Demoy-Schneider Marina
email: marina.demoy-schneider@upf.pf

Schmitt Nelly

Le Pennec Gaël

Suquet Marc

Jacky Cosson

Quality assessment of cryopreserved black-lip pearl oyster Pinctada margaritifera spermatozoa

Keywords: Spermatozoa quality, Cryopreservation, Pinctada margaritifera, Motility, Metabolism, CASA

High quality of sperm is essential to a high fertilization rate, especially post-cryopreservation. Assessment of sperm integrity, motility and energy reserves before cryopreservation is necessary for selection of milt with optimal fertilizing potential. We describe the effect of cryopreservation on the quality of black-lip pearl oyster, Pinctada margaritifera var. cumingii sperm. Evaluated quality indices of fresh and frozen/thawed P. margaritifera spermatozoa, included morphology, ultrastructure and motility characteristics relative to the energy content (ATP) and its capacity to be sustained by mitochondrial respiration. Morphology and ultrastructure were quantitatively evaluated using images obtained by optical microscopy assisted by the Image J software and TEM, respectively. Sperm motility was assessed using Image J software combined with a computer assisted sperm analysis plugin adapted for assessing P. margaritifera spermatozoa. Other sperm quality parameters evaluated included O 2 consumption, ATP content, and creatine kinase activity. Frozen/thawed spermatozoa exhibited damage to the head but retained a compact spherical shape. Sperm motility indicators showed a significant decrease in quality resulting from the freeze/thaw process. The percent of motile cells was 54% compared to 84% in fresh sperm, O 2 consumption was 4.8 compared to 44 nanomol min -1 , ATP content was 0.72 nmol/10 9 spermatozoa in the activating medium compared to 4.54 nmol/10 9 spermatozoa, and creatine kinase activity was 9.06 × 10 -5

IU mg

-1 protein compared to 12.5 × 10 -5

IU mg -1 protein.

The cryopreservation protocol allowed obtaining an acceptable motility rate after thawing, confirming the predictive value of sperm motility measurements before cryopreservation in terms of their ability to withstand freezing process.

Introduction

In French Polynesia, the black pearls produced by the endemic black-lip pearl oyster Pinctada margaritifera, are valued for characteristics such as their highly polished aspect and variety of colors. This economic asset needs to be preserved and perpetuated through development of pearl oyster culture and sustainable production. In French Polynesia, black pearl production currently relies almost exclusively on the collection of spat of wild P.

margaritifera, making aquaculture and marketing dependent on natural resources.

Gamete cryopreservation is a potentially powerful technique for preservation of black-lip pearl oyster lineages to safeguard its biodiversity. Several research programs have been developed to define biological and physiological parameters of black-lip pearl oyster spermatozoa in various swimming media. These studies have shown that P. margaritifera spermatozoa possess characteristics of movement, motility, mitochondrial respiration rates, and ATP utilization similar to those of other bivalve species [START_REF] Demoy-Schneider | Motility activation and metabolism characteristics of spermatozoa of the black-lip pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)[END_REF], 2014). Motility of black-lip pearl oyster spermatozoa develops gradually when spermatozoa are transferred into alkaline sea water with the percentage of motile cells increasing and motility lasting for several minutes. The fully motile spermatozoa exhibit planar and sinusoidal flagellar waves in a homogeneous fashion. The swimming tracks are almost circular with head trajectories describing arcs interspersed with short linear segments.

Spermatozoa held at 4° C retain potent motility for several days and can be subsequently activated by alkaline swimming medium. Mitochondrial respiration rates and ATP utilization are closely bound to the activation of motility. The respiration rates are strongly increased and ATP is more rapidly consumed immediately after motility activation [START_REF] Demoy-Schneider | Motility activation and metabolism characteristics of spermatozoa of the black-lip pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)[END_REF], 2014).

Cryopreservation technologies should be studied in this species using techniques applied to teleost [START_REF] Martínez-Páramo | Cryobanking of aquatic species[END_REF], sea urchins (Adams et al. 2004a) and bivalve mollusks such as Crassostrea gigas [START_REF] Bougrier | Cryopreservation of spermatozoa of the Japanese oyster, Crassostrea gigas[END_REF]Adams et al. 2004b;[START_REF] Dong | Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific oysters, Crassostrea gigas[END_REF], Crassostrea virginica (Paniagua-Chavez and Tiersch 2001;[START_REF] Yang | High-thoughput sperm cryoconservation of eastern oyster Crassostrea virginica[END_REF], Crassostrea tulipa [START_REF] Yankson | Cryopreservation of the spermatozoa of Crassostrea tulipa and other three oysters[END_REF], Haliotis diversicolor supertaxa [START_REF] Gwo | Semen cryopreservation of small abalone (Haliotis diversicolor supertexa)[END_REF], Pinctada fucata martensii [START_REF] Kawamoto | Effects of cryopreservation methods on post thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii[END_REF][START_REF] Narita | Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii[END_REF][START_REF] Narita | Fertility of cryopreserved spermatozoa of the Japanese pearl oyster, Pinctada fucata martensii[END_REF],

Mytilus galloprovincialis [START_REF] Di Matteo | Cryopreservation of the Mediterranean mussel (Mytilus galloprovincialis) spermatozoa[END_REF][START_REF] Liu | Cryopreservation of stripped spawned sperm using non-programmable freezing technique in the blue mussel Mytilus galloprovincialis[END_REF], Argopecten purpuratus [START_REF] Espinoza | Morphological alterations in cryopreserved spermatozoa of scallop Argopecten purpuratus[END_REF], Ostrea edulis [START_REF] Horváth | Cryopreservation of sperm and larvae of the European flat oyster (Ostrea edulis)[END_REF], Pecten maximus [START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF], and Pinctada angulata [START_REF] Riesco | First study in cryopreserved Crassostrea angulata sperm[END_REF]. [START_REF] Lyons | Cryopreservation of black-lip pearl oyster (Pinctada margaritifera) spermatozoa : effects of cryoprotectants on spermatozoa motility[END_REF] and Acosta-Salmon et al. (2007) reported the effect of several cryoprotectant additives on P. margaritifera sperm motility after freeze/thawing and Hui et al.

