer of follicle cells that surroundthe egg, by means of a glycoside–glycosidase binding mechanism[reviewed by 38].

During cryopreservation, the spermatozoon faces various stres-ses that may lead to functional impairment or death [59,60]. Sys-tematic studies were conducted in order to describe the damagesuffered by the sperm at the time of freezing-thawing [36]. Evenin presence of cryoprotective agents (CPA), damages to the integ-rity of spermatozoa are visible at mitochondrial, chromatin ormembrane levels [10]. Mitochondrial damage mainly affects inten-sity, percentage and duration of sperm motility after thawing; irre-versible chromatin alterations may impede the process offertilization and affect embryonic development; modifications ofthe plasma membrane may influence the ability to fuse with theegg cells [2,10,11,17,36,42,49]. As a consequence, post-thaw pro-cesses of sperm motility and fertilization success are not tightly

0011-2240/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.cryobiol.2013.11.005

q Statement of funding: This work was supported by Assemble (EU-FP7).⁄ Corresponding author at: Stazione Zoologica Anton Dohrn, Villa Comunale,

I-80121 Naples, Italy. Fax: +39 081 7641355.E-mail address: paolo.sordino@szn.it (P. Sordino).

Cryobiology 68 (2014) 43–49

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associated. Also, the choice of CPA and of its optimal concentrationdepends on the delicate balance between toxicity and capacity toprotect the cells from the damage caused by freezing and thawingprocesses. Since no universal CPA has been identified, cryopreser-vation procedures are therefore species-specific [26].

Information on sperm cryopreservation in liquid nitrogen(�196 �C) is available for the tunicate species Ciona savignyi (Ascid-iacea) and Oikopleura dioica (Larvacea) [25,40]. Although bothreports are based on the use of dimethyl sulfoxide as CPA, nomethodological detail is available for C. savignyi, while the secondmethod refers to a member of a different class of tunicates.Therefore, a species-specific understanding of a cryostoragemethod at each stage is required in C. intestinalis sp. A.

In this study, cryopreservation toxicity and concentration of fivecommonly used CPAs (dimethyl sulfoxide, ethylene glycol, 1,2-pro-pylene glycol, glycerol and methanol) [16,21,29] were tested onsperm and in the fertilization environment. Then, two chilling(�1 �C/min and �2 �C/min) and three freezing (�7 �C/min,�10 �C/min and �13 �C/min) rates were applied on selected CPAs.With the aim to develop a successful procedure that guaranteesrecovery of the morphological and functional characteristics ofthe cells, morphology and motility of tadpole larvae at each stage,and sperm motility at the beginning and at the end of the overallprocedure, were used as endpoints for the evaluation of spermquality.

Materials and methods

Sampling and acclimatization

C. intestinalis sp. A specimens (10–15 cm total length, 100–120 gweight) were collected by hand at Castellammare di Stabia harbor(Gulf of Naples, Tyrrhenian Sea – 40� 410 3400 N – longitude: 014�280 2500 E). Total length of the main body axis was measured witha vernier calliper from the base to the edge of the oral siphon. Spec-imens were kept in seawater during transport to laboratory facili-ties, where they were acclimatized for 2–3 days in semi-opensystem tank setups at �20 �C. Ripe individuals were then exposedto continuous illumination for 2–3 more days in order to preventgamete spawning [9].

Gamete sample preparation

C. intestinalis sp. A specimens were dissected at the base of theatrial siphon with a sterile blade in order to expose gonoducts. Toavoid self-fertilization, only one kind of gamete was collected fromeach specimen (‘‘Sperm sampling, activation and motility’’, ‘‘Eggsampling and preparation’’).

Sperm sampling, activation and motilitySperm was collected as dry as possible (at a concentration of

�108 sperm/mL [3]) from the spermiduct by using sterile Pasteurpipettes. To avoid sperm contamination with eggs from the adja-cent oviduct, each sperm sample was examined under a stereomi-croscope. Workable sperm samples were then transferred intopre-chilled 1.5 mL vials in ice. Ten micro litres of each spermsample were activated in 10 mL 0.22 lM Millipore filtered naturalsea-water (MFSW) at 18 �C (environmental temperature) and 38practical salinity units (psu) (dilution 1:1000 = �105 sperm/mL)in 15 mL Falcon tubes. Ten micro litres of activated sperm weredropped onto a glass microscope slide (without cover slip covering)and analyzed using an Olympus ITM2 microscope with phase-con-trast lens (200� total magnification). Semen quality was evaluatedvisually for motility classes (0–5) based on the percentage of rapid,vigorous and forward (RVF) motile spermatozoa [16]. About 15–25lL of at least 5 individual dry sperm samples, whose 1:1000

suspension showed motility class P4, were used to generate spermpools of �100 lL to be used each for both experimental procedureand corresponding controls.

Egg sampling and preparationEggs were collected with Pasteur pipettes and transferred in a

9 cm Petri dish filled with 0.22 lM MFSW at 18 �C and 38 psu.Egg maturity and possible sperm contamination were examinedunder a stereomicroscope. Workable egg samples from 5 individu-als were pooled in a single 9 cm Petri dish where they wereallowed to expand the chorion and the follicle cells, which makethe eggs floating and improve fertilization, and were then usedfor experimental procedures.

Optimization of the fertilization protocol and fertilization success

Fertilization tests were carried out in 9 cm Petri dishes filledwith 10 mL 0.22 lM MFSW at 18 �C and 38 psu [9,31]. Eggs wereadded to a final concentration of �6 egg/mL (�60 eggs per Petridish). This experimental setup constitutes the ‘‘fertilization envi-ronment’’ (FE), and it will be named hereafter as such. In order todetermine the optimal egg/sperm ratio, sperm was first activatedin a 1:1000 dilution (10 lL dry sperm in 10 mL MFSW: �105

sperm/mL) in 15 mL Falcon tubes at 18 �C, and then further dilutedto 1:10,000, 1:100,000 and 1:1,000,000 in the FE. Petri dishes werestored at 18 �C, and fertilization success was evaluated in terms ofpercentage of eggs in the FE that developed in normal tadpole lar-vae after 15–18 h with a stereomicroscope [27]. Final sperm con-centration that allowed the highest percentage of larvae withregular morphology and motility was selected for the subsequentexperimental phases.

Toxicity tests in the fertilization environment

In order to assess the potential toxicity of CPAs on development,dimethyl sulfoxide (Me2SO), ethylene glycol (EG), 1–2 propyleneglycol (PG), methanol (MetOH) and glycerol (GlOH) (Sigma–Aldrich) were added to the FE at the highest concentration to beused in this study (20% Me2SO, EG, PG and GlOH; 10% MetOH) withrespect to activated sperm at the selected final dilution, soon fol-lowed by the addition of sperm (‘‘Optimization of the fertilizationprotocol and fertilization success’’). Fertilization success wascompared with a control consisting of activated sperm readily usedat the selected concentration (‘‘Optimization of the fertilizationprotocol and fertilization success’’) in a CPA-free FE.

