Gamete cryopreservation, an asset for a durable pearl farming in French Polynesia

Abstract

Cryopreservation is a useful tool for genetic improvement which has been applied to several bivalve mollusk species. It would allow to keep the gametes of selected individuals and to preserve ex-situ the biodiversity of wild populations of the black-lip pearl oyster Pinctada margaritifera threatened of standardization by the seed transfers between the islands in French Polynesia. The ability to cryopreserve spermatozoa would bring about significant benefits to the cultured black pearl industry. Sperm freezing supposes the control of different steps: preparation of breeders, sperm collection, evaluation of sperm quality and the freezing process itself. Broodstock conditioning in hatchery allows to obtain gametes from the pearl oyster. Sperm quality is evaluated using a motility index and bioluminescence ATP measurements of spermatozoa. The following cryoprotectant additives (CPA) mixture were evaluated : (1) 0.41 M trehalose and 0, 0.45, 0.91, 1.36 M dimethyl sulfoxide (Me2SO), (2) 0.7 M trehalose and 0.8 M Me2SO and (3) 0.10 M trehalose. Freezing was made in two steps, in nitrogen vapor followed by direct plunging of spermatozoa in liquid nitrogen. Total motility was best retained when spermatozoa were cryopreserved with 0.41 M trehalose and 0, 0.45, 0.91, 1.36 M Me2SO and 0.7 M trehalose and 0.8 M Me2SO. Bioluminescence ATP measurements showed that spermatozoa conserved their energetic stock. Moreover, fertilization efficiency of thawed sperm was demonstrated using artificially matured oocytes, which allowed the development of embryos and D larvae.