(2011) reported reduced viability of spermatozoa after freeze/thawing. However, other studies focused cryopreservation aspects in different species leading to varied sperm survival rates after thawing, and suggested limitations of using only a single trait to define sperm quality [START_REF] Holt | Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality[END_REF]. Complementary studies such as those investigating movement tracking using computer assisted sperm analysis (CASA), cell integrity, and activity of enzymes involved in the regeneration of ATP, are expected to lead to better understanding of P. margaritifera sperm quality prior to cryopreservation. Sperm motility is one of the most important criteria in sperm quality, especially in predicting its fertilization ability (Cosson et al., 2008a and b).

In French Polynesia, hatchery practices have been developed in the same oyster species, and some fertilization assays using frozen/thawed spermatozoa show fertilization rates similar to those obtained using fresh spermatozoa [START_REF] Hui | Gamete cryopreservation, an asset for a durable pearl farming in French Polynesia[END_REF][START_REF] Hui | Hatchery-scale trials using cryopreserved spermatozoa in black-lip pearl oyster (Pinctada margaritifera)[END_REF]. In this context, P.

margaritifera sperm cryopreservation will prove beneficial to black pearl commercial interest.

The goals of sperm preservation are to maintain the gametes of individuals selected for high-growth potential and/or the color of their pearls (Ky et al. 2015) along with easier availability and lower risks compared to those incurred in maintenance of broodstock. In addition, the freezing of gametes assures the ex-situ preservation of genetic diversity of the populations of wild black-lip pearl oysters threatened with standardization by the transfer of the juveniles among Polynesian islands [START_REF] Arnaud-Haond | Spatio-temporal variation in the genetic composition of wild populations of pearl oyster (Pinctada margaritifera cumingii) in French Polynesia following 10 years of juvenile translocation[END_REF]). Sperm freezing thus present a means of maintaining the economic potential and safety of black-lip pearl oyster culture, knowing that the black pearl represents the second economic resource after tourism in French

Polynesia.

The objective of the study was to describe the quality of fresh and frozen/thawed black-lip pearl oyster spermatozoa assessing parameters such as percent of intact spermatozoa and spermatozoa ultrastructure, membrane integrity, motility and, metabolism.

Material and methods

Oysters and sperm collection

All experiments were carried out using black-lip pearl oysters reared in the Takapoto Atoll, Tuamotu Archipelago, French Polynesia, located 560 km east of Tahiti (14°32' S 145°14' W) until the age of 24 months and an average diameter of 10.5 ± 2.1 cm [START_REF] Pouvreau | Growth of the black-lip pearl oyster, Pinctada margaritifera, in suspended culture under hydrobiological conditions of Takapoto lagoon (French Polynesia)[END_REF]. During the natural reproduction period, pearl oysters were collected from suspended long-line culture and shipped at 25 ° C on a 1 h flight to the laboratory (UMR 241 EIO, University of French Polynesia, Tahiti, French Polynesia). Immediately upon arrival, oysters were immersed in filtered seawater tanks (25 ° C, pH 8.1) for 24 h without feeding, prior to experimental use.

The shell valves of each oyster were opened, using a knife to disrupt the shell muscle. Milt was manually collected by micropipette after natural release from the gonopore.

The semen (dry sperm) was stored for a maximum of 2 h in test tubes at 4.0 ° C ± 0.5 ° C before measurements.

Swimming medium

For a rapid evaluation of sperm motility, an aliquot of sperm from each male was transferred at a dilution of 1:2000 (v/v) in a saline activating medium composed of NaCl, 19.5 g L -1 ; glycine, 6.25 g L -1 ; CaCl 2 , 0.15 g L -1 ; MgSO 4 , 0.19 g L -1 and tris-aminomethane, 2.42 g L -1 (DCSB 4 ) (adapted from Paniagua-Chavez et al. 1998) with final pH adjusted to 8.2 according to [START_REF] Bougrier | Cryopreservation of spermatozoa of the Japanese oyster, Crassostrea gigas[END_REF]. Chemical compounds were purchased from Fisher Scientific Labosi (Elancourt, France), VWR-Prolabo (Fontenay sous Bois, France) and Sigma-Aldrich (Saint Quentin Fallavier, France).

The analyses were performed on 28 males. The sperm of 18 presenting sperm motility  70%, was used for cryopreservation.

Cryopreservation

The cryoprotectant additive mixture was adapted from the method of Hui et al.

(2009), using a solution of 0.7 M trehalose, 0.8 M dimethyl sulfoxide and 10% oyster hemolymph collected from the mantle cavity. According to Acosta-Salmon et al. (2007), milt was slowly mixed into the cryoprotectant additive mixture at a 1:10 (v/v) dilution and 350 μL of the mixture was placed in 500 µL semen straws (IMV, France). Samples remained on ice less than 10 min between milt dilution and initiation of the cooling process. Cooling was conducted in liquid nitrogen vapor: straws were placed on a floating tray 3 cm above the liquid nitrogen surface for 12 min before immersion. The straws remained at least 2 h in the liquid nitrogen. Subsequently, they were thawed by incubation in a water bath for 30 sec 25 ° C.