Toxicity tests on sperm

In order to assess potential toxicity on sperm, different CPA con-centrations (5%, 10%, 15% and 20% Me2SO, EG, PG and GlOH; 4%, 6%,8% and 10% MetOH) were tested each in 1 mL activated sperm(1:1000 dry sperm) in 1.5 mL vials by adding corresponding CPAamounts all at once, followed by gentle mixing. CPA-treated spermsamples were incubated for 10 min and 20 min at 18 �C, and thenwere added to the FE at the selected final sperm concentration(‘‘Optimization of the fertilization protocol and fertilization suc-cess’’). Fertilization success was compared with three CPA-freecontrols consisting of activated sperm (i) readily used (Ct0), (ii)used after 10 min (Ct10) and (iii) used after 20 min (Ct20), at theselected concentration (‘‘Optimization of the fertilization protocoland fertilization success’’).

Chilling tests

In order to assess the combined effect of CPAs and chilling, 1 mLactivated sperm (‘‘Optimization of the fertilization protocol andfertilization success’’) containing selected CPAs and CPA concentra-

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tions (‘‘Toxicity tests on sperm’’) was inserted in cryopaillettes byPasteur pipettes and immediately chilled from 18 �C to 5 �C in aNESLAB RTE-211 water bath (Thermo) according to two differentrates: �2 �C/min (�7 min total exposure to CPAs) and �1 �C/min(�13 min total exposure to CPAs). Then, different volumes ofchilled sperm (according to CPA concentration) for each assay weredirectly used in the FE to reach the selected sperm concentration(‘‘Optimization of the fertilization protocol and fertilization suc-cess’’), and compared with three CPA-free controls consisting ofactivated sperm (i) readily used, and (ii–iii) chilled at the two dif-ferent rates herein analyzed, all at the selected concentration(‘‘Optimization of the fertilization protocol and fertilization suc-cess’’). Fertilization success was measured as in ‘‘Optimization ofthe fertilization protocol and fertilization success’’. The best chill-ing rate was selected for freezing trials.

Freezing tests

Sperm chilled at the selected conditions (‘‘Chilling tests’’) wasimmediately frozen at �80 �C using a CRYOMED 8023 freezer (For-ma Scientific Inc.) according to three different freezing rates(�7 �C/min, �10 �C/min and �13 �C/min). Cryopaillettes were thenplunged into liquid nitrogen for cryopreservation. The day after,different volumes of sperm (according to CPA concentration) foreach assay were thawed for 10 s in a 30 �C water bath and usedin the FE to reach the selected sperm concentration (‘‘Optimizationof the fertilization protocol and fertilization success’’). Fertilizationsuccess was compared with five CPA-free controls consisting ofactivated sperm (i) readily used, (ii) chilled at the selected rate,and (iii–v) chilled as in (ii) and frozen at the three different ratesherein analyzed, all at the selected concentration (‘‘Optimizationof the fertilization protocol and fertilization success’’). The (iii–v)controls were designed in order to evaluate whether sperm of C.intestinalis sp. A is able to recover from low temperatures withoutcryoprotectants.

Sperm motility after chilling and freezing rates

To examine the effect of selected chilling (‘‘Chilling tests’’) andfreezing (‘‘Freezing tests’’) rates on CPA-added sperm motility clas-ses, sperm samples were examined using the semen quality evalu-ation procedure described in ‘‘Sperm sampling, activation andmotility’’. Sperm was dropped onto a glass microscope slide soonafter chilling or the day after freezing by previously thawing for10 s in a 30 �C water bath. Results were compared with those ofthree CPA-free controls consisting of activated sperm (i) readilyused, (ii) chilled at the selected rate, and (iii) chilled as in (ii) and

frozen at the selected rate, all at the selected concentration (‘‘Opti-mization of the fertilization protocol and fertilization success’’).The rationale of the (iii) control is the same as for the (iii–v) con-trols in ‘‘Freezing tests’’.

Statistical analysis

Each experiment was run nine times (n = 9) in each experimen-tal phase. Statistical analysis was performed using BioEstat 5.0. Apaired sample Student’s t test was used to search for significantdifferences between two sample groups. One-way ANOVA wasused to evaluate the effect of different CPA concentrations withthe same chilling rate, while two-way ANOVA was used for com-paring different concentrations with different chilling and freezinggradients. Results of these statistical tests were expressed asmean ± standard deviation. The Mann–Whitney test was used toanalyze motility classes, and the results were expressed as median(interquartile range). In each figure, statistical significance of col-umn values is indicated by same or different superscripts.p < 0.05 was chosen as threshold for significance [14,56,57].

Results

Optimization of fertilization protocol

Percentages of normal larvae obtained by fertilizing eggs withthe three sperm dilutions (1:1,000,000, 1:100,000, 1:10,000) arereported in Table 1 (Supplementary data) and Fig. 1. A good fertil-ization ratio was obtained by using 1:1,000,000 semen dilution(94.49 ± 2.63% normal larvae). The other two tested dilutionsshowed lower fertilization outcomes (24.32 ± 6.34% and4.02 ± 1.14% normal larvae with 1:100,000 and 1:10,000 dilutions,respectively). Therefore, the 1:1,000,000 dilution ratio was chosenfor the fertilization procedure.

Toxicity tests in the fertilization environment

Following CPA addition in the FE (‘‘Toxicity tests in the fertiliza-tion environment’’), percentages of normal larvae obtained by fer-tilizing eggs with diluted sperm (‘‘Optimization of fertilizationprotocol’’) are reported in Table 2 (Supplementary data) andFig. 2. Slight differences were noted between the control(91.80 ± 3.55%) and the five CPAs tested (89.40 ± 4.20% to90.91 ± 2.04%) (p > 0.05) (Table 3, Supplementary data), suggestinglow or no CPA toxicity at the tested final dilutions on gamete inter-action and larval development, and therefore making superfluousCPA removal before fertilization.

Fig. 1. Effect of three different sperm dilutions on fertilization success. Dilution rates refer to dry sperm. Results are expressed in terms of fertilization success (% normallarvae). n = 9 replicates.

