Cryopreservation of turbot ( scophthalmus maximus ) spermatozoa

ELSEVIER

CRYOPRESERVATION OF TURBOT (scophthalmus maximus) SPERMATOZOA

C. Dreant~,“~ M. Suq~et,~ L. Quemet~,~ J. C~sson,~ F. Fierville,’ Y. Normant,’ and R. Billard’

‘Laboratoire dlchtyologie, Museum National d’Histoire NaturelIe, 7523 1 Paris, France

‘Laboratoire de Physiologie des Poissons, IFREMER, 29280 Plouzane, France

%aboratoire de Biologie Celhdaire, CNRS, URA 671,06230 Villefranche sur mer, France

Received for publication: Augus t 2 3, 1 g g 6

Accepted: March 21, 1997

ABSTRACT

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of fiozen- thawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modiied Mounib extender, supplemented with 100/o BSA and loo/o DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30°C for 5 sec. Use of this simple method resulted in a 60 to 800/r reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was signiticantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of ctyopreservation significantly decreased intracelhdar ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration. 0 1997 by Else&r Science Inc.

Key words: cryopreservation, spermatozoa, marine fish, sperm viability, Scoohthalmus maximus

INTRODUCTION

Sperm cryopresetvation has been investigated mainly in salmonids and in some other freshwater fish species (35, 20, 30). Most methods of cryopreservation result in a decrease in the percentage of motile spermatozoa, sperm velocity, duration of motility, fertilization capacity and in structural damage (24, 39, 12). In frozen-thawed spermatozoa, the reduction in fertilizing capacity and in motility characteristics have been largely attributed to the alteration of membrane structure and fimction during the processes of cooling, t&zing and thawing (26). Temperature changes affect membrane integrity as do water and cryoprotectant movement through the membrane, which leads to cell volume and osmolarity changes; all together, these factors result in damages to the diierent cell compartments (22). Adjustment of the various parameters such as extender composition, cryoprotectant, dilution ratio, and freezing and thawing rate should minimize the cellular injuries.

Acknowledgments The authors thanks G. Maisse (INRA) for fruitful discussions on the cryopreservation protocol and A. Salaun (IFREMER) for documentation research. This work was supported by grants from IFREMER, Museum National d’Histoire Naturelle and CNRS (URM n03).

90 whom correspondence should be addressed.

Thenogenology 48:589-603. 1997 0093-691X/97/517.00 0 1997 by Elsevier Science Inc. PII SOO93-691X(97)00276-8

590 Theriogenology

In turbot @coohthalmus maximus), some knowledge of sperm biology exists in the literature (4, 7,37). So far, no research has been conducted on sperm cryopreservation in this species.

Thus, the aim of this work was 1) to establish a simple, practical and efficient method for cryopresetving turbot semen; 2) to assess the quality of cryopreserved spermatozoa by studying motility characteristics, sperm metabolism and fertilization capacity. Motility parameters, including the percentage of motile spermatozoa and velocity (Computer-Assisted Sperm Analysis), intracellular ATP concentrations, respiratory rate, fertilization capacity and short-term storage capacity were examined concomitantly as an expression of integrity of frozen-thawed spermatozoa.

MATERIALS AND METHODS

Gamete Collection

Turbot brooodstock was handled according to the methods of Omnes et al. (25). Semen was collected by stripping ripe males a&r careti~l cleaning of the genital area. To avoid any sperm contamination by urine, the ureter was catheterized and the urinary bladder was emptied by gently squeezing the fish belly. Sperm was then caretidly sucked into a syringe and stored at 4°C until use. The percentage of motile spermatozoa was quickly determined as described below, and only sperm samples presenting a high initial motility (>70% at 10 set post activation) were used in these experiments. Batches of eggs were collected by abdominal pressure of the females. The quality of eggs was estimated from their morphological features under a dissecting microscope. Batches of eggs showing more than 2O?h abnormal eggs were discarded.