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Gamete cryopreservation, an asset for a durable pearl farming
in French Polynesia
Bélinda Hui a, Marina Demoy-Schneiderb, Vincent Vonaua, Gilles Le Moullaca, Jacques Moriceaua,
Marcel Le Pennecb and Jean-Claude Cocharda.
a Ifremer - Département d’Aquaculture en Polynésie Centre du Pacifique, BP 7004, 98719 Taravao, French Polynesia
bUniversité de la Polynésie Française, Equipe Biologie Marine, Laboratoire BIOTEM - EA 4239, BP 6570 Faa’a,
French Polynesia
Correspondence : Belinda.Hui@ifremer.fr
ABSTRACT
Cryopreservation is a useful tool for genetic improvement which
has been applied to several bivalve mollusk species. It would
allow to keep the gametes of selected individuals and to preserve
ex-situ the biodiversity of wild populations of the black-lip pearl
oyster Pinctada margaritifera threatened of standardization by the
seed transfers between the islands in French Polynesia. The
ability to cryopreserve spermatozoa would bring about significant
benefits to the cultured black pearl industry. Sperm freezing
supposes the control of different steps: preparation of breeders,
sperm collection, evaluation of sperm quality and the freezing
process itself. Broodstock conditioning in hatchery allows to
obtain gametes from the pearl oyster. Sperm quality is evaluated
using a motility index and bioluminescence ATP measurements of
spermatozoa. The following cryoprotectant additives (CPA)
mixture were evaluated : (1) 0.41 M trehalose and 0, 0.45, 0.91,
1.36 M dimethyl sulfoxide (Me2SO), (2) 0.7 M trehalose and 0.8
M Me2SO and (3) 0.10 M trehalose. Freezing was made in two
steps, in nitrogen vapor followed by direct plunging of
spermatozoa in liquid nitrogen. Total motility was best retained
when spermatozoa were cryopreserved with 0.41 M trehalose and
0, 0.45, 0.91, 1.36 M Me2SO and 0.7 M trehalose and 0.8 M
Me2SO. Bioluminescence ATP measurements showed that
spermatozoa conserved their energetic stock. Moreover,
fertilization efficiency of thawed sperm was demonstrated using
artificially matured oocytes, which allowed the development of
embryos and D larvae.
Keywords
Pearl
Spermatozoa, Fertilization.
oyster, Pinctada margaritifera, Cryopreservation,
1. INTRODUCTION
The black pearl oyster, Pinctada margaritifera, is used for pearl
production. Production relies almost exclusively on the collection
of wild spat, which makes the activity very dependent on natural
resources. Recruitment is highly variable in space and time and so
spat is very often transferred by the farmers between atolls or
even between archipelagos in French Polynesia. Cryopreservation
techniques for gamete could greatly assist in preserving black
pearl oyster lineages in the pearl farming industry, and selective
breeding for desirable traits in the future.
Spermatozoa cryopreservation is a useful tool for genetic
improvement and have been applied to several bivalve mollusc
[2][5]. Exploration of the development of gametes
cryopreservation techniques from the black-lip pearl oyster
P.margaritifera showed the significance of cryoprotectant
additives (CPA) on spermatozoa motility [8]. Acosta-Salmón et al
(2007) has evaluated the toxicity and the effects of the molar
concentration of trehalose and dimethyl sulfoxide (Me2SO) and
the freezing protocol on motility of P.margaritifera spermatozoa.
Previous work on cryopreserving black pearl oyster sperm has
found that motility was retained when spermatozoa were
cryopreserved in 0.45 M trehalose and 0, 0.64, 1.02 and 1.53 M
dimethyl sulfoxide (Me2SO) [1][8], but the fertility of these
cryopreserved spermatozoa was unknown to date.
The present study is to carry on with this work to estimate the
fertility of cryopreserved spermatozoa and to complete with the
effect of some other molar concentration of CPA on spermatozoa
motility.
2. MATERIALS AND METHODS
2.1 Collection of gametes
P. margaritifera used in this study were from Vairao lagoon,
Tahiti, French Polynesia. Oyster were transferred from the culture
long-line to a conditioning place during 1 month to obtain mature
oyster. Sperm end and eggs were obtained by “strip spawning”.
Eggs were collected by lacerating the gonad wall with a scalpel
and gently scraping and washing the gonad contents. Eggs were
released into 100 ml seawater containing 2mM NH3 to induce
oocyte maturation [7][11].
Spermatozoa were manually stripped and collected with a Pasteur
pipette., Spermatozoa were obtained from 3 oysters.
2.2 Experiments
2.2.1 Experiment 1: effect of CPA’s on post-thaw
motility of cryopreserved sperm
We evaluated 6 CPA mixtures at the following in straw
concentrations: (1) 0.41M trehalose (Sigma, France) alone and in
combination with 0.45, 0.91, 1.36 M Me2SO (Sigma, France) , (2)
0.7 M trehalose and 0.8 M Me2SO and (3) 0.10 M trehalose alone.
Cryoprotectant solutions were prepared in distilled water. The
concentrated spermatozoa was diluted in the appropriate CPA on
a 1:10 (v/v) then drawn by suction into 500 µl semen straws
(IMV, France). Equilibration period, the time between sperm
dilution and the start of cooling, was less than 10 min at room
temperature (27 °C). The straws were left in liquid nitrogen (LN)
steam during 10 min and then immersed in the LN. For thawing,
the straws were immersed in seawater at room temperature (27°C)
for 25s. Fresh and post-thaw spermatozoa were diluted with

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seawater and DCSB4 solution [3] on a 5:5 ? (v/v) at a final
dilution ratio 1:500. A 6 µl aliquot of the activated spermatozoa
was transferred to a glass slide and motility was observed without
a coverslip. Sperm motility was measured using the Motility
Index by Christen and al. (1987).
2.2.2 Experiment 2: ATP content of sperm
ATP content of the whole sperm was evaluated using
bioluminescence (ATP lite Luminescence ATP detection Assay
System; Perkin Elmer Life and Analytical Sciences B.V.) as
following.