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Toxicity tests on sperm

Percentages of normal larvae obtained by fertilizing eggs in theFE with sperm exposed to different CPA concentrations and incu-bation times are reported in Table 4 (10 min sperm exposure toCPAs), Table 5 (20 min) (Supplementary data) and Fig. 3. As to con-trols, a slightly reduced fertilization success was found when com-paring activated sperm readily used (92.13 ± 2.65%) and used after10 min incubation time (90.64 ± 2.09%) (p > 0.05), while a moresubstantial reduction was observed in the control with sperm usedafter 20 min incubation time (78.88 ± 1.94%) (p < 0.05) (Table 6,Supplementary data). PG, MetOH and GlOH were highly toxic atall tested concentrations and incubation times, with the most se-vere effects on spermatozoa and fertilization observed after20 min exposure (0 to 5.19 ± 0.48%) (p < 0.05) (Tables 4, 5, 7, 8,Supplementary data). These three CPAs were therefore excludedfrom the subsequent analyses. On the other hand, while percent-ages of normal larvae in Me2SO and EG tests were very low after20 min exposure (0.32 ± 0.64% to 17.63 ± 1.21% for Me2SO;2.02 ± 0.67% to 15.04 ± 0.63% for EG), 10 min exposure to 5%, 10%and 15% Me2SO and to 5% and 10% EG allowed high percentagesof normal development (83.58 ± 1.16% to 93.11 ± 2.30%) (Table 4,Supplementary data). Of these, only the percentage of normaldevelopment obtained with 5% EG was statistically significant(p > 0.05) (Table 7, Supplementary data). Higher Me2SO (20%) and

EG (15% and 20%) concentrations had clearly toxic effects(p < 0.05). Following these results, only 2 CPAs (Me2SO and EG)and 2 concentrations each (5% and 10%) were selected for the sub-sequent experimental phases.

Chilling

A small but significant difference was observed between per-centages of regular development in controls with activated spermreadily used (90.92 ± 2.84%) and used after �1 �C/min chilling(83.83 ± 3.47%) (p < 0.05) (Table 10, Supplementary data) (Fig. 4).The two selected CPAs were both compatible with �80% larvaldevelopment when a �1 �C/min chilling rate was applied (Table 9,Supplementary data) (Fig. 4). Conversely, faster chilling(�2 �C/min) caused a remarkable reduction of normal larval devel-opment both in the corresponding control (13.27 ± 2.80%) and withCPAs, with percentages of successful development ranging from10.76 ± 2.88 to 22.72 ± 3.20 (p < 0.05) (Table 10, Supplementarydata) (Fig. 4). Thus, the �1 �C/min chilling rate was selected forfreezing rate tests.

Freezing

Percentages of normal larvae obtained by fertilizing eggs in theFE with sperm chilled at �1 �C/min rate and frozen at the three

Fig. 2. Effect of cryoprotectants on the fertilization environment. Results are expressed in terms of fertilization success (% normal larvae). n = 9 replicates. Me2SO: dimethylsulfoxide, EG: ethylene glycol, PG: 1–2 propylene glycol, MetOH: methanol, GlOH: glycerol.

Fig. 3. Effect of cryoprotectants on sperm fertility after 10 and 20 min of incubation. Results are expressed in terms of fertilization success (% normal larvae). n = 9 replicates.Me2SO: dimethyl sulfoxide, EG: ethylene glycol, PG: 1–2 propylene glycol, MetOH: methanol, GlOH: glycerol.

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different slow rates (�7 �C/min, �10 �C/min and �13 �C/min) arereported in Table 11 (Supplementary data) and Fig. 5. Fertilizationsuccess was null in CPA-free frozen sperm controls. All freezingrates tested induced reductions in the percentage of normal larvaewith respect to environmental control (p < 0.05) (Table 12). High-est normal larvae percentages were obtained when applying�13 �C/min freezing rate (61.42 ± 4.69% to 80.32 ± 0.89%) (Table 11,Supplementary data). In particular, 5% EG and 10% Me2SO bothprovided �80% normal larvae, although differences with respectto the chilled control were not statistically significant only in thecase of 10% Me2SO (Table 12, Supplementary data). Lower fertiliza-tion success was obtained with 5% Me2SO (61.42 ± 4.69%) and 10%EG (55.54 ± 2.31%) at �13 �C/min freezing rate. Lower freezingrates (�7 �C/min and �10 �C/min) were always harmful to sperm,with a disabled capacity of fertilization (�7 �C/min: 31.94 ± 5.20%with 5% Me2SO, 14.99 ± 1.71% with 10% EG; �10 �C/min:37.75 ± 3.33% with 5% Me2SO, 18.47 ± 2.17% with 10% EG)(Table 11, Supplementary data).

Sperm motility at the selected chilling and freezing rates

Motility classes of thawed spermatozoa following chilling(�1 �C/min) and freezing (�13 �C/min) treatments at the selectedCPA concentrations (5% and 10% Me2SO, 5% and 10% EG) arereported in Table 13 (Supplementary data) and Fig. 6. The absenceof CPAs induced a motility reduction (p < 0.05) in post-chilling con-trol (4) relative to the environmental control (4.5) (Tables 13 and14, Supplementary data). Sperm motility after chilling with 5%and 10% Me2SO is similar (class 4) (p > 0.05) to the post-chillingcontrol (Table 14, Supplementary data). A slight motility class

reduction was noted when using 5% and 10% EG (3.5 and 3, respec-tively) (Table 13, Supplementary data). After freezing, no spermsurvived in the control without CPAs, while a drastic reduction ofsperm motility was observed in 5% and 10% EG (1 and 1.5, respec-tively), with RVF values always lower than class 2. On the contrary,sperm motility of samples that were treated with 5% and 10% Me2-

SO belonged to class 3, with no difference between the two con-centrations (p > 0.05) (Tables 13 and 14, Supplementary data).

Discussion

Cryopreservation of sperm in C. intestinalis sp. A appears to be apromising method for preserving cell function and for providingmale gametes compatible with normal development. All phasesof the cryopreservation procedure were studied in relation to thetype of cell under examination, as a requisite to restore the mor-phological and physiological characteristics of fresh semen afterthawing. Moreover, the sperm density used to fertilize the eggsshould be high enough to guarantee fertilization but not too highto cause polispermy, that may generate abnormal embryos. Indeed,egg/sperm ratio tests revealed that the most satisfactory results interms of percentage of fertilization success were obtained with thelowest sperm density (102 sperm/mL). This result disagrees with aprevious report that recommended higher sperm density (108–107

sperm/mL) [3], a discrepancy likely due to differences in gametequality and processing details.

The results obtained in the incubation trials of C. intestinalis sp.A spermatozoa to CPAs showed low toxicity at 615% Me2SO con-centration for 10 min incubation time (Table 3, Supplementarydata); this CPA was used also for sperm cryopreservation in the

Fig. 4. Effect of different chilling rates on sperm fertility. Controls consist of cryoprotectant-free activated sperm readily used, chilled at �1 �C/min and at �2 �C/min. Resultsare expressed in terms of fertilization success (% normal larvae). n = 9 replicates. Me2SO: dimethyl sulfoxide, EG: ethylene glycol.

Fig. 5. Effect of different freezing rates on sperm fertility. Controls consist of cryoprotectant-free activated sperm readily used, chilled at the selected condition, or chilled atthe selected condition (�1 �C/min) and frozen at �7 �C/min, �10 �C/min and �13 �C/min. Results are expressed in terms of fertilization success (% normal larvae). Last threecontrols were incompatible with fertilization. n = 9 replicates. Me2SO: dimethyl sulfoxide, EG: ethylene glycol.

