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Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis
Journal Pre-proof Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis Yibing Liu, Mark Gluis, Penny Miller-Ezzy, Jianguang Qin, Jiabo Han, Xin Zhan, Xiaoxu Li PII:
S0044-8486(19)31599-6
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734554
Reference:
AQUA 734554
To appear in:
Aquaculture
Received Date: 27 June 2019 Revised Date:
28 September 2019
Accepted Date: 29 September 2019
Please cite this article as: Liu, Y., Gluis, M., Miller-Ezzy, P., Qin, J., Han, J., Zhan, X., Li, X., Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734554. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis Yibing Liu a, Mark Gluis b, Penny Miller-Ezzy b, Jianguang Qin c, Jiabo Han a, Xin Zhan d,*, Xiaoxu Li b,*
a
Dalian Key Laboratory of Conservation Biology for Endangered Marine mammals,
Liaoning Ocean and Fisheries Science Research Institute, Dalian, 116023, China b
South Australian Research and Development Institute - Aquatic Sciences Centre, Adelaide,
5024, Australia c
College of Science and Engineering, Flinders University, Adelaide, 5042, Australia
d
The Ocean College, Hainan University, Haikou, 570228, China
* Corresponding author: Xin Zhan Email: [email protected]
* Corresponding author: Xiaoxu Li Tel: ++ 61 8 8429 0504 Fax: ++ 61 8 8207 5481 Email: [email protected]
Highlights
This study developed a programmable freezing technique to cryopreserve larvae in Mytilus galloprovinvialis. The post-thaw survival rate has been improved significantly by the application of Ficoll and polyvinylpyrrolidone together. The technique can facilitate breeding programs and hatchery management in the mussel aquaculture industry.
Abstract This study investigated the factors important to the development of a programmable larval cryopreservation technique in Mytilus galloprovinvialis, including (1) larval developmental stages; (2) cryoprotectant agents (CPAs); (3) thawing temperatures; (4) sucrose concentrations to remove CPA after thawing and (5) straw volumes, using D-larval rate as the post-thaw survival indicator. The results showed that larvae at 25 h post-fertilization (PF) had the highest resistance to cryopreservation. A post-thaw D-larval rate higher than 80% was achieved when the larvae were cryopreserved with 10% ethylene glycol + 7.5% Ficoll + 0.2% polyvinylpyrrolidone, thawed at 28 °C and used 9% sucrose solution as the medium to remove CPA after thawing. No significant difference in post-thaw D-larval rates (P > 0.05) was found between larvae cryopreserved in 0.25 mL and 0.5 mL straws. The performance comparison experiment showed that although a significantly lower survival rate (P < 0.05) was found in cryopreserved larvae than that in fresh larvae at day 2 PF (D larvae), no significant difference (P > 0.05) was shown on relative mortality rate from day 8 PF (early umbonal larvae) to day 32 PF (spat) and on shell length at day 8 PF and day 32 PF between fresh and cryopreserved larvae. Therefore, the larval cryopreservation technique developed in this study would enhance the breeding program and all year-round hatchery production in this species.
Keywords: Mytilus galloprovinvialis, larval cryopreservation, Ficoll, polyvinylpyrrolidone
1
1. Introduction
2
Larval cryopreservation in aquaculture has been acknowledged as an effective and reliable
3
technique to preserve superior genetic resources, facilitate breeding design flexibility, provide
4
a reference family for selective breeding and supply larvae without seasonal limitation
5
(Zhang, 2004; Paredes et al., 2013; Labbé et al., 2018). However, larval cryopreservation in
6
marine bivalves is very difficult with few competent pediveliger larvae produced, < 3% in
7
oysters, clams and mussels (Chao et al., 1997; Lin et al., 1999; Usuki et al., 2005; Wang et al.,
8
2011; Paredes et al., 2012, 2013; Suneja et al., 2014; Labbé et al., 2018; Simon and Yang,
9
2018). The reasons causing this phenomenon are still unclear, may be due to that larvae in
10
marine bivalves are large in size and have large propositions of yolk, thus a low surface area
11
to volume ratio, leading to high sensitivity to cryopreservation (Ushijima et al., 1999; Seki et
12
al., 2007; Gosling, 2015).