Evaluation of Motility

Sperm motility was quantified after activation using a two-step dilution procedure. Fresh spermatozoa were tirst transferred at a 1:24 dilution rate in a nonactivating medium (NAM; composed of NaCl 70 n&l, KCI 1.5 mM, MgCls 6.1 mM, CaCls 2.7 mM, Glucose 0.4 mM, BSA 10 a, Tris HCI 20 mM adjusted to pH 8.2, osmolality 200 mOsmoFkg) maintaining spermatozoa quiescent. Then, 1 uL of this sperm suspension was transferred in a 24-uL drop of activating medium (AM, composed of natural sea water, BSA 10 g/L, Tris HCI 20 mM adjusted to pH 8.2, osmolality 1100 mOsmol/kg) previously placed on a glass slide on the microscope stage. The tinal dilution was thus 1:624. For 6ozen-thawed semen, a direct dilution method was used, and the amount of semen was adjusted accordmg to the initial dilution ratio in the extender. Sperm motility was observed using a dark field microscopy (Olympus BH-L) attached to a camcorder (Canovision EX 1 Hi, Canon). A dark field condenser was combiied with a x 10 non- immersion lens). Spermatozoa were visual&d on a video monitor (Panasonic BT-Ml420PY) at a final magnification of x 200.

The Percentage of motile cells was always estimated by the same 2 observers and was expressed by 6 arbitrary scores, with Score 0 representing no motile spermatozoa, 1 > 0 to 2O??, 2 > 20% to 40%, 3 > 40% to 60%, 4 > 60% to 80%, 5 > 80% to 100% of spermatozoa showing progressive movement. The percentage of motile spermatozoa was determined at 10 set and 60 set post activation. All the observations were run at room temperature (18 to 2O’C). To validate the motility measurements, preliminary tests were carried out by the 2 observers using 3 sperm

Theriogenology 591

samples. The percentage of motile spermatozoa was measured 10 times for each sample. No significant difference was observed between the data recorded by the 2 observers.

Spermatozoa velocity was determined using the modcell Computer-Assisted Sperm Analysis (CASA) system developed by Sch&vaert et al., (33). It was used for measurement of turbot sperm motion endpoints. The software was calibrated to turbot spermatozoa. The calibration settings are presented in Table 1. The straight line velocity is detined as the average velocity of the sperm head along its actual trajectory and is expressed in micrometer per second (pm/s).

Table 1. Set parameters for CASA analysis of turbot semen

Setting parameters Frame Frame rate Number of consecutive frames to analyze Minimum number of consecutive frames to analyze Field of view Maximum burst speed Minimum motile speed Cell size range Minimum number of cells

video 50 (frame/s) 32 1s total video image 300 pm/s 5 urn/s 5 to 30 urn2 30

Development of Cryopreservation Method

The general procedure of cryopreservation was the following: for each experiment, semen was collected from 6 males during the spawning period of the females. Semen was transferred into various extenders and cryoprotectants at different dilution rates. No equilibration time was allowed, The diluted semen was sucked into 200~uL straws (Instruments de m&iecine veterinaire IMV, L’Aigle, France) and placed on a tray in nitrogen vapour (NV) at various distances above the surface of liquid nitrogen (LN) in a Styrofoam box covered by a lid. After a 15-min period, the straws were transferred into LN and stored until thawing. Straws were thawed in a water bath for 5 set, and the percentage of motile spermatozoa was immediately determined.