Sperm preparation and processing

Sperm concentration

The sperm concentration of each frozen/thawed sample was evaluated by counting the cells at 400x (Leica, DM 2000) magnification after 1:2000 (v/v) dilution in filtered seawater. Images were recorded using a numeric digital video camera (Leica, DFC 425C)

followed by image analysis. The sperm suspension was introduced in a Malassez's hemacytometer and six 0.05 mm 2 rectangular areas were randomly photographed and analyzed using ImageJ 1.45 software (https://imagej.nih.gov/ij/) under the parameters described in Table 1 to calculate the mean concentration as spermatozoa mL -1 .

Sperm morphology and ultrastructure

Morphological characteristics of fresh and frozen/thawed spermatozoa, including area, Crofton perimeter, Feret diameter and circularity were determined using Image J software. In an intact particle with an uninterrupted surface, the mean Feret diameter is proportional to the particle perimeter. Circularity provides indications of the particle shape and is defined as 4π x a / (p) 2 , in which "a" is the particle area and "p" is the particle perimeter. This parameter ranges between 0 and 1, the latter corresponding to a perfect round particle. The calibration parameters were the same as for the evaluation of sperm concentration (Table 1).

Transmission electron microscopy (TEM) was used to assess ultrastructure of fresh and frozen/thawed sperm samples from three oysters randomly selected. Fresh sperm was diluted 1:20 (v/v) in regular filtered seawater. Frozen/thawed as well as fresh spermatozoa were allowed to settle at room temperature (25 ° C) for 30 min to eliminate the cryoprotectant additive mixture without centrifugation. The cryoprotectant additive supernatant was discarded and the frozen/thawed spermatozoa were diluted 1:20 (v/v) in regular filtered seawater. Motility was assessed in the DCSB 4 activating medium as previously described [START_REF] Demoy-Schneider | Motility activation and metabolism characteristics of spermatozoa of the black-lip pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)[END_REF]. Then, 1 mL of fixative (5% glutaraldehyde in filtered seawater) was slowly added under gentle stirring, to 1 mL of the diluted sperm sample. After complete homogenization, the samples were incubated at 4 ° C overnight to achieve optimal fixation. Fixative supernatant was removed and samples were washed twice with 2 mL sodium cacodylate buffer (pH 7.3, 0.1 M in filtered seawater) with overnight settling down at 4 ° C after each washing. After the second washing, sodium cacodylate supernatant was removed and 2 mL of 70% ethanol was added to the fixed spermatozoa. After a complete dehydration in ascending series of alcohol, spermatozoa were embedded in Spurr's resin following manufacturer's recommendations (EM0300, Sigma-Aldrich, Saint Quentin Fallavier, France). Ultra-thin sections (60 nm) were contrasted with uranyl acetate and lead citrate before observations using a JEOL JEM-1400 transmission electron microscope.

Sperm motility assessment

Motility characteristics of fresh and frozen/thawed spermatozoa were obtained from video records. Fresh sperm was diluted 1:100 (v/v) while frozen/thawed sperm was diluted 1:10 (v/v), using DCSB 4 as activating solution in test tubes for 4 min, at the end of the capacitation phase, when maximum values of sperm movement are observed [START_REF] Boulais | Change of pH triggers sperm motility in the Pacific oyster (Crassostrea gigas)[END_REF]) and reach full sperm motility. Subsequently, 2 µL of the suspension was immediately transferred into a 50 µL drop of DCSB 4 onto a glass slide and covered with a cover-slip (25 000x final dilution) at room temperature. Microscopy observations were conducted, at the level of the cover-slip, 4 min post-activation using a LEICA DM2000 phase contrast microscope under 400x magnification. Ten-second video records were filmed with a LEICA DFC425C video camera (25 frames sec -1 ). Sperm movement was analyzed using the computer assisted sperm analysis (CASA) plug-in for ImageJ software (Wilson-Leedy and Ingermann 2007) initially developed for fish spermatozoa and adapted to the black-lip pearl oyster spermatozoa. Sperm motility percentage, straight line velocity (VSL, µm sec -1 ), velocity of average path (VAP, µm sec -1 ) and linearity (LIN, as a measure of a curvilinear path, 100 x VSL/VAP) were analyzed. The calibration parameters used for motility measurements are presented into the Table 2. A minimum of 50 spermatozoa were tracked for each video record.

Sperm metabolism and energy stores

Mitochondrial respiration rates of the sperm suspensions were assessed for fresh and frozen/thawed spermatozoa, by continuous recording of the oxygen concentration, during 10 min in filtered seawater or DCSB 4 with a highly sensitive calibrated Clark electrode (Rank Brothers, Cambridge, England) according to [START_REF] Pacey | Agonists of sperm maturation in Arenicola marina (Annelida: Polychaeta) increase the oxygen consumption of sperm suspensions in vitro[END_REF]. To determine the O 2 consumption, 50 µ L of dry black-lip pearl oyster sperm (15x10 9 spermatozoa mL -1 ) was diluted in 1 mL of the swimming solution (DCSB 4 ) to a final concentration of 7.5x10 8 spermatozoa mL -1 in a tightly closed chamber. The sperm suspension was kept as homogenous as possible using a magnetic stirrer. A 100% reading in O 2 concentration (253 nmol mL Yvette, France). The ATP concentration was evaluated at 4 min post-activation, when spermatozoa were fully motile. Sperm dilution was, adjusted to a final concentration of 75x10 6 spermatozoa mL -1 . Luminescence intensity was assessed using a Spectrafluor Plus luminometer (Tecan Group Ltd., Maennedorf, Switzerland) with the XFluor4 software (Genios Pro, Tecan Group Ltd., Maennedorf, Switzerland). The emitted light was in direct proportion to the ATP content; the latter was calculated using a standard curve [relative luminescence units versus ATP content (nmol mL -1 )].