13
Major steps in marine bivalve larval cryopreservation include (1) larval collection and
14
preparation; (2) cryoprotectant agent (CPA) stock solution preparations; (3) preparation of
15
CPA and larval suspension, and equilibration; (4) transfer CPA and larval suspension into
16
cryo-containers; (5) cooling by liquid nitrogen (LN) vapour; (6) storage in LN; (7) thawing
17
and CPA removal; and (8) post-thaw larval quality evaluation. In the development of a larval
18
cryopreservation technique, controlling the formation of intracellular ice is of importance.
19
One of the most effective strategies is to optimize CPAs or their combination to improve the
20
post-thaw survival rate (Massip et al., 2001; Jain and Paulson, 2006; Paredes et al., 2012;
21
Veleva et al., 2013; Liu and Li, 2015). Normally, CPA is divided into permeable and non-
22
permeable types depending on their ability to penetrate the cell membrane, a combination of
23
these two types is commonly used for the embryo cryopreservation in humans and livestock
24
species (Michelmann and Nayudu, 2006; Pereira and Marques, 2008; Youngs, 2011). In
25
Mytilus galloprovincialis larval cryopreservation, ethylene glycol has been shown as a
26
suitable permeable CPA, whereas a suitable non-permeable CPA is yet to be determined,
27
although sugars, such as polyvinylpyrrolidone and trehalose, have been evaluated (Wang et
28
al., 2011; Paredes et al., 2013). Recently, Ficoll has been found as an effective non-permeable
29
CPA for the oocyte cryopreservation in M. galloprovincialis (Liu and Li, 2015) which also
30
has shown beneficial effects in embryo cryopreservation of other species (Kuleshova et al.,
31
2001; Pereira and Marques, 2008). Nevertheless, Ficoll has not been evaluated on larval
32
cryopreservation in marine bivalves.
33
Methods to remove CPA after thawing are important for the success of embryo
34
cryopreservation, as inappropriate CPA removal could compromise embryo quality due to
35
osmotic pressure imbalance (Michelmann and Nayudu, 2006; Pereira and Marques, 2008). In
36
humans and livestock species, these adverse osmotic effects have been mitigated by using
37
sucrose solution to remove the CPA after thawing (Michelmann and Nayudu, 2006; Pereira
38
and Marques, 2008; Youngs, 2011). However, this method has not been evaluated in marine
39
bivalves.
40
M. galloprovincialis is one of the most important bivalve species farmed in the world
41
(Pettersen et al., 2010; Paredes et al., 2013). In addition, their larvae have also been
42
extensively utilized as a biological indicator in environmental monitoring programs (Jha et al.,
43
2000; Geffard et al., 2002; Beiras et al., 2003). Therefore, development of a M.
44
galloprovincialis larval cryopreservation technique could be beneficial for providing
45
progenies without seasonal limitations not only for the aquaculture productions, but also for
46
environmental monitoring programs (Pettersen et al., 2010; Sánchez-Lazo and Martínez-Pita,
47
2012a, b). In our previous study in this species, we have found that the combination of Ficoll
48
and ethylene glycol as CPA and using 9% sucrose solution to remove CPA after thawing
49
were suitable for oocyte cryopreservation (Liu and Li, 2015). Therefore, in order to achieve
50
the post-thaw larvae survival level that could potentially be used in M. galloprovincialis
51
commercial production or other applications, such as, selective breeding, environment
52
monitoring, etc., the protocol developed from oocyte cryopreservation was investigated in
53
this study with the purpose to develop a programmable freezing technique for larval
54
cryopreservation in M. galloprovincialis.