Four extenders (for composition see Table 2) were tested: the medium described by Mounib (23) modified by the addition of 10% BSA (mod&d Mounib medium: MMM); a ginger medium adapted to fish by Peleteiro et al. (modified ginger medium: MRM; 27) two variants of artificial seminal liquid (ASL) diiering in the buffer composition and mimicking turbot seminal fluid (37). Since bicarbonate buffers have been described previously as more successful than Tris buffer for sperm preservation (34) the effects of these 2 buffers were compared. Sperm samples were diluted at a 1:2 ratio in various extenders containing 10% DMSO. The freezing capacity of samples deprived of any diluent but supplemented with only 10% DMSO was also tested. The modiied Mounib medium (MMM) was used 1) to compare the effects of 4 permeating cryoprotectants (DMSO, glycerol, ethylene glycol, methanol) at various concentrations (5, 10 and 15%); 2) to study the effects of simultaneous addition to MMM of a nonpermeating cryoprotectant (BSA, egg yolk) with a permeating cryoprotectant (DMSO): egg yolk (10%) BSA (10%) or a combination of both cryoprotectants (5% + 5%); 3) to define the optimum dilution rate (semen was diluted at 4 spermextender ratios: l:l, 1:2, 1:4, 1:9); 4) to test 3

592 Theriogenology

cooling rates (straws were placed at various distances above the LN surface (2 cm, 6.5 cm and 13 cm)); and 5) to investigate 3 thawing temperatures (20,30 and 40°C).

To test for individual variations in sperm cryopreservation success, the semen of 15 turbots was gozen.

Table 2. Composition of extenders for turbot semen’

NaCl ASLl ASL2 70 n&I 7omM 74 mlu

KCI 1.5 mlu 1.5 mM 26.8 mM CaCIZ 2.7 mh4 2.7 mM 1.8mM MN2 _ 6.1 mlU 6.1 mM - NaHCOs - 25 mlu 1.59mM KI-ICO, 1OOmM - Tris-HCI 2omM Glucose 0.4 mlu 0.4 mM sucrose 125 mM _ Reduced 6.5 n&I gluthatione BSA 10 m&nl 10 mg/ml 10 mg/ml 10 mgAnl PH 7.8 8.2 8.0 8.0 0sm01ality 310 200 200 200 (mOsmol/kg) ‘Chemical compounds manufactured by Sigma Chemical Co, St Louis, MO. h4MM: modified Mounib medium; ASL: artificial seminal liquid; MRM: modified Ringer medium.

Assessment of the Frozen-Thawed Sperm Quality

The quality of frozen-thawed spermatozoa was studied in terms of velocity, respiratory rate and fertilizing capacity. Velocity of spermatozoa was determined using the modcell CASA at various times post activation. According to the method previously described by Perchec et al. (28), ATP spermatozoa concentration was measured by bioluminescence (kit from Pertstorp S.A., Division Lumac, Bezons, France) using a biocounter BM2010A lumac/3M after ATP extraction. The respiratory rate of the sperm suspension was assessed polarographically with an oxygen consumption recorder (SI instrument, Oxygen meter model 781). Fresh semen was diluted at a ratio 1:2 with NAM. Then, either 50 )tL of this suspension or of cozen-thawed sperm was added in 1 mL of either AM or in NAM to the closed container of the oxymeter adjusted at 20°C. The respiratory rate was expressed as the oxygen uptake in pL O2 consumed by 1 O9 spermatozoa per min.

The fertilization capacity of fresh spermatozoa was compared with that of frozen-thawed sperm cells using the method of Suquet et al. (38) on 15 individual sperm samples. Triplicate samples of eggs were inseminated using 3 diierent spermatozoa:egg ratios (3,000; 6,000; 20,000). Fertiliition success (fertilization rate = number of 4-cell stage eggs/number of eggs)

Theriogenology 593

was evaluated 4 h later on 200 eggs for each batch. The embryo survival beyond the 4-&l stage was not evaluated.

Immediately after thawing, short-term storage capacity of the spermatozoa was studied using samples maintained on crushed ice following 1 of the 3 procedures: 1) dilution 1:9 in ASL2 medium since this d&rent was succes&lly used for short-term storage of gesh sperm (Cheriguini et al., unpublished data); 2) dilution 1:9 in MMM or 3) undiluted, control semen. The percentage of motile spermatozoa was determined at various time periods after the freezing-thawing procedure.