According to [START_REF] Dawson | Creatine kinase: evidence for a dimeric structure[END_REF], creatine kinase activity is measured by three successive coupled enzyme reactions, with the final measured compound being nicotinamide adenine dinucleotide phosphate (NADPH). In the present study, NADPH production was measured spectrophotometrically at 340 nm, using the ECPK-100 kit (EnzyChrom TM CK Assay Kit, Bioassay Systems, Hayward, CA, USA) and was proportional to the creatine kinase activity. As a positive control, a piece of chicken breast muscle freshly excised was used. One gram of chicken muscle was homogenized using an Ultraturax homogenizer in a threefold volume of lysis buffer (Tris hydroxyl amino methane 0.1 M, EDTA 1 mmol) at 4 ° C for 1 min. A 500 µL volume of fresh or frozen/thawed sperm was diluted in 1.5 mL of lysis buffer and homogenized in the same conditions as previously described for the chicken muscle. The homogenized tissue was centrifuged at 20 000 x g for 30 min at 4 ° C (adapted from [START_REF] Bacou | Evolution quantitative et qualitative de la créatine kinase chez le lapin au cours des périodes foetale et néonatale[END_REF], and 10 µL of each supernatant were used for creatine kinase activity quantification. Enzyme reactions were performed in 96-well flat bottom micro-plates. A 100 µL volume of kit reaction medium was prepared and immediately added to 10 µL of supernatant. The micro-plates were shaken for 5 min at 25 ° C and then incubated at 37 ° C for 10 min. Measurements were performed in triplicate, 10 min and 40 min after mixing, at 340 nm (UV) using an Infinite M200 spectrophotometer (TECAN, Austria).

We chose to express creatine kinase activity as IU mg -1 of protein, to facilitate comparison with published results of studies in other species. Sperm total proteins were prepared with the lysis buffer provided with the kit and quantified using the method of [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF].

Data analysis

Data are presented as mean±SEM obtained from the analysis of independent sperm samples. Percentages were arcsine square root transformed before further analysis. To quantify the effect of cryopreservation on the percentage of motile spermatozoa, on the spermatozoa movement and on the morphological characteristics, means were compared using a two-way analysis of variance (ANOVA). When significant differences were observed (P  0.05) Shapiro Wilk and Fisher tests were used.

Results

Sperm concentration

The total volume of dry sperm collected from each animal was individual-dependent with a mean of 1.00±0.25 mL. The mean sperm concentration was 16.1±1.2 x 10 9 spermatozoa mL -1 (n = 28).

Effect of cryopreservation on spermatozoa morphology and ultrastructure

Most assessed morphological parameters of frozen/thawed sperm showed a significant difference (P < 0.05) from those of fresh (Table 3). Area, Crofton perimeter, and Feret diameter values were 61%, 39% and 35% lower, respectively. No significant differences were observed in the circularity parameter and the Feret diameter/Crofton perimeter ratio. Fifty TEM images were analyzed. The black-lip pearl oyster spermatozoon possessed a spherical head and a short mid-piece. The dense spherical nucleus occupied most of the head and exhibited an anterior depression located in the sub-acrosomal zone and a posterior depression at the basal pole. The proximal centriole was inserted centrally into a ring (Fig 1a) comprising four mitochondria (Fig. 1b). The distal centriole was located in the mid-piece, near the base of the flagellum (Fig. 1a). A flat cap was apparent over the acrosome, and the sub-acrosomal space was filled with a dense material (Fig. 1c). The axoneme comprises 9 peripheral + 2 central microtubules (Fig. 1d,1f).

Damage to the plasma membrane of frozen/thawed spermatozoa was observed. The membrane appeared swollen on some portions of the head (Fig. 1e,1f). Minimal differences from fresh spermatozoa were noted in the acrosome (Fig. 1g) and mitochondria (Fig. 1h). The nucleus and chromatin did not differ from those of fresh spermatozoa. The most prevalent difference was related to the flagellum: the latter was not visible in nearly 80% of frozen/thawed spermatozoa TEM images possibly because of the fragilization of the head/tail junction resulting from the treatment applied for TEM visualization (Fig. 1e). When the flagellum was observable, proximal and distal centrioles were located in the same position as in fresh spermatozoa. The axoneme appeared to have a similar pattern that of the fresh spermatozoa, with peripheral and central microtubules (Fig. 1h).

Effect of cryopreservation on sperm motility and movement

Fresh, as well as frozen/thawed spermatozoa were non-motile in regular seawater, but motility was activated in a large proportion of cells in DCSB 4 : 84.7% of fresh cells and 54.4% of frozen/thawed spermatozoa (Fig. 2). In addition, the motility of fresh and frozen/thawed spermatozoa showed a significant linear correlation (P  0.05) (R 2 = 0.59). The more motile the spermatozoa are before cryopreservation, the more motile they are after thawing (Fig. 3). Velocity parameters for fresh sperm were 66.6 µm sec -1 for VSL and 165.7 µm sec -1 for VAP. Linearity was calculated at 40.6%. After freeze/thaw, 35% lower sperm motility (P  0.05) and 20% lower VAP (P  0.05) compared to fresh was observed. No significant difference was observed in VSL and LIN values of fresh and frozen sperm for any treatment (Table 4).