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tunicate species Oikopleura dioica and C. savigny [25,37,40]. On thecontrary, sperm of C. intestinalis sp. A showed a rather high sensi-tivity to PG, GlOH and MetOH even at low concentrations and after10 min exposure (Table 3, Supplementary data). As previouslyreported for other aquatic organisms, these results suggest thatGlOH is toxic to C. intestinalis sp. A gametes. They also reveal a highsensitivity of the model tunicate spermatozoa to PG and MetOH,especially when compared to other aquatic species [14,22,29,39].However, results obtained with increased time of incubation, evenat the lowest CPA concentrations (Table 4, Supplementary data),suggest the overall sensitivity of this biological system to the timeof contact/interaction with CPAs. C. intestinalis sp. A sperm showeda good adaptability to slow chilling at �1 �C/min rate (�13 min,18 �C to 5 �C), with or without cryoprotectants (Table 5, Supple-mentary data). In comparison with spermatozoa of other aquatic

invertebrate species (e.g., molluscs, echinoderms and crustaceans),C. intestinalis sp. A sperm cryopreservation appears to require shortadaptation and long chilling times [1,14,15,28,41,56–58]. In thisreport, 10% Me2SO provided the best results in terms of percent-ages of normal tadpole larvae (�60–80%) and semen motility (class3, 50% RVF spermatozoa). 5% EG provided comparable results interms of fertilization success (�55–70%), confirming the cryopro-tective value of this compound [56,57]. However, EG-based freez-ing leads to lower post-thaw RVF values, lending support to thehypothesis that sperm motility in C. intestinalis sp. A is not strictlyimportant for fertilization success, and therefore is not an impecca-ble indicator of the physiological status of spermatozoa after thaw-ing. This is in agreement with observations on spermatozoa ofother aquatic species [41,43,56].

A flowchart summarizing the experimental design is reported inFig. 7. Based on our results, a protocol for C. intestinalis sp. A semencryopreservation is here proposed: addition of 10% Me2SO to1:1000 diluted semen in 0.22 lM MFSW at 18 �C and 38 psu, chill-ing from 18 �C to 5 �C at �1 �C/min rate, freezing from 5 �C to�80 �C at �13 �C/min rate, followed by storage in liquid nitrogen(�196 �C); thawing for 10 s in a 30 �C water bath.

Acknowledgments

The authors thank C. Paetzold (University of Prince EdwardsIsland) and R. Marino (SZN) for valuable advices, and two anony-mous reviewers for constructive comments on an earlier versionof this paper.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.cryobiol.2013.11.005.

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Fig. 6. Effect of selected chilling and freezing rates on sperm motility. Controls consist of cryoprotectant-free activated sperm readily used, chilled at the selected condition,chilled and frozen at the selected conditions. Results are expressed in terms of motility class (from 0 to 5). Last control was incompatible with fertilization. n = 9 replicates. Nobox is reported when q1 = q3. Me2SO: dimethyl sulfoxide, EG: ethylene glycol, q1: quartile 1, q3: quartile 3, Min: minima, Max: maxima, CH: chilling, FR: freezing.

Fig. 7. Step-by-step investigation of sperm cryopreservation in Ciona intestinalis sp.A. The endpoints for the evaluation of sperm quality were morphology and motilityof tadpole larvae at each stage, and sperm motility at the beginning and at the endof the overall procedure. n = 9 replicates. CPA: cryoprotectant, Me2SO: dimethylsulfoxide, EG: ethylene glycol, PG: 1–2 propylene glycol, MetOH: methanol, GlOH:glycerol.

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G. Sorrenti et al. / Cryobiology 68 (2014) 43–49 49

Supplementary material

Table 1. Effect of sperm dilution on fertilization success.

1:1000000 1:100000 1:10000

EN NL % EN NL % EN NL %

1 61 58 95.08 65 17 26.15 59 2 3.38

2 67 63 94.03 66 20 30.30 62 4 6.45

3 65 62 95.38 70 14 20.00 65 3 4.61

4 59 54 91.52 72 24 33.33 56 2 3.57

5 62 60 96.77 66 12 18.18 62 2 3.22

6 65 62 95.38 60 12 20.00 63 3 4.76

7 58 57 98.27 54 10 18.51 69 2 2.89

8 55 52 94.54 58 19 32.75 70 3 4.28

9 57 51 89.47 61 12 19.67 67 2 2.98

Ma 94.49 24.32 4.02

σ 2.63 6.34 1.14

Data are presented as average and standard deviation. Abbreviations: EN, number of eggs; NL,

number of normal larvae; %, percentage of normal larvae; Ma, average; σ, standard deviation.

Table 2. Effect of cryoprotectants in the fertilization environment.

Ct0 20% Me2SO 20% EG

EN NL % EN NL % EN NL %

1 66 57 86.36 63 57 90.47 65 60 92.30

2 65 59 90.76 57 50 87.71 59 52 88.13

3 61 58 95.08 64 58 90.62 66 59 89.39

4 46 41 89.13 51 49 96.07 58 49 84.48

5 43 38 88.37 48 42 87.50 49 43 87.75

6 50 48 96.00 59 51 86.44 53 48 90.56

7 74 67 90.54 70 66 94.28 67 59 88.05

8 72 69 95.83 67 60 89.55 78 73 93.58

9 68 64 94.11 61 50 81.96 63 59 93.65

Ma 91.80 89.40 89.77

σ 3.55 4.20 3.05

20% PG 10% MetOH 20% GlOH

EN NL % EN NL % EN NL %

1 64 59 92.18 59 53 89.83 58 52 89.65

2 69 60 86.95 65 59 90.76 62 57 91.93

3 71 65 91.54 69 61 88.40 51 46 90.19

4 62 54 87.09 54 50 92.59 53 47 88.67

5 57 53 92.98 59 53 89.83 54 50 92.59

6 47 41 87.23 49 46 93.87 50 45 90.00

7 71 69 97.18 69 64 92.75 68 60 88.23

8 69 57 82.60 73 65 89.04 71 67 94.36

9 62 56 90.32 70 62 88.57 67 62 92.53

Ma 89.79 90.63 90.91

σ 4.29 1.99 2.04

Data are presented as average and standard deviation. Control consists of cryoprotectant-free

activated sperm readily used (Ct0). Abbreviations: C, control; Me2SO, dimethylsulfoxide; EG,

ethylene glycol; PG, 1–2 propylene glycol; MetOH, methanol; GlOH, glycerol; EN, number of

eggs; NL, number of normal larvae; %, percentage of normal larvae; Ma, average; σ, standard

deviation.

Table 3. Statistical significance of the differences in cryoprotectant toxicity in the fertilization

environment.