Statistical Analysis

Data are expressed as mean f SEM Motilities of sperm samples were compared using a one- or two-way analysis of covariance (Ancova) atIer angular transformation of the mean percentage of each motility score. The male factor was included as a covariance in Ancova. When diierences were significant, a Tukey a posteriori multiple range test was used for comparison. Respiratory rates were analyzed using a two-way analysis of variance (Anova). After angular transformation, fertilization rates were tested using a two-way anova. Individual variation in sperm motility after cryopreservation were compared by one-way Anova considering the diierence between the motility of fresh and frozen-thawed spermatozoa. Concentrations of ATP were compared by a Student’s t-test. Short-term storage capacity of frozen-thawed spermatozoa was evaluated by linear regression of the percentage of motile spermatozoa versus storage time. Linear regressions from various treatments were compared for slope and intercept (40). A value of P<O.OS was taken as statistically significant.

RESULTS

Development of the Sperm Cryopreservation Method

The effect of diierent extenders on the motility of 6ozen-thawed spermatozoa is shown in Table 3. No sign&ant difference was observed between the diierent extenders. However, lower variability of sperm motility was recorded in MMM. Motility of frozen-thawed spermatozoa was not signiRcantly altered when DMSO was added to semen devoid of extender.

The effect of permeating cryoprotectants is summadzed in Table 4. The highest motility score was observed with 15% DMSO and the lowest with 5%. The addition of DIMS0 improved the sperm motility score compared with that of the other cryoprotectants whatever the concentration used. Addition of glycerol resulted in low motility after freezing and thawing. Methanol and ethylene glycol were totally inefficient. Neither of the 2 nonpermeating cryoprotectants (egg yolk or BSA) significantly improved post-thaw motility (Table 4) nor was there a significant effect on the various sperm dilution rates (Table 5). The highest motility results were obtained when straws were placed at 6.5 cm above the LN surface (-99“C at I5 mitt) compared with 2 cm (-14VC at I5 min) and 13 cm (-46°C at I5 min; Table 6). The thawing temperature (20, 30 and 4O’C) did not intluence the motility of frozen-thawed sperm samples (Table 6).

The motility of frozen-thawed spermatozoa collected from I5 males differed significantly (Figure I). No linear correlation between the motility of fresh and frozen-thawed spermatozoa at

594 Theriogenology

10 set post activation was observed. Siiar results were recorded when motility was checked at 60 set post activation (data not shown).

Table 3. Effect of extenders on the post-thaw sperm motility. Semen was mixed at a 1:2 ratio with various extenders supplemented with loo/o DMSO, frozen at 6.5 cm above the surface of liquid nitrogen (LN). Straws were plunged in LN and thawed at 3O“C for 5 SecondS.

ExtenderS Percentage of motile spermatozoa 10 seconds 60 seconds

post activation post activation 69.9 f 15.6 53.9 f 12.2

ASLl 70.3 f 15.6 58.3 f 13.3 ASL2 59.9 *11.8 50.6 f 8.9

78.3 f 3.2 61.1 l 2.4 No extender 55.2 f 13.9 43.4 f 10.4

MRM: modified Ringer medium, ASL: artificial seminal liquid, MMM: modified Mounib medium.

Table 4. Effect of type and concentration of cryoprotectant on post-thaw sperm motility.