Effect of cryopreservation on sperm metabolism and energy stores

Fresh sperm activation using DCSB 4 was associated with significantly higher (P  0.05) O 2 consumption compared to cryopreserved (44.4 vs. 6.7 nmol min -1 /10 9 spermatozoa).

Respiration in activated fresh spermatozoa was 44.4 nmol min -1 /10 9 spermatozoa and was 4.8 nmol min -1 /10 9 after freeze/thaw, a difference of 89% (P < 0.05) (Fig. 4a).

Cryopreservation was associated with significantly (P < 0.05) lower sperm ATP content, with and without activation. Motility activation makes the ATP content decrease from 34.50 to 4.54 µmol/10 9 spermatozoa for fresh sperm and from 13.30 to 0.72 µmol/10 9 spermatozoa for frozen/thawed sperm (Fig. 4b). For frozen/thawed spermatozoa, this represents a difference of 61.4% and 84.1%, in natural seawater and DCSB 4 , respectively, compared to fresh cells.

The EnzyChrom TM CK Assay Kit was calibrated in chicken muscle, in which creatine kinase activity was in the range of 20.95±1.35 IU mg -1 of muscle protein. The mean protein content of the spermatozoa was 151.70±23.70 g L -1 , corresponding to 9.48±1.48 mg - 1 /10 9 spermatozoa, with mean sperm concentration of 16x10 9 spermatozoa mL -1 .

Compared to fresh spermatozoa, significantly lower (P < 0.05) creatine kinase activity was observed after freeze/thawing, at 12.5 IU mg -1 in fresh spermatozoa and at 9.06 IU mg -1 in frozen/thawed cells (Fig. 4c).

Discussion

Fertilization of an oocyte is dependent on the quality of spermatozoa, specifically their motility (Cosson et al. 2008a, b) and metabolic activity (mitochondrial respiration, ATP hydrolysis) [START_REF] Boulais | Involvement of mitochondrial activity and OXPHOS in ATP synthesis during the motility phase of spermatozoa in the Pacific oyster, Crassostrea gigas[END_REF][START_REF] Boulais | ATP content and viability of spermatozoa drive variability of fertilization success in the Pacific oyster (Crassostrea gigas)[END_REF][START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF]. It has been reported that cryopreservation may induce oxidative stress (Li et al. 2010a;[START_REF] Shaliutina-Kolešová | The influence of cryoprotectants on sturgeon (Acipenser ruthenus) sperm quality, DNA integrity, antioxidant responses, and resistance to oxidative stress[END_REF] damaging spermatozoon structure [START_REF] Gwo | Evaluation of damage in Pacific oyster (Crassostrea gigas) spermatozoa before and after cryopreservation using comet assay[END_REF]Paniagua-Chavez et al. 2006;[START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF]) and decreasing motility [START_REF] Dong | Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific oysters, Crassostrea gigas[END_REF][START_REF] Gwo | Semen cryopreservation of small abalone (Haliotis diversicolor supertexa)[END_REF]Kawamoto et al. 2007;[START_REF] Riesco | First study in cryopreserved Crassostrea angulata sperm[END_REF][START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF]) and fertilizing capacity [START_REF] Boulais | ATP content and viability of spermatozoa drive variability of fertilization success in the Pacific oyster (Crassostrea gigas)[END_REF][START_REF] Dong | Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific oysters, Crassostrea gigas[END_REF][START_REF] Gwo | Semen cryopreservation of small abalone (Haliotis diversicolor supertexa)[END_REF][START_REF] Narita | Fertility of cryopreserved spermatozoa of the Japanese pearl oyster, Pinctada fucata martensii[END_REF]Paniagua-Chavez and Tiersch. 2001;[START_REF] Riesco | First study in cryopreserved Crassostrea angulata sperm[END_REF].

The black-lip pearl oyster spermatozoon has the typical morphological characteristics of oysters spermatozoa: a round shape head and a short mid-piece containing four mitochondria, a flat acrosome, an axoneme with 9+2 microtubules and, in contrast to mammalian sperm, no accessory structures. These observations are in accordance with the spermatozoa ultrastructure described in many other oyster species [START_REF] Yurchenko | Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast[END_REF].

Area and Crofton perimeter have been previously used to estimate the quality of frozen/thawed European eel spermatozoa [START_REF] Asturiano | Effect of sperm cryopreservation on the european eel sperm viability and spermatozoa morphology[END_REF]) and frozen-thawed Japanese oyster Crassostrea gigas larvae (Suquet et al. 2012a). No significant variation was observed in the circularity parameter, suggesting that the spermatozoa kept a circular contour shape after the freeze/thawing process. The Feret diameter/Crofton perimeter ratio did not significantly differ from fresh, indicating that the particle shape remained intact after thawing.

On the other hand, we observed that the area of frozen/thawed spermatozoa was 61% lower than that of fresh sperm. Crofton perimeter was 39% lower and Feret diameter 35 % lower, probably associated with spermatozoon head damage (Table 3). The TEM micrographs revealed that the plasma membrane was disrupted at the head-tail junction, in the mitochondria region. We observed the absence of visible flagella, for a high number of frozen/thawed spermatozoa. The complete flagellum may not have been included in the photographic frame. Flagella may also have become detached during sample processing. In addition, the accumulation of swimming spermatozoa near surfaces, obscuring the flagella should be considered, as has been shown in several species [START_REF] Cosson | How spermatozoa come to be confined to surfaces[END_REF]. This accumulation may partly explain, why the mean % of motile cells remains of 54% in motility trials when about 80% of spermatozoa have lost their flagellum after freezing.