55
2. Materials and methods
56
2.1. Larval collection
57
Mature M. galloprovincialis were supplied by Kinkawooka Mussels in Port Lincoln, South
58
Australia and transported in a refrigerated container overnight to South Australian Research
59
and Development Institute (SARDI) - Aquatic Sciences Centre. Upon arrival, the animals
60
were washed with 1 µm filtered seawater (FSW) prior to spawning induction. Mussels were
61
spawned individually by thermal shock (increasing water temperature from 17 to 20 °C for
62
30 min) and the gametes were collected as described by Liu and Li (2015). In each
63
experiment, fresh eggs and sperm were collected from at least 10 and 5 individuals,
64
respectively. Fresh eggs were fertilized in a 2 L beaker at a sperm to egg ratio of 20:1. At 10
65
min post-fertilization, the fertilized eggs were gently washed on a 35 µm screen and then
66
cultured in 50 L tanks at a concentration of approximate 20 individuals mL-1 at 17 °C. When
67
the fertilized eggs were cultured for a predetermined time post-fertilization (PF), the larvae
68
were collected from the top of tanks on a 35 µm screen. The larvae were then transferred into
69
10 mL tubes and stored on ice, which would minimize the potential temperature shock on
70
larvae when mixed with cold CPAs. The larvae density was counted and diluted to 4 x 105
71
individuals mL-1 for the subsequent experiments. The larvae stored on ice were used within
72
30 min in each experiment.
73
2.2. Chemicals and equipment
74
All chemicals, ethylene glycol (EG), sucrose, Ficoll PM 70 (FIC) and polyvinylpyrrolidone
75
(PVP) were purchased from Sigma-Aldrich Pty Ltd (St. Louis, MO, USA). The
76
cryoprotective stock solution was prepared in Milli-Q water at a concentration two times as
77
high as that required in the experiments. Therefore, when the same volume of stock solution
78
and larvae suspension were mixed, the required final chemical concentration was produced.
79
The programmable controller applied in Liu and Li (2015) was used in this study. The
80
required temperatures in the thawing bath were produced by mixing ambient and boiled
81
seawater. The recovery bath (18 ºC) was produced by mixing ambient and cold seawater.
82
2.3. Experiments
83 84
2.3.1. Effects of different larval developmental stages on post-thaw D-larval rates
85
In this study, 10% EG + 7.5% FIC was selected as CPA because this combination has
86
produced the highest post-thaw D-larval rates in oocyte cryopreservation for this species (Liu
87
and Li, 2015). The larvae were collected at 10, 20, 25 and 30 h post-fertilization and were
88
mixed with CPA for 10 min on ice. The mixtures were then transferred into 0.25 mL straws
89
and maintained at 0 °C for 5 min in the programmable freezer. The straws were then cooled
90
at a rate of -1 °C/min from 0 to -10 °C and at -0.3 °C/min from -10 to -34 °C before being
91
plunged into LN. After at least 12 h storage in LN, the straws were thawed individually in a
92
28°C water bath until the ice melted. They were then moved into an 18 °C seawater bath for
93
recovery for approximately 5 min. The content in each straw was then expelled into a 4 mL
94
tube and diluted for 10 min using 0.25 mL medium consisting of 9% sucrose. The CPA
95
concentration was further diluted twice by adding a same volume of FSW as that in the tube
96
at 10 min intervals. The larvae were then cultured in 500 mL containers until reaching D-
97
larval stage (48 h post-fertilization). The D-larval rate was calculated as the percentage of
98
larvae that develop into D-larvae. Controls were established and cultured in the same way as
99
those cryopreserved. Each treatment was replicated three times.