Cryoprotectants Concentration Percentage of motile spermatozoa

(W 10 seconds 60 seconds post activation post activation

Permeating DMSO 5 40.0 f 10.0” 32.2 f 7.8b” 10 55.6 f 9.4& 40.6 f 5.5* 15 63.3 f 4.6’ 46.7 f 6.78

Glycerol 5 26.1 f 6.4& 6.7 f l.4d 10 26.7 f 7.4cd 7.2 f 1.7d 15

2;2;04.$ 10 f l.6d

Ethylene glycol 5 0.0 l O.Od 10 0.0 f o:O@ 0.0 f o.od 15 0.0 f o.osh 0.0 f o.od

Methanol 5 0.6 0. f lg 0.0 f o.od 10 2.8 f 2.08 3.9 f 2.9 15 0.0 f o.@ 0.0 f O.Od

Non-penneating BSA 10 66.7 f 6.4 54.4 l 5.3 egg yak 10 71.1 l 5.7 58.9 f 4.8 BSA + yolk egg 10 71.1 f 5.2 62.2 f 4.9

Permeating cryoprotectants: semen was diluted at a ratio of 1:2 in modiied Mounib extender. Nonpermeating cryoprotectants: semen was diluted at a ratio of I:2 in the modified Mounib medium added 1% DMSO. Straws were 6ozen at 6.5 cm above the surface of liquid nitrogen (LN), plunged in LN and thawed at 3O“C for 5 seconds. tiValues within columns followed by diierent superscripts are diierent (P<O.OS).

Theriogenology 595

Table 5. Effect of dilution rate on post-thaw sperm motility. Semen was diluted at 4 different rates (1: 1, 1:2, 1:4 and 1:9) in modiied Mounib extender supplemented with 10% DMSO, frozen at 6.5 cm above the surface of liquid nitrogen (LN). Straws were plunged in LN and thawed at 30°C for 5 seconds.

Dilution rate (sperm volume: extender volume)

1:l 1:2 1:4 1:9

Percentage of motile spermatozoa 1oseconds 60 seconds

post activation post activation 64.4 f 2.2 54.4 f 4.1 75.6 f 3.9 63.3 f 4.2 71.1 f 2.2 59.7 f 3.2 70.0 l 4.7 61.1 f 3.4

Table 6. Effect of cooling and thawing temperatures on the post-thaw sperm motility. Semen was diluted at the rate 1:2 in modified Mounib extender supplemented with 10% DMSO.

Percentage of motile spermatozoa 10 seconds 60 seconds

post activation post activation Distance above liquid nitrogen surface (cm) 2 56.7 f 4.6’ 44.4 f 3.sc

6.5 81.1 f 3.6b 73.3 f 5.9*

13 74.7 f 4.9b 50.3 i 2.5b

Thawing temperature (“C) 20 75.6 M.8 61.1 f 5.9 30 75.6 f 6.4 66.7 f 7.2 40 78.9 f 4.1 65.6 f 6.4

Distances above the liquid nitrogen surface: straws were thawed at 30°C for 5 seconds. Thawing temperature: straws were frozen at 6.5 cm above the surface of liquid nitrogen (LN), plunged in LN and thawed for 5 seconds. a-b Values within columns followed by diierent superscripts are different (P<O.OS).

Assessment of Frozen-Thawed Sperm Quality

The ATP content of immotile frozen-thawed spermatozoa was significantly lower than that recorded for immotile fresh spermatozoa (Table 7). On the other hand, no significant difference was recorded between motile tksh and frozen&awed spermatozoa when the ATP concentration was measured at 60 set post activation. The ATP level of spermatozoa diluted in MMM was similar to that of tiesh spermatozoa in NAM. When diluted in NAM, the respiratory rate of fresh and thawed spermatozoa was not significantly dierent from that of the frozen-thawed spermatozoa (Table 7). When spermatozoa were diluted in AM, a significant increase in the respiration rate was observed; while following dilution in MMM, sperm respiration ceased

The velocity of fresh spermatozoa did not differ signiticantly from that of frozen-thawed sperm cells (Figure 2) while the percentage of motile frozen-thawed spermatozoa was significantly lower until 60 set post activation (Figure 3).

596 Theriogenology

Figure 1.