Ct0 20% Me2SO 20% EG 20% PG 10% MetOH 20% GlOH

Ct0 - >0.05 >0.05 >0.05 >0.05 >0.05

20% Me2SO - >0.05 >0.05 >0.05 >0.05

20% EG - >0.05 >0.05 >0.05

20% PG - >0.05 >0.05

10% MetOH - >0.05

20% GlOH -

Data are presented as p-value. Control consists of cryoprotectant-free activated sperm readily used

(Ct0). Abbreviations: C, control; Me2SO, dimethylsulfoxide; EG, ethylene glycol; PG, 1–2

propylene glycol; MetOH, methanol; GlOH, glycerol.

Table 4. Effect of cryoprotectants on sperm (10 min incubation time).

Ct0 Ct10 5% Me2SO 10% Me2SO 15% Me2SO 20% Me2SO 5% EG 10% EG 15% EG 20% EG 5% PG

EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL %

1 71 66 92.95 70 65 92.85 67 60 89.55 66 58 87.87 65 54 83.07 66 14 21.21 57 54 94.73 58 52 89.65 57 33 57.89 55 2 3.63 68 15 22.05

2 66 62 93.93 67 60 89.55 68 61 89.70 69 59 85.50 68 57 83.82 68 15 22.05 58 53 91.37 57 51 89.47 55 31 56.36 56 2 3.57 69 15 21.73

3 60 56 93.33 57 52 91.22 71 64 90.14 72 62 86.11 69 56 81.15 67 15 22.38 56 53 94.64 60 54 90.00 59 34 57.62 52 2 3.84 66 14 21.21

4 67 63 94.02 69 63 91.30 68 62 91.17 65 58 89.23 64 54 84.37 60 13 21.66 63 57 90.47 61 55 90.16 63 36 57.14 65 3 4.61 73 16 21.91

5 69 65 94.20 71 62 87.32 65 59 90.76 67 57 85.07 68 57 83.82 62 14 22.58 67 61 91.04 63 56 88.88 67 38 56.71 68 3 4.41 72 16 22.22

6 65 61 93.84 68 63 92.64 64 57 89.06 63 55 87.30 67 56 83.58 64 11 17.18 66 60 90.90 66 60 90.90 68 39 57.35 70 3 4.28 75 17 22.66

7 59 54 91.52 58 51 87.93 69 58 84.05 68 56 82.35 69 59 85.50 68 17 25.00 70 66 94.28 69 62 89.85 69 39 56.52 78 3 3.84 60 13 21.66

8 63 55 87.30 57 53 92.98 71 63 88.73 72 60 83.33 68 57 83.82 69 16 23.18 75 70 93.33 72 65 90.27 70 40 57.14 75 3 4.00 64 15 23.43

9 67 59 88.05 60 54 90.00 70 63 90.00 69 61 88.40 71 59 83.09 64 15 23.43 72 70 97.22 71 65 91.54 65 37 56.92 74 3 4.05 65 14 21.53

Ma 92.13 90.64 89.24 86.13 83.58 22.07 93.11 90.08 57.07 4.02 22.05

σ 2.65 2.09 2.08 2.31 1.16 2.14 2.30 0.78 0.50 0.35 0.66

10% PG 15% PG 20% PG 4% MetOH 6% MetOH 8% MetOH 10% MetOH 5% GlOH 10% GlOH 15% GlOH 20% GlOH

EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL %

1 75 2 2.66 73 2 2.73 74 2 2.70 62 24 38.70 62 16 25.80 67 10 14.92 63 8 12.69 65 6 9.23 58 1 1.72 62 1 1.61 62 0 0

2 71 1 1.40 72 2 2.77 73 2 2.73 65 25 38.46 65 17 26.15 66 10 15.15 66 8 12.12 66 7 10.60 57 1 1.75 65 1 1.53 65 0 0

3 69 1 1.44 72 3 4.16 74 2 2.70 63 25 39.68 62 16 25.80 60 9 15.00 61 7 11.47 62 6 9.67 55 1 1.81 63 1 1.58 63 0 0

4 75 2 2.66 70 2 2.85 59 1 1.69 69 24 34.78 65 17 26.15 67 10 14.92 68 8 11.76 70 7 10.00 76 2 2.63 69 1 1.44 69 0 0

5 74 2 2.70 71 2 2.81 60 1 1.66 71 25 35.21 65 17 26.15 66 10 15.15 69 8 11.59 72 7 9.72 78 2 2.56 71 1 1.40 71 0 0

6 75 2 2.66 69 2 2.89 63 1 1.58 68 24 35.29 73 19 26.02 68 10 14.70 71 8 11.26 69 7 10.14 77 2 2.59 68 1 1.47 68 0 0

7 58 1 1.72 69 2 2.89 57 1 1.75 55 22 40.00 58 15 25.86 55 8 14.54 57 7 12.28 57 6 10.52 59 1 1.69 55 0 0 55 0 0

8 54 1 1.85 63 2 3.17 55 1 1.81 58 23 39.65 58 15 25.86 60 9 15.00 59 7 11.86 59 6 10.16 62 1 1.61 58 0 0 58 0 0

9 59 1 1.69 64 2 3.12 59 1 1.69 61 24 39.34 61 16 26.22 59 9 15.25 62 8 12.90 54 5 9.25 60 1 1.66 61 0 0 61 0 0

Ma 2.09 3.05 2.04 37.90 26.00 14.96 11.99 9.92 2.00 1.00 0

σ 0.56 0.44 0.51 2.16 0.17 0.22 0.55 0.49 0.44 0.75 0

Data are presented as average and standard deviation. Controls consist of cryoprotectant-free activated sperm readily used (Ct0), and used 10 min

after collection (Ct10). Abbreviations: C, control; Me2SO, dimethylsulfoxide; EG, ethylene glycol; PG, 1–2 propylene glycol; MetOH, methanol;

GlOH, glycerol; EN, number of eggs; NL, number of normal larvae; %, percentage of normal larvae; Ma, average; σ, standard deviation.

Table 5. Effect of cryoprotectants on sperm (20 min incubation time).

Ct20 5% Me2SO 10% Me2SO 15% Me2SO 20% Me2SO 5% EG 10% EG 15% EG 20% EG 5% PG

EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL %

1 68 54 79.41 67 12 17.91 66 9 13.63 65 6 9.23 66 0 0 57 8 14.03 58 7 12.06 57 3 5.26 55 1 1.81 68 3 4.41

2 66 53 80.30 68 12 17.64 69 9 13.04 68 6 8.82 68 1 1.47 58 9 15.51 57 7 12.28 55 2 3.63 56 1 1.78 69 3 4.34

3 60 49 81.66 71 13 18.30 72 10 13.88 69 7 10.14 67 0 0 56 8 14.28 60 8 13.33 59 3 5.08 52 1 1.92 66 2 3.03

4 68 54 79.41 68 13 19.11 65 8 12.30 64 6 9.37 60 0 0 63 10 15.87 61 8 13.11 63 3 4.76 65 2 3.07 73 3 4.10