We observed swelling in the plasma membrane after the freeze/thaw process. Similar damage was reported in the Chilean scallop Argopecten purpuratus [START_REF] Espinoza | Morphological alterations in cryopreserved spermatozoa of scallop Argopecten purpuratus[END_REF], blue mussel

Mytilus galloprovincialis [START_REF] Liu | Cryopreservation of stripped spawned sperm using non-programmable freezing technique in the blue mussel Mytilus galloprovincialis[END_REF]) and great scallop Pecten maximus [START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF].

Damage caused by the freeze-thaw process may differ with species. Studies of other species reported that alterations in morphology of frozen/thawed spermatozoa are clearly visible. [START_REF] Kurokura | Lesions of spermatozoa by cryopreservation in oyster Crassostrea gigas[END_REF] observed abnormalities of the pacific oyster acrosome and mid-piece and [START_REF] Gwo | Evaluation of damage in Pacific oyster (Crassostrea gigas) spermatozoa before and after cryopreservation using comet assay[END_REF] observed alterations inside the chromatin. [START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF] noted variable damage in samples of the great scallop: in some spermatozoa, little or no damage was observed at the level of acrosome and mitochondria while for others, alterations were considerable.

The anomalies we observed in the black-lip pearl oyster morphological parameters (area, diameter and perimeter), suggests alterations in the shape of spermatozoa resulting from the freeze/thaw process, although the circularity parameter and Feret diameter/Crofton perimeter ratio did not differ significantly from fresh. These results confirmed that, despite the addition of a cryoprotectant agent, spermatozoa undergo significant morphological and ultrastructure damage with freezing, which could influence their quality after thawing.

For several marine species, sperm membrane integrity is closely related to motility Aberkane and Iguer-ouada 2016; [START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF][START_REF] Boulais | ATP content and viability of spermatozoa drive variability of fertilization success in the Pacific oyster (Crassostrea gigas)[END_REF]. We observed that movement of frozen/thawed black-lip pearl oyster spermatozoa differed from that of fresh. This is in accordance with Acosta-Salmon et al. (2007), who reported that motility of frozen/thawed sperm was 65% lower than the values recorded for fresh sperm, in the same species. Many other studies of mollusk species have also shown significantly lower sperm motility as a result of the freeze/thawing process. In 2016, Suquet et al. assessed the effect of cryopreservation on the percentage of motile frozen/thawed spermatozoa and reported motility values versus controls, of 65% in the Pacific oyster; from 50 and 60% in the blue mussel Mytilus galloprovincialis, eastern oyster Crassostrea virginica or Chilean scallop

Argopecten purpuratus; and from 20 and 35% in the pearl oyster Pinctada fucata martensii, great scallop Pecten maximus, and flat oyster Ostrea edulis. We did not observe any significant difference in the motility duration (~10 min) in frozen/thawed black-lip pearl oyster spermatozoa compared to fresh, in contrast to results obtained by [START_REF] Kawamoto | Effects of cryopreservation methods on post thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii[END_REF] for P. fucata martensii who reported significantly lower motility duration in frozen/thawed spermatozoa. Our results suggest that when fresh spermatozoa are fully motile, they remain still motile after thawing, which shows the predictive value of motility measurements of sperm before freezing in their ability to resist freezing process. This indicates that the spermatozoa that survived the freeze/thaw process possess sufficient energy reserves to show activity similar to fresh sperm.

In the active spermatozoa, we observed 20% lower VAP in frozen/thawed sperm compared to fresh. [START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF] reported 75% lower velocity in frozen/thawed spermatozoa of the great scallop compared to fresh. [START_REF] Linhart | Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos[END_REF] [START_REF] Figueroa | Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology[END_REF]), 97 µm s -1 to 134 µm s -1 in Prochilodus lineatus [START_REF] Viveiros | Sperm dilution ratio affects post-thaw motility rate and velocity of Prochilodus lineatus (Characiformes) sperm[END_REF][START_REF] Viveiros | Storage and transportation of Prochilodus lineatus (Characiformes) sperm prior to cryopreservation[END_REF]), 57 µm s -1 in rainbow trout Oncorhynchus mykiss (Suquet et al. 2012b) and 150 µm s -1 in Siberian sturgeon Acipenser baerii spermatozoa [START_REF] Sarosiek | Inhibition of β-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii)[END_REF]. VSL of sea urchin spermatozoa was shown to be 170-240 µm sec -1 in Hemicentrotus pulcherrimus [START_REF] Hiramoto | A quantitative analysis of flagellar movement in echinoderm spermatozoa[END_REF] and 150-200 µm sec -1 in Paracentrotus lividus [START_REF] Fabbrocini | Motility of sea urchin Paracentrotus lividus spermatozoa in the post-activation phase[END_REF]. The factors contributing to the relatively low velocity observed for black-lip pearl oyster in our study may be twofold: firstly, the activation of oyster sperm motility and its duration is associated with internal concentration of cAMP (Demoy-Schneider et al. 2014) under internal pH control [START_REF] Boulais | Change of pH triggers sperm motility in the Pacific oyster (Crassostrea gigas)[END_REF]. Maximum sperm velocity may not have been reached in our assay conditions, although only 4 min separated the transfer to the swimming solution and the first video records. Secondly, the movement of oyster spermatozoa is erratic [START_REF] Swann | The propulsion of the non-rotating ram and oyster spermatozoa[END_REF]) and cells frequently change the plane of their swimming path [START_REF] Demoy-Schneider | Biological characteristics of sperm in two oyster species: the Pacific Oyster, Crassostrea gigas, and the black-lip-pearl o yster, Pinctada Margaritifera[END_REF]). These features contribute to challenges and possible underestimation of velocity values. However, the conditions for velocity estimates were the same for fresh and frozen/thawed sperm samples.