2 3 4 5 6 7 8 9 101112131415

Turbot males (number)

-

Individual variation in cryopreservation success. The percentage of fresh and frozen- thawed motile spermatozoa was measured at 10 seconds post activation (the bars with no line present standard errors from the replicate measurements of sperm motility below 0.2 ). I Fresh Spermatozoa 0 Frozen-thawed spermatozoa.

Table 7. Intracellular ATP content and respiratory rate of tiesh and frozen-thawed spermatozoa. In activating medium, ATP content was measured at 60 seconds post activation (i.e., after the transfer of spermatozoa into the activating medium).

ATP content (nmole/109 spermatozoa)

Respiratory rate (uL 0,/min/109 spermatozoa)

Fresh Frozen-thawed Fresh Frozen-thawed spermatozoa spermatozoa spermatozoa spermatozoa

Non activating medium 260 f 3gW 158 f 22”‘b 1.61 * O.Oy 1 45 f 0.12%” Activating medium 72 f lSb’ 69 f gb” 2.99 f 0.28b” 3:24 f 0.20bg Modified Mounib medium 244 f 28’ 0.38 f 0.03- 0.36 f O.O4cs

8-slalues followed by diierent superscripts are significantly different (WO.05). The tlrst letter corresponds to results of statistical analysis within a column. The second letter represents the results of statistical treatment between the columns.

Theriogenology 597

Figure 2.

Figure 3

100 -

80 -

60 I I I I I 10 20 30 40 50 60

Time post activation (seconds)

Changes in 6esh and frozen-thawed spermatozoa velocity with time after dilution in activating medium. -o- Fresh spermatozoa -e- Frozen-thawed spermatozoa.

100 -

0 I 1 I I I I 0 50 100 150 200 250 300

Time post activation (seconds)

Changes in the percentage of fresh and frozen-thawed motile spermatozoa with time a&r dilution in activating medium. Asterisks indicate a percentage of motile spermatozoa significantly Merent at various time post activation between iksh and frozen-thawed. -o- Fresh spermatozoa -o- Frozen-thawed spermatozoa.

598 Theriogenology

The fertilization rate obtained with frozen-thawed spermatozoa was significantly lower (58.5 f 4.2) than that observed with t&h spermatozoa (66.9 f 2.8) (Table 8). Moreover, for both fksh and frozen-thawed spermatozoa, a significantly higher fertilization rate was recorded using 20,000 than 6,000 and 3,000 spermatozoa per egg.

Table 8. Fertilization rate (%) of f&h and fiozen-thawed spermatozoa using various spermatowa:egg ratios.

Ratios (spermatowa:ena) 3,000 6,000

Fresh spermatozoa 63.7 f 5.2b 66.7 l 4.7b

Frozen-thawed spermatozoa 52.6 f 6.Sb 52.3 f 6.2b

20,000 70.2 f 4.6’ 67.5 f 3.8’

a ’ Values within cohunns followed by different superscripts are different (WO.05).

The percentage of motile spermatozoa decreased as a function of the duration of the storage period (Figure 4). When frozen-thawed spermatozoa were diluted right after thawing, a decrease in the initial percentage of motile spermatozoa was observed compared with that of undiluted frozen-thawed spermatozoa. No significant difference was found between the slopes of the regression line of frozen-thawed, nondiluted spermatozoa and frozen-thawed spermatozoa previously diluted in MYMM. On the contrary, the regression line of the frozen-thawed sperm diluted in ASL2 was not parallel to the 2 other regression lines (RO.05).

loo 1

\ ti=Oo.58

0 10 20 30 40 50 60 70

Time post thawing (minutes)

- 0.678x

- 0.782x

Figure 4. Short term storage at 4’C of spermatozoa a&r f&zing-thawing . o modiied Mounib medium (undiluted semen) n modiied Mounib medium (1:9) l artificial seminal liquid 2 (1:9).

Theriogenology 599

DISCUSSION

The present work demonstrates that turbot spermatozoa can be successfilly frozen and thawed while preserving good fertilizing ability.