100
2.3.2. Comparison of different CPAs on post-thaw D-larval rates
101
The highest post-thaw D-larval rates were achieved when the larvae were cryopreserved at 25
102
h post-fertilization in the previous experiment. Therefore, this stage of larvae was used for
103
this and subsequent experiments. In this experiment, different CPAs, 10% EG, 10% EG + 7.5%
104
FIC, 10% EG + 0.2% PVP and 10% EG + 7.5% FIC + 0.2% PVP were evaluated for the
105
post-thaw D-larval rates. The other procedures were the same as Experiment 2.3.1
106 107 108 109
2.3.3. Effects of polyvinylpyrrolidone (PVP) concentrations on post-thaw Dlarval rates The CPA combination, 10% EG + 7.5% FIC + 0.2% PVP produced the highest post-thaw D-
110
larval rates in the previous experiment. In this experiment, different PVP concentrations (0.05,
111
0.1, 0.2 or 0.4%) were compared for post-thaw D-larval rates. The other procedures were the
112
same as Experiment 2.3.2.
113 114 115
2.3.4. Effects of thawing temperatures on post-thaw D-larval rates The highest post-thaw D-larval rates were achieved when the 0.2% PVP was added in 10%
116
EG + 7.5% FIC in the previous experiment. Therefore, this CPA combination was used for
117
this and subsequent experiments. In this experiment, thawing temperatures at 18, 28, 38, 48
118
and 58 °C were evaluated on post-thaw D-larval rates. The other procedures were the same as
119
Experiment 2.3.3.
120 121 122 123
2.3.5. Effects of sucrose CPA dilution medium concentrations on post-thaw D-larval rates The highest post-thaw D-larval rates were achieved when a 28 °C thawing temperature was
124
used in the previous experiment. Thus, this temperature was used for this and subsequent
125
experiments. In this experiment, 6, 9, 12 and 15% sucrose solution were evaluated to dilute
126
the CPA after thawing. The other procedures were the same as Experiment 2.3.4.
127 128 129
2.3.6. Effects of straw volumes on post-thaw D-larval rates Sucrose at concentration of 9% produced the highest post-thaw D-larval rates in the previous
130
experiment and this medium was used for this and subsequent experiments. In this
131
experiment, straw volume of 0.25 mL (Minitube, Germany) and 0.5 mL (IMV, France) was
132
evaluated on post-thaw D-larval rates. The other procedures were the same as Experiment
133
2.3.5.
134 135 136 137
2.3.7. Performance comparison between progenies produced with cryopreserved and fresh larvae The cryopreservation protocol (larvae: 25 h PF; CPA: 10% EG + 7.5% FIC + 0.2% PVP;
138
post-thaw CPA removal medium: 9% sucrose; thawing temperature: 28 °C; straw volume: 0.5
139
mL) developed in this study was applied to compare the performance between progenies
140
produced with fresh and cryopreserved larvae. After the assessment of D-larval rates, D
141
larvae were transferred into 10 L tanks and stocked at a density of ～10 individuals mL-1 for
142
cryopreserved larvae (3 tanks) and fresh larvae (3 tanks). Methods for larval culture and
143
settlement were the same as those used by Wang et al. (2011) and Pettersen et al. (2010). The
144
survival rate (%) was calculated in terms of dividing the number of alive on the sample
145
collection date (PF) by the number of larvae initially stocked in the tank. The relative
146
mortality rate (%) was calculated by dividing the difference in survival rate between adjacent
147
sampling collection dates with the survival rate at the start of this period. At day 8 and day 32
148
PF, 30 larvae from each tank were randomly selected to measure shell length.