Linearity in cryopreserved black-lip pearl oyster spermatozoa was not significantly different from fresh. This is in accordance with the results obtained for Acipenser ruthenus, in which LIN decreased slightly from 0.55 to 0.51 [START_REF] Psenicka | Acrosome staining and motility characteristics of sterlet spermatozoa after cryopreservation with use of methanol and DMSO[END_REF]) and for Atlantic cod Gadus morhua L., in which LIN was not significantly modified by cryopreservation [START_REF] Butts | Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua[END_REF]. Our results indicated that sperm cells that survived the freeze/thaw process presented some defects that induce slight perturbations in swimming ability compared to the non-treated samples.

The metabolic activity of the black-lip pearl oyster spermatozoa, including respiratory activity, ATP content and creatine kinase activity was significantly affected by the cryopreservation process. Compared to fresh sperm, a significant 88% lower respiration rate was observed in frozen/thawed sperm. This difference may be explained by the discrepancy in the percent of active cells. Motile spermatozoa should present a high mitochondrial respiration rate, allowing high ATP production. It was demonstrated in sea urchin (Tombes andShapiro 1985, 1989;[START_REF] Tombes | Creatine kinase-dependent energy transport in sea urchin spermatozoa. Flagellar wave attenuation and theoretical analysis of high energy phosphate diffusion[END_REF]Walliman et al. 2011;[START_REF] Ingermann | Metabolism of motile zebrafish sperm[END_REF] that creatine-phosphate stores and creatine kinase activity are related the flagellar motility [START_REF] Saudrais | The use of phosphocreatine plus ADP as energy source for motility of membrane-deprived trout spermatozoa[END_REF][START_REF] Dzyuba | Energetics of fish spermatozoa: the proven and the possible[END_REF][START_REF] Fedorov | Development and application of LC/HRPS for quantification of adenine nucleotides, creatine phosphate, and creatine in sturgeon spermatozoa[END_REF][START_REF] Van Dorsten | Activation of sea-urchin sperm motility is accompanied by an increase in the creatine kinase exchange flux[END_REF] showed that, in sea urchin sperm, creatine kinase activity is related to energy metabolism (O 2 consumption and ATP production).

The initial ATP content was low in black-lip pearl oyster (5 nmol/10 9 spermatozoa) compared to Pacific oyster (45 nmol/10 9 spermatozoa) [START_REF] Suquet | Changes in motility, ATP content, morphology and fertilization capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm[END_REF]) and great scallop (203 nmol/10 9 spermatozoa) [START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF]. This low ATP content may partly explain the low velocity values observed for black-lip pearl oyster sperm. The cryopreservation process resulted in 85% lower ATP content in thawed spermatozoa using DCSB 4 as activating medium, as previously observed [START_REF] Demoy-Schneider | Biological characteristics of sperm in two oyster species: the Pacific Oyster, Crassostrea gigas, and the black-lip-pearl o yster, Pinctada Margaritifera[END_REF]. This was in correlation with the reduction in mitochondrial respiration. One interpretation is that most of the initial ATP content is used to sustain sperm swimming, even for frozen/thawed spermatozoa which contained low ATP levels. A similar decrease was reported in Pacific oyster [START_REF] Suquet | Changes in motility, ATP content, morphology and fertilization capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm[END_REF]), but in great scallop, no significant difference in intracellular sperm ATP content was observed between fresh and frozen/thawed sperm [START_REF] Suquet | The quality of great scallop (Pecten maximus) sperm after thawing[END_REF]. We found cryopreservation to be associated with a 27.5% lower creatine kinase activity black-lip pearl oyster spermatozoa compared to fresh. It was also shown that enzyme activity, including creatine kinase activity, was involved in flagellar mobility and thus represents an indicator of sperm quality, as reported in sea urchin (Tombes andShapiro 1985, 1989;[START_REF] Wallimann | Creatine kinase isoenzymes in spermatozoa[END_REF][START_REF] Tombes | Creatine kinase-dependent energy transport in sea urchin spermatozoa. Flagellar wave attenuation and theoretical analysis of high energy phosphate diffusion[END_REF][START_REF] Wallimann | Creatine kinase in non-muscle tissues and cells[END_REF][START_REF] Van Dorsten | Activation of sea-urchin sperm motility is accompanied by an increase in the creatine kinase exchange flux[END_REF][START_REF] Wallimann | The creatine kinase system and pleiotropic effects of creatine[END_REF], rainbow trout O. mykiss [START_REF] Saudrais | The use of phosphocreatine plus ADP as energy source for motility of membrane-deprived trout spermatozoa[END_REF]) and sturgeon A. ruthenus [START_REF] Fedorov | Development and application of LC/HRPS for quantification of adenine nucleotides, creatine phosphate, and creatine in sturgeon spermatozoa[END_REF]. Cryopreservation has been reported to affect enzyme activity in marine fish.

Actually, Li et al. (2010b) [START_REF] Cosson | Adenosine Triphosphate: chemical properties, biosynthesis and functions in cells[END_REF] in fish spermatozoa.