Turbot spermatozoa either treated with any of the tested extenders or undiluted exhibited good motility parameters afbx freezing-thawing. For instance, attempts to cryopreserve undiluted turbot semen show little success, especially because aggregats have been observed in fiozen- thawed spermatozoa. As a consequence, undiluted sperm samples could not be successWly used for subsequent experimentation and artificial fertilization. Compared with modified Ringer and ASL, the modiied Mounib extender presented a smaller variation in post-thaw motility, which could be due to the presence of reduced gluthatione in the later medium. This compound is thought to preserve the activity of several enzymes involved in COZ fixation in sahnonid and cod sperm (34). Moreover, KHCOs could contribute to inhibiting turbot sperm motility before freezing, as NaHC03 does (S), and the mitochondrial respiration.

To preserve energy required for fertilization, a suitable extender must not induce sperm motility before t&zing (35, 32). Addition of 10% DMSO to the different extenders tested resulted in increased osmolality up to 1100 mOsmol/kg. The movement of turbot spermatozoa is initiated by a rise in osmotic pressure of the surrounding medium (4), thus the inclusion of DMSO serves to induce sperm motility. When semen was diluted in modified Mounib medium complemented with DMSO, the spermatozoa were motile,although only for a period of less than 1 min. This is a short period compared with that of several minutes observed by Chauvaud et al. (4) using sea water. This short duration of motility observed in modified Mounib extender probably corresponds to the period of time needed to reach an equilibrium between the extracellular and intracellular concentration of DMSO as well as to the time needed for the inhibition of respiration by KHCOJ. This transient activation before freezing would appear to be less deleterious in turbot than in other species because turbot spermatozoa are able to synthesize most of their energy (ATP) by de novo oxydative respiration during motion (28). As a consequence, an extender able to protect mitochondrial function should be used for turbot spermatozoa. Since intracellular ATF’ content decreases during the process of freezing and thawing, this technique is energy-consuming for sperm stores. On the other hand, mitochondrial respiratory activity was not altered when fresh and frozen-thawed sperm respiratory rates were compared. Oxydative phosphorylation was triggered at the same time as motility of cozen- thawed spermatozoa was induced, showing that ATP synthesis occurs de novo. Moreover, even if ATF’ concentration had decreased after frting and thawing, its level stayed high enough to allow for the initiation and maintenance of the motion phase.

The DMSO extender has been successtidly used in many fish species (20). In the present work, we compared various cryoprotectants and demonstrated that DMSO was more efficacious for turbot spermatozoa than glycerol, ethylene glycol and methanol. According to Gwo (10, 12) the effects of DMSO are concentration-dependent. In the case of turbot spermatozoa, we did not observe any toxic effects for concentrations in the range of 5 to 15%. Glycerol is considered to be less toxic than DMSO for most types of cells (19); however it appears to be more toxic for the semen of most fish species (34, 35). Since glycerol is slow to permeate membranes and is osmotically active, an equilibration time is usually needed prior to freezing. Nevertheless, as shown by Gwo (lo), an equilibration period did not improve the viability of frozen yellowtin

600 Theriogenology

seabream spermatozoa. Glycerol induced some irreversible damage such as the modification of membrane fluidity, reduction of membrane electricrd capitance and alteration in polymerization and depolymerization of microtubules... (26, 13). Ethylene glycol and methanol are also frequently used as cryoprotectants for their abiity to penetrate the cell structure very rapidly (20), but these do not appear to be suitable for turbot semen, as shown by our results. Using ethylene glycol, poor results were also reported in striped bass (Morone saxatilia; 16) and Pacific herring (CluDea u; 29). Methanol was shown to be better than DMSO and glycerol for preserving tilapia (Sarotherodott mossambicus; 14, 31), zebra tish (BrachvdaniQ ti; 15) and carp spermatozoa (Cyprin~ & 21). However, methanol does not protect spermatozoa of most marine species i.e., black grouper (Bpinephel r&tbaricus; 9), Atlantic croaker @4icropo8Qtr& undulatua; 12), and barramundi Q&s car&if&; 18) against cryoinjuries.