5 72 58 80.55 65 12 18.46 67 9 13.43 68 6 8.82 62 0 0 67 10 14.92 63 8 12.69 67 3 4.47 68 2 2.94 72 3 4.16

6 70 54 77.14 64 12 18.75 63 8 12.69 67 6 8.95 64 0 0 66 10 15.15 66 9 13.63 68 4 5.88 70 1 1.42 75 3 4.00

7 63 48 76.19 69 11 15.94 68 8 11.76 69 6 8.69 68 0 0 70 11 15.71 69 9 13.04 69 4 5.79 78 2 2.56 60 2 3.33

8 59 45 76.27 71 12 16.90 72 9 12.50 68 6 8.82 69 1 1.44 75 11 14.66 72 10 13.88 70 4 5.71 75 1 1.33 64 3 4.68

9 62 49 79.03 70 11 15.71 69 9 13.04 71 7 9.85 64 0 0 72 11 15.27 71 9 12.67 65 3 4.61 74 1 1.35 65 3 4.61

Ma 78.88 17.63 12.92 9.19 0.32 15.04 12.97 5.02 2.02 4.07

σ 1.94 1.21 0.67 0.51 0.64 0.63 0.60 0.73 0.67 0.56

R 10% PG 15% PG 20% PG 4% MetOH 6% MetOH 8% MetOH 10% MetOH 5% GlOH 10% GlOH 15% GlOH 20% GlOH

EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL %

1 75 3 4.00 73 4 5.47 74 2 2.70 62 2 3.22 62 1 1.61 67 1 1.49 63 3 4.76 65 1 1.53 58 1 1.72 62 0 0 62 0 0

2 71 3 4.22 72 4 5.55 73 2 2.73 65 3 4.61 65 1 1.53 66 0 0 66 3 4.54 66 1 1.51 57 1 1.75 65 0 0 65 0 0

3 69 3 4.34 72 4 5.55 74 2 2.70 63 2 3.17 62 1 1.61 60 0 0 61 3 4.91 62 0 0 55 1 1.81 63 0 0 63 0 0

4 75 3 4.00 70 4 5.71 59 1 1.69 69 3 4.34 65 2 3.07 67 1 1.49 68 4 5.88 70 1 1.42 76 2 2.63 69 0 0 69 0 0

5 74 3 4.05 71 3 4.22 60 1 1.66 71 3 4.22 65 2 3.07 66 0 0 69 4 5.79 72 1 1.38 78 2 2.56 71 0 0 71 0 0

6 75 3 4.00 69 3 4.34 63 1 1.58 68 3 4.41 73 2 2.73 68 0 0 71 4 5.63 69 1 1.44 77 2 2.59 68 0 0 68 0 0

7 58 2 3.44 69 3 4.34 57 1 1.75 55 2 3.63 58 1 1.72 55 0 0 57 3 5.26 57 1 1.75 59 1 1.69 55 0 0 55 0 0

8 54 2 3.70 63 3 4.76 55 1 1.81 58 3 5.17 58 1 1.72 60 0 0 59 3 5.08 59 0 0 62 1 1.61 58 0 0 58 0 0

9 59 3 5.08 64 3 4.68 59 1 1.69 61 3 4.91 61 1 1.63 59 0 0 62 3 4.83 54 0 0 60 1 1.66 61 0 0 61 0 0

Ma 4.09 4.96 2.04 4.19 2.08 0.33 5.19 1.00 2.00 0 0

σ 0.45 0.60 0.51 0.70 0.67 0.65 0.48 0.76 0.44 0 0

Data are presented as average and standard deviation. Control consists of cryoprotectant-free sperm used 20 min after collection (Ct20).

Abbreviations: C, control; Me2SO, dimethylsulfoxide; EG, ethylene glycol; PG, 1–2 propylene glycol; MetOH, methanol; GlOH, glycerol; EN,

number of eggs; NL, number of normal larvae; %, percentage of normal larvae; Ma, average; σ, standard deviation.

Table 6. Statistical significance of differences between controls.

Ct0 Ct10 Ct20

Ct0 - >0.05 <0.05

Ct10 - <0.05

Ct20 -

Data are presented as p-value. Controls consist of cryoprotectant-free activated sperm readily used

(Ct0), and used 10 min (Ct10) and 20 min after collection (Ct20). Abbreviation: C, control.

Table 7. Statistical significance of differences in cryoprotectant effects on sperm (10 min incubation time).

Ct0 Ct10 5%

Me2SO

10%

Me2SO

15%

Me2SO

20%

Me2SO

5%

EG

10%

EG

15%

EG

20%

EG

5%

PG

10%

PG

15%

PG

20%

PG

4%

MetOH

6%

MetOH

8%

MetOH

10%

MetOH

5%

GlOH

10%

GlOH

15%

GlOH

20%

GlOH

Ct0 - >0.05 <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

Ct10 - >0.05 <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% Me2SO - <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

15% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

20% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% EG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% EG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

15% EG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

20% EG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% PG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% PG - >0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05

15% PG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

20% PG - <0.05 <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05

4% MetOH - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

6% MetOH - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

8% MetOH - <0.05 <0.05 <0.05 <0.05 <0.05

10% MetOH - <0.05 <0.05 <0.05 <0.05

5% GlOH - <0.05 <0.05 <0.05

10% GlOH - <0.05 <0.05

15% GlOH - <0.05

20% GlOH -

Data are presented as p-value. Controls consist of cryoprotectant-free activated sperm readily used (Ct0) and used 10 min after collection (Ct10).

Abbreviations: C, control; Me2SO, dimethylsulfoxide; EG, ethylene glycol; PG, 1–2 propylene glycol; MetOH, methanol; GlOH, glycerol.

Table 8. Statistical significance of differences in cryoprotectant effects on sperm (20 min incubation time).

Ct20 5%

Me2SO

10%

Me2SO

15%

Me2SO

20%

Me2SO

5%

EG

10%

EG

15%

EG

20%

EG

5%

PG

10%

PG

15%

PG

20%

PG

4%

MetOH

6%

MetOH

8%

MetOH

10%

MetOH

5%

GlOH

10%

GlOH

15%

GlOH

20%

GlOH

Ct20 - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% Me2SO - <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% Me2SO - <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

15% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

20% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% EG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% EG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

15% EG - <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05

20% EG - <0.05 <0.05 <0.05 >0.05 <0.05 >0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05

5% PG - >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% PG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

15% PG - <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

20% PG - <0.05 >0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05

4% MetOH - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

6% MetOH - <0.05 <0.05 <0.05 >0.05 <0.05 <0.05

8% MetOH - <0.05 <0.05 <0.05 <0.05 <0.05

10% MetOH - <0.05 <0.05 <0.05 <0.05

5% GlOH - <0.05 <0.05 <0.05

10% GlOH - <0.05 <0.05

15% GlOH - >0.05

20% GlOH -

Data are presented as p-value. Controls consist of cryoprotectant-free sperm used 20 min after collection (Ct20). Abbreviations: C, control; Me2SO,

dimethylsulfoxide; EG, ethylene glycol; PG, 1–2 propylene glycol; MetOH, methanol; GlOH, glycerol.