Mg2+
ATP + D-Luciférine + O2
CO2 + Photons
Oxy-luciférine + Ppi + AMP +
Luminescence was read with a Spectrafluor Plus luminometer
(TECAN Group Ltd, Maennedorf, Switzerland) [10].
2.3 Evaluation of fertility of fresh and
cryopreserved spermatozoa
Fertilization assays were used to evaluate the fertility of sperm
before and after cryopreservation. Eggs were fertilized with fresh
and cryopreserved spermatozoa at four sperm/egg ratio : 20
000/1, 40 000/1, 90 000/1 and 380 000/1. The fertilization rates
were evaluated by counting normally cleaved eggs under
stereomicroscope 4h after fertilization. Approximately 100 eggs
in each well were examined microscopically to determine the
percentage of fertilized eggs.
3. RESULTS
3.1 Effects of CPAs on post-thaw motility of
cryopreserved sperm
The total motility can be successful retained when cryopreserved
P. margaritifera spermatozoa in 0.41 M trehalose and 0, 0.45,
0.91, 1.38 M Me2SO and was particularly best retained for
spermatozoa cryopreserved in 0.7 M trehalose and 0.8 M Me2SO
(Fig. 1).
0
1
2
3
0 M 0,45 M0,91 M 1,38 M 0,7M trehalose
Cryoprotectants additives
I
n
d
e
x
m
o
t
i
l
i
t
y
Figure 1. Mean motility after thawing of P. margaritifera
spermatozoa with each CPA: 0 M: 0.41M trehalose; 0.45 M:
0.41 M trehalose in 0.45 M Me2SO, 0.91 M: 0.41 M trehalose in
0.91 M Me2SO, 1.38 M: 0.41 M trehalose in 1.38 M Me2SO, 0.8
M: 0.7 M trehalose in 0.8 M Me2SO.
3.2 ATP measurements
Bioluminescence ATP measurements showed that spermatozoa
conserved their energetic stock is changing sample to sample (Fig.
2).
0,1
0,15
0,2
0,25
mol/million spz)
[ATP] (n
0
0,05
12345
Sperm of different males
Luciférase
Figure 2. Bioluminescence ATP measurements
3.3 Fertilization essays
Fertilization rates of more than 80% were obtained in both fresh
and post-thaw spermatozoa with a ratio of 3.8 spermatozoa per
egg (Fig. 3). Fertilization efficiency of thawed sperm (quel CPA
?) was demonstrated using artificially matured oocytes which
allowed the development of embryos and D larvae.
0%
10%
20%
0,20,40,9 3,83,8
Ratio sperm per egg (x 105)
30%
40%
50%
60%
70%
80%
90%
100%
Fertilization rate
Cryopreserved sperm Fresh sperm
Figure 3. Effects of spermatozoa density on fertilization rates
using fresh and cryopreserved sperm.
4. DISCUSSION
This study has confirmed the importance of CPA on motility and
for the first time shown fertility of cryopreserved spermatozoa of
P. margaritifera. Trehalose and Me2SO were confirmed to be the
most effective CPA in preserving the motility and the fertilization
ability of freezed P. margaritifera [1][8], and Crassostrea gigas
[2] spermatozoa. We have shown that cryopreserving
spermatozoa in 0.7 M trehalose and 0.8 M Me2SO coupled with a
medium rapid cooling rate resulted in the best retention of
motility. The higher concentrations of cryoprotectants help reduce
damage due to cryoinjuries but also increase biological toxicity to

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cells [9]. In this study, high fertilization rates were obtained by
adding 15x108 sperm ml-1to 2x103 eggs ml-1 . Embryos and D
larvae development were obtained with low rates due to in-vitro
maturation of oocytes. Fertilization test with natural mature
oocytes are required. For pratical application in pearl oyster
aquaculture, larger-volume cryopreservation procedures must be
developed in future studies.
5. ACKNOWLEDGMENTS
This work was funded within an integrated research program
Perdur “Perliculture Durable” financed jointly by the national
agency of research (ANR) of the French government and by the
Service de la Perliculture of French Polynesia.
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