Proteins and phospholipids (e.g., egg yolk, BSA, etc.) have been commonly added to extenders either to prevent damage to the plasma membrane during cryopresewation or as energy sources (34). These compounds act by lowering the f&zing point, raising the glass transformation temperature of the extracelllular solution (19) and stabilizing membranes (26). No significant difference was observed between egg yolk and BSA extender in the frozen-thawed motility of turbot spermatozoa. However, BSA was selected for the extender composition since it also prevents sperm aggregation (4).

Little information is available on the freezing and thawing rates used for processing fish spermatozoa. The estimated optimal freezing rate of turbot spermatozoa was similar to the rates reported for other marine fish species (9, 10, 12). Rapid thawing is often used to minimize recrystallization. The post-thaw motility of turbot spermatozoa was similar at all the thawing rates tested. In halibut (HiDDoalossus ~~DDO~~OSSU$ Bolla et al. (2) found that the mean fertilization rate following thawing at 10 or 40°C did not differ significantly. In carp, Leveroni et al. (21) obtained the best results with a 4°C thawing temperature. The choice of thawing temperature needs to be based on the cryoprotectant used, because its toxicity is influenced by temperature (8).

In turbot, the percentage of motile frozen-thawed spermatozoa was signiticantly lower than for fresh spermatozoa while the velocity and the duration of motion were not significantly mod&d. In amago salmon (Onchorhvnchus masou ishikawae), the velocity as well as the percentage of motile frozen-thawed spermatozoa decreased through the process of freezing and thawing (24).

The fertilization rate was significantly influenced by spermatozoa concentration. On the other hand any excess in spermatozoa concentration would render insensitive the tests for differences in fertiliiion capacity. A minima) sperm density must be detined for fertilization trials (12). Jn turbot, the fertilization capacity of frozen-thawed spermatozoa is expected to be lower than that of fresh spermatozoa because of the decrease in the number of viable sperm cells. Nevertheless, increasing the concentration of spermatozoa up to 20,006 sperm cells:ovule, appears to compense for the decreased capacity.

When stored at 4”C, frozen-thawed turbot spermatozoa needs to be used rapidly. The percentage of motile spermatozoa decreases significantly within a 30-min period. In rainbow trout, a decrease in fertilization was observed after a 30-set delay following thawing (36). In turbot, the dilution of frozen-thawed spermatozoa with a thawing solution leads to a decrease in

Theriogenology 601

the initial percentage of motile cells. Nevertheless, the short-term storage capacity was higher compared with results obtained using modified Mounib medium.

Individual variation in cryopreservation success of spermatozoa was observed in turbot and in rainbow trout (17). Many factors could be involved in this variation: individual factors, sperm quality, age of the breeders, stage of the reproductive season and stripping frequency (1, 3).

Spermatozoa of marine fish species are easier to cryopreserve than those of freshwater species (1). This fact could be explained by the specific composition of membranes and especially by the cholesterol:phospholipid ratio. Cholesterol modulates bilayer fluidity through steric interaction with membrane phospholipids (26). The cholesterol:phospholipids ratio is 2 to 3 times higher in marine. fish than in tieshwater fish (6). Because phosphatidylcholine protects cells from osmotic and cold stress, the high level of phosphatidylcholine found in marine spermatozoa could also explain the higher capacity of cryoresistance (6).

In conclusion, the simple, practical method of cryopreserving turbot spermatozoa described here yields high post-thaw motility and fertilization rates after freezing and thawing. The percentage of motile frozen-thawed spermatozoa was lower than for fresh spermatozoa, but no alteration in velocity, fertilizing capacity or metabolic functions was recorded after freezing. We recommend this simple method for application in aquacultural programs,

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