Table 9. Effect of chilling rates on the fertilization success under selected cryoprotectant conditions.

-1 °C/min

Ct0 Ct0 - CH 5% Me2SO 10% Me2SO 5% EG 10% EG

EN NL % EN NL % EN NL % EN NL % EN NL % EN NL %

1 67 64 95.52 61 53 86.88 61 50 81.96 63 53 84.12 69 58 84.05 64 59 92.18

2 64 59 92.18 63 54 85.71 66 52 78.78 67 51 76.11 59 50 84.74 66 60 90.90

3 59 51 86.44 68 60 88.23 60 51 85.00 62 47 75.80 64 51 79.68 70 62 88.57

4 75 70 93.33 74 58 78.37 73 61 83.56 68 49 72.05 71 62 87.32 69 60 86.95

5 77 70 90.90 69 56 81.15 74 57 77.02 73 55 75.34 68 58 85.29 73 62 84.93

6 73 65 89.04 70 56 80.00 69 58 84.05 78 61 78.20 70 57 81.42 71 63 88.73

7 71 66 92.95 67 55 82.08 70 57 81.42 70 61 87.14 67 58 86.56 63 54 85.71

8 69 61 88.40 65 56 86.15 68 50 73.52 64 54 84.37 65 59 90.76 65 58 89.23

9 67 60 89.55 64 55 85.93 65 53 81.53 72 50 69.44 70 60 85.71 68 62 91.17

Ma 90.92 83.83 80.76 78.06 85.06 88.71

σ 2.84 3.47 3.69 5.97 3.23 2.49

-2°C/min

Ct0 - CH 5% Me2SO 10% Me2SO 5% EG 10% EG

EN NL % EN NL % EN NL % EN NL % EN NL %

1 9.83 67 10 14.92 69 13 18.84 63 10 15.87 65 7 10.76 59 92.18

2 11.11 66 8 12.12 73 17 23.28 61 9 14.75 65 6 9.23 60 90.90

3 8.82 68 7 10.29 63 16 25.39 68 10 14.70 69 9 13.04 62 88.57

4 14.86 59 9 15.25 50 13 26.00 58 9 15.51 56 5 8.92 60 86.95

5 14.49 60 4 6.66 56 10 17.85 55 8 14.54 54 5 9.25 62 84.93

6 12.85 56 5 8.92 52 14 26.92 59 10 16.94 50 6 12.00 63 88.73

7 14.92 64 6 9.37 74 15 20.27 68 11 16.17 63 6 9.52 54 85.71

8 15.38 56 6 10.71 68 15 22.05 72 9 12.50 73 8 10.95 58 89.23

9 17.18 70 6 8.57 67 16 23.88 65 10 15.38 75 10 13.33 62 91.17

Ma 13.27 10.76 22.72 15.15 10.78

σ 2.80 2.88 3.20 1.26 1.69

Data are presented as average and standard deviation. Controls consist of cryoprotectant-free activated sperm readily used (Ct0), chilled at -1

°C/min and at -2 °C/min (Ct0 - CH) rates. Abbreviations: C, control; Me2SO, dimethylsulfoxide; EG, ethylene glycol; EN, number of eggs; NL,

number of normal larvae; %, percentage of normal larvae; Ma, average; σ, standard deviation.

Table 10. Statistical significance of differences between chilling rates on the fertilization success

under selected cryoprotectant conditions.

Ct0 -1 °C/min -2 °C/min

Ct0 - CH 5%

Me2SO

10%

Me2SO

5%

EG

10%

EG

Ct0 - CH 5%

Me2SO

10%

Me2SO

5%

EG

10%

EG

Ct0 - <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05

-1 °C/min

Ct0 - CH - >0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% Me2SO - >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% EG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% EG - <0.05 <0.05 <0.05 <0.05 <0.05

-2 °C/min

Ct0 - CH - >0.05 <0.05 >0.05 <0.05

5% Me2SO - <0.05 <0.05 >0.05

10% Me2SO - <0.05 <0.05

5% EG - <0.05

10% EG -

Data are presented as p-value. Controls consist of cryoprotectant-free activated sperm readily used

(Ct0), chilled at -1 °C/min and at -2 °C/min (Ct0 - CH) rates. Abbreviations: C, control; Me2SO,

dimethylsulfoxide; EG, ethylene glycol.

Table 11. Effect of freezing rates on the fertilization success under selected cryoprotectant

conditions.