Conclusion

The present work was an opportunity to use a panel of quality criteria to ensure integrity, motility and the energy stores prior to cryopreservation to assist in selection of the welladapted samples of black-lip pearl oyster sperm to cryopreservation. The descriptive analysis is essential to optimize the success rate of cryopreservation and in vitro fertilization. The simplest and most rapid parameter to be studied is the percent motility in the alkaline activating medium DSCB 4 . The results we obtained showed that motility measurements can be used as a predictive possibility for future application of cryopreservation in this species.

Ultimately, fertilization tests must be conducted to confirm the success of sperm cryopreservation in P. margaritifera.

Minimum VCL for motile sperm (µm sec

(

  Paniagua-Chavez et al. 2006;[START_REF] Merino | Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: Stability of mitochondrion as criterion of effectiveness[END_REF]. The CASA plug-in developed for Image J (Wilson-Leedy and Ingermann 2007) has long been used to describe sperm motion and provides information for quality assessment of semen of many species, including oysters[START_REF] Wilson-Leedy | Development of a novel C.A.S.A. system based on open source software for characterization of zebrafish sperm motility parameters[END_REF][START_REF] Amann | Computer-assisted sperm analysis (CASA): capabilities and potential developments[END_REF][START_REF] Boulais | Involvement of mitochondrial activity and OXPHOS in ATP synthesis during the motility phase of spermatozoa in the Pacific oyster, Crassostrea gigas[END_REF]

  Figure 4a

  -1) indicated to the swimming medium in equilibrium with the air at 25 ° C while 0% O 2 concentration in the swimming medium was obtained by addition of few crystals of sodium dithionite at the end of the measurement period[START_REF] Demoy-Schneider | Motility activation and metabolism characteristics of spermatozoa of the black-lip pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)[END_REF]).

	Adenosine 5' triphosphate (ATP) content of the milt was estimated as we
	previously described (Demoy-Schneider et al. 2012). Briefly, ATP content was assessed in
	triplicate for fresh and frozen/thawed sperm, by bioluminescence (ATPlite-M kit according
	to the manufacturer instructions, Perkin Elmer Life and Analytical Sciences, Villebon sur

  observed alterations of enzyme activity involved in metabolism of C. carpio frozen/thawed spermatozoa.[START_REF] Butts | Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua[END_REF] reported a decrease in anti-trypsin activity in Atlantic cod frozen/thawed spermatozoa and[START_REF] Zilli | Adenosine triphosphate concentration and β-D glucuronidase activity as indicators of sea bass semen quality[END_REF] observed an increase of malate dehydrogenase activity in sea bass Dicentrarchus labrax frozen/thawed spermatozoa, but did not observe change in activity of β-aspartate aminotransferase, isocitrate dehydrogenase or β-D glucuronidase. This decrease in enzyme activity, combined with lower mitochondrial respiration could explain why the ATP content is lower in frozen/thawed blacklip pearl oyster spermatozoa, as these metabolic processes could be limiting for an efficient cellular ATP supply

Table 3 Effect of the cryopreservation on morphological characteristics of black-lip pearl oyster spermatozoa.

 3 Values represent mean±SEM calculated for n = 28 fresh samples and n = 18 frozen/thawed samples. Different superscripts indicate significant differences (P  0.05) between fresh and frozen/thawed spermatozoa.

	-1 )	8

Table 4 Effect of cryopreservation on black-lip pearl oyster sperm movement.

 4 Motility parameters were measured using computer-assisted sperm analyzer (CASA) plug-in, for Image J software (Wilson-Leedy and Ingermann 2007) and adapted to P. margaritifera spermatozoa (see methods). Values represent means±SEM, calculated for n =28 fresh samples and n = 18 frozen/thawed samples. Different superscripts indicate significant differences (P  0.05) between fresh and frozen/thawed spermatozoa. VSL = straight line velocity, VAP = average path velocity and LIN = linearity.

Acknowledgments This work was supported by the French Ministère des Outremers and conducted under the research program "La cryoconservation des spermatozoïdes de l'huître perlière Pinctada margaritifera, un atout pour une perliculture durable et la sauvegarde de sa biodiversité". Black-lip pearl oysters P. margaritifera were kindly provided by the Direction des Ressources Marines et Minières of French Polynesia. The ATP concentration measurements were carried out in the Laboratoire d'Analyses Médicales of the Centre Hospitalier de la Polynésie française in French Polynesia with the helpful collaboration of Rodolphe BADIN. The authors are grateful to Michel RODIÈRE (UPF) for his help regarding the statistical analysis and to Kathleen HILLS and Alan PIKE (Lucidus Consultancy) for reviewing the English. 7.

Effect of cryopreservation on P. margaritifera spermatozoon motility.

Measurements were performed using the CASA. Values represent means±SEM calculated for n = 28 fresh samples and n = 18 frozen/thawed samples. «a» indicates significant difference (P  0.05) between fresh and frozen-thawed sperm. Values represent means±SEM calculated for 28 fresh samples and 18 frozen/thawed samples.

«a» = significant difference ( P 0.05) with/without activation, between fresh and frozenthawed spermatozoa. «b» = significant difference (P  0.05) between fresh and frozen/thawed spermatozoa.

4b ATP content with and without activation in DCSB 4 .

Values represent means±SEM calculated for 28 fresh sperm samples and 18 frozen/thawed samples. «a» = significant difference ( P 0.05) in ATP content depending on swimming media in fresh and frozen/thawed spermatozoa. «b» = significant difference (P  0.05) between fresh and frozen/thawed sperm.

4c Creatine kinase activity.

Values represent means±SEM calculated for 28 fresh sperm samples and 18 frozen/thawed samples. «a» = significant difference (P  0.05) between fresh and frozen/thawed spermatozoa.