149 150
2.4. Statistical analysis
151
The D-larval rate was normalized against the controls before data analysis. Data was
152
normalized to the control mean percentage of larval abnormality using Abbot’s formula: P =
153
(Pe-Pc)/(100-Pc) x 100, where Pc and Pe are control and experimental percentages of response,
154
respectively (Paredes et al., 2013). The data were presented as mean ± standard deviation (SD)
155
and was arcsine transformed for statistical analyses using SPSS 22. One-way analysis of
156
variance (ANOVA) was applied to analyze the data on the effects of different larval stages,
157
different CPAs, different PVP concentrations, sucrose medium concentrations and thawing
158
temperatures on post-thaw D-larval rate. The Least-Significant Difference (LSD) comparison
159
test was used when significance was observed. A t-test was applied to compare the straw
160
volumes on D-larval rate after cryopreservation and a paired sample t-test was applied to
161
compare the performances (survival rate, relative mortality rate and spat size) between
162
progenies produced with fresh and cryopreserved larvae. Differences were considered
163
statistically significant at P < 0.05.
164
3. Results
165
3.1. Effects of different larval developmental stages on post-thaw D-larval rate
166 167
The highest post-thaw D-larval rate of 62.6 ± 4.1% was achieved when the 25 h PF larvae
168
were cryopreserved, which was significantly higher than those collected at other periods (P <
169
0.05; Fig. 1).
170
3.2. Comparison of different CPAs on post-thaw D-larval rate
171 172
The addition of 7.5% FIC + 0.2% PVP in 10% EG significantly improved the post-thaw D-
173
larval rate to 78.0 ±7.7% in comparison with 10% EG, 10% EG +7.5% FIC and 10% EG +
174
0.2% PVP treatments (P < 0.05; Fig. 2). The addition of 7.5% FIC or 0.2% PVP in 10% EG
175
did not affect the post-thaw D-larval rate in comparison with 10% EG alone (P > 0.05).
176
3.3 Effects of polyvinylpyrrolidone (PVP) concentrations on post-thaw D-larval
177
rate
178 179
The addition of 0.2% PVP in 10% EG + 7.5% FIC resulted in the highest post-thaw D-larval
180
rate of 84.0 ± 1.5% in comparison with 10% EG + 7.5% FIC and the addition of PVP at
181
other concentrations evaluated (P < 0.05; Fig. 3).
182 183 184
3.4. Effects of thawing temperatures on post-thaw D-larval rate The highest post-thaw D-larval rate of 80.5 ± 6.0% was produced when a 28 °C thawing
185
temperature was used (Fig. 4). This thawing temperature produced significantly higher post-
186
thaw D-larval rate than an 18 °C thawing temperature (P < 0.05), whereas no significant
187
difference was found in comparison with other thawing temperatures (P > 0.05).
188
3.5. Effects of sucrose CPA dilution medium concentrations on post-thaw D-
189
larval rate
190 191
Significantly higher post-thaw D-larval rate was produced when the 9% sucrose was used as
192
a medium to dilute the CPA after thawing in comparison with either the lower or higher
193
sucrose concentrations evaluated (P < 0.05; Fig. 5).
194 195 196
3.6. Effects of straw volumes on post-thaw D-larval rate No significant difference was found when the larvae were cryopreserved in 0.25 and 0.5 mL
197
straws (P > 0.05), resulting in 78.4 ± 6.3% and 78.0 ± 5.8% post-thaw D-larval rates,
198
respectively.
199 200
3.7. Performance comparison between progenies produced with cryopreserved and fresh larvae
201 202
Table 1 demonstrated that the survival rate of fresh larvae was significantly higher than that
203
of cryopreserved larvae at all the periods PF evaluated (P < 0.05). However, after day 8 PF,
204
there was no significant difference in relative mortality rate between fresh and cryopreserved
205
larvae. Moreover, no significant difference (P > 0.05) in shell length was found between
206
fresh and cryopreserved progenies at day 8 PF (fresh larvae: 146.7 ± 5.8 µm; cryopreserved
207
larvae: 143.9 ± 2.5 µm, n = 90) and day 32 PF (fresh larvae: 380.0 ± 8.7 µm; cryopreserved
208
larvae 371.7 ± 9.1 µm, n = 90).
209 210
4. Discussion
211
This study has developed a programmable larval cryopreservation technique for M.