-1 °C/min -7 °C/min

Ct0 Ct0 - CH Ct0 - FR 5% Me2SO 10% Me2SO 5% EG 10% EG

EN NL % EN NL % EN NL % EN NL % EN NL % EN NL % EN NL %

1 65 61 93.84 70 54 77.14 65 0 0 66 25 37.87 58 12 20.68 65 10 15.38 64 10 15.62

2 64 60 93.75 69 56 81.15 62 0 0 65 20 30.76 59 10 16.94 61 9 14.75 65 9 13.84

3 64 58 90.62 66 53 80.30 64 0 0 69 17 24.63 65 8 12.30 59 11 18.64 69 10 14.49

4 72 66 91.66 64 54 84.37 73 0 0 72 29 40.27 74 9 12.16 70 13 18.57 69 8 11.59

5 75 68 90.66 64 55 85.93 65 0 0 74 21 28.37 71 11 15.49 73 12 16.43 71 11 15.49

6 64 60 93.75 65 56 86.15 68 0 0 69 20 28.98 68 10 14.70 69 10 14.49 75 12 16.00

7 61 56 91.80 66 58 87.87 59 0 0 70 25 35.71 59 8 13.55 66 11 16.66 72 11 15.27

8 69 63 91.30 65 57 87.69 61 0 0 66 22 33.33 65 11 16.92 63 9 14.28 68 10 14.70

9 65 58 89.23 75 58 77.33 66 0 0 69 19 27.53 68 10 14.70 61 10 16.39 67 12 17.91

Ma 91.84 83.10 0 31.94 15.27 16.18 14.99

σ 1.63 4.23 0 5.20 2.66 1.62 1.71

-10 °C/min

Ct0 - FR 5% Me2SO 10% Me2SO 5% EG 10% EG

EN NL % EN NL % EN NL % EN NL % EN NL %

1 65 0 0 65 23 35.38 67 14 20.89 63 12 19.04 64 14 21.87

2 59 0 0 68 28 41.17 63 11 17.46 64 10 15.62 65 13 20.00

3 66 0 0 66 22 33.33 65 10 15.38 67 14 20.89 68 10 14.70

4 69 0 0 69 27 39.13 67 13 19.40 72 12 16.66 70 14 20.00

5 62 0 0 65 25 38.46 68 12 17.64 68 11 16.17 67 11 16.41

6 63 0 0 73 29 39.72 68 9 13.23 74 13 17.56 69 12 17.39

7 72 0 0 72 30 41.66 70 13 18.57 63 13 20.63 62 11 17.74

8 61 0 0 65 21 32.30 73 13 17.80 65 11 16.92 65 12 18.46

9 68 0 0 70 27 38.57 69 12 17.39 69 11 15.94 66 13 19.69

Ma 0 37.75 17.53 17.71 18.47

σ 0 3.33 2.21 2.00 2.17

-13 °C/min

Ct0 - FR 5% Me2SO 10% Me2SO 5% EG 10% EG

EN NL % EN NL % EN NL % EN NL % EN NL %

1 73 0 0 65 45 69.23 64 51 79.68 54 45 83.33 63 34 53.96

2 69 0 0 64 40 62.50 56 45 80.35 67 50 74.62 67 38 56.71

3 61 0 0 69 38 55.07 67 53 79.10 65 55 84.61 69 37 53.62

4 65 0 0 62 37 59.67 68 56 82.35 59 48 81.35 69 39 56.52

5 66 0 0 68 39 57.35 71 57 80.28 62 49 79.03 71 43 60.56

6 67 0 0 71 41 57.74 72 58 80.55 69 47 68.11 73 41 56.16

7 70 0 0 69 42 60.86 70 56 80.00 71 56 78.87 66 35 53.03

8 72 0 0 68 46 67.64 67 54 80.59 72 55 76.38 63 34 53.96

9 66 0 0 67 42 62.68 65 52 80.00 73 56 76.71 65 36 55.38

Ma 0 61.42 80.32 78.11 55.54

σ 0 4.69 0.89 4.98 2.31

Data are presented as average and standard deviation. Controls consist of cryoprotectant-free

activated sperm readily used (Ct0), used after chilling at the selected condition (Ct0 - CH), or used

after chilling at the selected condition and frozen at -7 °C/min, -10 °C/min and -13 °C/min (Ct0 -

FR). Abbreviations: C, control; CH, chilling; Me2SO, dimethylsulfoxide; EG, ethylene glycol; EN,

number of eggs; NL, number of normal larvae; %, percentage of normal larvae; Ma, average; σ,

standard deviation.

Table 12. Statistical significance of differences between freezing rates under selected cryoprotectant conditions.

Ct0

-1° C/min

-7 °C/min -10 °C/min -13 °C/min

Ct0 - CH

Ct0 - FR 5%

Me2SO

10%

Me2SO

5%

EG

10%

EG

Ct0 - FR 5%

Me2SO

10%

Me2SO

5%

EG

10%

EG

Ct0 - FR 5%

Me2SO

10%

Me2SO

5%

EG

10%

EG

Ct0 - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

-1 °C/min Ct0 - CH - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05

-7 °C/min

Ct0 - FR - <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05

5% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% Me2SO - >0.05 >0.05 <0.05 <0.05 >0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% EG - >0.05 <0.05 <0.05 >0.05 >0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% EG - <0.05 <0.05 >0.05 >0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

-10 °C/min

Ct0 - FR - <0.05 <0.05 <0.05 <0.05 >0.05 <0.05 <0.05 <0.05 <0.05

5% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% Me2SO - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% EG - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

10% EG - <0.05 <0.05 <0.05 <0.05 <0.05

-13 °C/min

Ct0 - FR - <0.05 <0.05 <0.05 <0.05

5% Me2SO - <0.05 <0.05 <0.05

10% Me2SO - >0.05 <0.05

5% EG - <0.05

10% EG -

Data are presented as p-value. Controls consist of cryoprotectant-free activated sperm readily used (Ct0), used after chilling at the selected condition

(Ct0 CH), or used after chilling at the selected condition and frozen at -7 °C/min, -10 °C/min and -13 °C/min (Ct0 - FR). Abbreviations: C, control;

CH, chilling; Me2SO, dimethylsulfoxide; EG, ethylene glycol.

Table 13. Sperm motility under selected cryoprotectant, chilling and freezing conditions.

Ct0 5% Me2SO 10% Me2SO 5% EG 10% EG

18 °C CH FR CH FR CH FR CH FR CH FR

1 4 4 0 4 3 4 3.5 3 1 3 1

2 4.5 3.5 0 4 3.5 4 3 3.5 1.5 3.5 1.5

3 4 4 0 4.5 3 4.5 3 3 1 3.5 1

4 4.5 3.5 0 3.5 3.5 3.5 2.5 3 1 2.5 1.5

5 4 4 0 3.5 3 3.5 2.5 4 1 3 2

6 4 3 0 3.5 3 4 3 3.5 1 3 2

7 4.5 4 0 4 2.5 4 3 4 1.5 3 1.5

8 5 4 0 3.5 3 4 3 4 1 3.5 1.5

9 4.5 3.5 0 4 3 4 3.5 3.5 1 3.5 1.5

q1 4 3.5 0 3.5 3 4 3 3 1 3 1.5

Min 4 3 0 3.5 2.5 3.5 2.5 3 1 2.5 1

Median 4.5 4 0 4 3 4 3 3.5 1 3 1.5

Max 5 4 0 4.5 3.5 4.5 3.5 4 1.5 3.5 2

q3 4.5 4 0 4 3 4 3 4 1 3.5 1.5

Data are presented as median, 1st - 3

rd interquartile range and minimum and maximum values.

Controls consist of cryoprotectant-free activated sperm readily used (Ct0), used after chilling at the

selected condition (CH), and used after chilling and freezing at the selected conditions (FR).

Abbreviations: C, control; Me2SO, dimethylsulfoxide; EG, ethylene glycol; CH, chilling; FR,

freezing; q1, first interquartile; Min, minimum value; Max, maximum value; q3, third interquartile.

Table 14. Statistical significance of differences between sperm motility classes under selected

cryoprotectant, chilling and freezing conditions.

Ct0 5% Me2SO 10% Me2SO 5% EG 10% EG

18 °C CH FR CH FR CH FR CH FR CH FR

Ct0

18 °C - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

CH - <0.05 >0.05 <0.05 >0.05 <0.05 >0.05 <0.05 <0.05 <0.05

FR - <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

5% Me2SO

CH - <0.05 >0.05 <0.05 >0.05 <0.05 <0.05 <0.05

FR - <0.05 >0.05 <0.05 <0.05 >0.05 <0.05

10% Me2SO

CH - <0.05 <0.05 <0.05 <0.05 <0.05

FR - <0.05 <0.05 >0.05 <0.05

5% EG

CH - <0.05 >0.05 <0.05

FR - <0.05 <0.05

10% EG

CH - <0.05

FR -

Data are presented as p-value. Controls consist of cryoprotectant-free activated sperm readily used

(Ct0), used after chilling at the selected condition (CH), and used after chilling and freezing at the

selected conditions (FR). Abbreviations: C, control; Me2SO, dimethylsulfoxide; EG, ethylene

glycol; CH, chilling; FR, freezing.