212
galloprovincialis. The post-thaw larval survival rate was improved to >80% when the 25 h
213
post-fertilization larvae were cryopreserved with 10% EG + 7.5% FIC + 0.2% PVP, thawed
214
at 28 °C and using 9% sucrose solution as medium to remove CPA after thawing.
215
It has been generally acknowledged that larval age is of importance for cryosurvival, as
216
larvae at different developmental stages have various ability to be cryopresered (Gwo 1995;
217
Nascimento et al., 2005; Paredes et al., 2012, 2013). In this study, M. galloprovincialis larvae
218
collected at 25 h post-fertilization had the highest resistance to cryopreservation, with 62%
219
post-thaw D-larval rates achieved. This developmental stage was later than Paredes et al.
220
(2013) study (20 h) on the same species with about 50% post-thaw D-larval rates produced.
221
This difference may be related to early larvae having more lipid, which is stored as an energy
222
reserve and used for larval development (Bayne et al., 1978; Gosling, 2015). An embryo with
223
high lipid content would be more sensitive to cryopreservation and this phenomenon has been
224
shown in livestock species, such as pigs (Nagashima et al., 1995; Dobrinsky 2002), cattle
225
(Massip 2001; Pryor et al., 2011) and sheep (Massip 2001).
226
Ethylene glycol has been shown as a suitable permeable CPA in larval cryopreservation in M.
227
galloprovincialis and Perna canaliculus (Paredes et al., 2012, 2013). For the non-permeable
228
CPAs, on the other hand, although some sugars such as trehalose and PVP have been
229
investigated in mussels and oysters, no spat has been produced in the published results
230
(Paredes et al., 2012, 2013). In our previous study, the non-permeable CPA, FIC,
231
demonstrated the ability to significantly improve the post-thaw oocyte quality in M.
232
galloprovincialis (Liu and Li, 2015). Although the PVP was not used for the larval
233
cryopreservation in this species (Paredes et al., 2013), it has been applied in mice and cattle
234
cryopreservation (Leibo and Oda, 1993; Saha et al., 1996; Titterington and Robinson, 1996;
235
Kim et al., 2008). Furthermore, some studies have shown that a combination of different non-
236
permeable CPAs could be more effective for improving the cryosurvival rate of larvae (Saha
237
et al., 1996; Titterington and Robinson, 1996). In this study, 10% EG + 7.5% FIC and 10%
238
EG + 0.2% PVP had the similar cryoprotective ability as 10% EG used alone (Fig. 2). This is
239
in agreement with the study by Paredes et al. (2013) on the same species where 10% EG with
240
or without trehalose produced a similar post-thaw D-larval rate. However, in the present
241
study, when the 7.5% FIC was applied with 0.2% PVP together, the post-thaw D-larval rate
242
was significantly improved (Fig 2, 3). This result indicates that the combined effects of FIC
243
and PVP could play an important role in M. galloprovincialis larval cryopreservation.
244
Reasons for this improvement are not clear, although it might be because the intercellular ice
245
formation is limited by the application of suitable non-permeable CPAs as suggested by Gao
246
and Critser (2000) in their review on cryoinjury mechanisms. In addition, further study would
247
be needed to determine if any correlation between the improvement in post-thaw survival rate
248
achieved in this study and the toxicity of CPAs used. Result in this study agrees with the
249
study reported by Saha et al. (1996) that the hatching rate was improved significantly when
250
cattle embryos were cryopreserved in EG (40%) plus two sugars (11.3% trehalose + 20%
251
PVP) in comparison with EG used alone or in combination with either sugars.
252
Thawing temperature is a critical factor affecting the success of larval cryopreservation. High
253
thawing temperature could inhibit recrystallization of the internal ice, while a low
254
temperature might prevent osmotic pressure on the larvae as extracellular medium melts fast.
255
The reduced pressure could result in a rapid shift of free water into the larvae if the
256
intracellular CPA cannot diffuse out quickly, leading to extreme larval swelling, and possible
257
rupture (Jain and Paulson, 2006). Therefore, controlling thawing temperature is necessary,
258
although studies in this area have been lacking in marine bivalves. In this study, 28 °C was
259
the optimal thawing temperature for M. galloprovincialis larval cryopreservation. This
260
temperature has also been applied for the oocyte and larval cryopreservation in the same
261
species (Paredes et al., 2013; Liu and Li, 2015), Crassostrea gigas (Lin et al., 1999; Paredes
262
et al., 2013; Suneja et al., 2014) and P. canaliculus (Paredes et al., 2012). This temperature is
263
higher than that applied in Pinctada fucata martensii (25 °C; Choi and Chang, 2003).
264
Besides thawing temperature, application of proper medium to remove CPA from the larvae
265
after thawing could also improve the success of cryopreservation (Woods et al., 2004; Jain
266
and Paulson, 2006). Medium containing sucrose has been widely used to remove CPA in
267
embryo cryopreservation in cattle (Mahmoudzadeh et al., 1993; Youngs, 2011), dogs
268
(Guaitolini et al., 2012), sheep (Youngs, 2011), mice (Fathi et al., 2012) and pigs (Castillo-
269
Martín et al., 2013). In the larvae cryopreservation of marine mollusc species, seawater and
270
bovine serum albumin are commonly used to remove CPA in oysters (Lin et al., 1999; Choi
271
and Chang, 2003; Paredes et al., 2013; Suneja et al., 2014) and mussels (Wang et al., 2011;
272
Paredes et al., 2012, 2013), although low or no spat productions were reported in these
273
studies. In the current study, 9% sucrose solution achieved the highest post-thaw D-larval
274
rates (Fig. 5) which has also been used on the oocyte cryopreservation in the same species
275
(Liu and Li, 2015).
276
Different straw volumes have different surface to volume ratio which is important for
277
cryosurvival. Higher surface to volume ratio could enable a uniform cooling rate and proper
278
heat exchange properties which would be advantageous for maintaining cell quality (Zhu et
279
al., 2014). In this study, 0.25 mL and 0.5 mL straws produced similar post-thaw D-larval
280
rates. This is in agreement with a study on P. canaliculus larvae cryopreservation where no
281
significant difference on post-thaw D-larval rate was found when larvae were cryopreserved
282
in 0.25 mL and 0.5 mL straws (Paredes et al., 2012).
283
After cryopreservation, the developmental capacity of larvae is reduced, resulting in a
284
significantly lower survival rate compared to fresh larvae (Table 1). This phenomenon has
285
been observed in other studies, as the mechanism of cell cleavage might be compromised
286
during cryopreservation (Wang et al., 2011; Paredes et al., 2012). Nevertheless, in the present
287
study, the relative mortality rates after day 8 PF remained the same between fresh and
288
cryopreserved larvae (Table 1). Moreover, the shell length of larvae at day 8 and spat at day
289
32 PF was similar (P > 0.05) between the fresh and cryopreserved larvae. Therefore, the
290
results of this study indicate that cryopreservation affects the larvae at an early developmental
291
stage in M. galloprovincialis. Similar phenomenon has also been observed in other studies on
292
the same species (Wang et al., 2011; Paredes et al., 2012, 2013; Liu and Li, 2015).
293
In conclusion, the larval cryopreservation technique developed in this study has significantly
294
improved the post-thaw D-larval rate to >80% in M. galloprovincialis with cryodamage being
295
expressed only at early development stages. Therefore, this technique could be applied to
296
guarantee a reliable year round supply of progenies for hatchery production and
297
environmental monitoring programs, and enhance the management efficiency of breeding
298
programs in the M. galloprovincialis aquaculture industry.
299
300
Acknowledgments
301
This research was funded by the Talent Project of Revitalizing Liaoning (Project No.
302
XLYC1807087), Department of Ocean and Fisheries of Liaoning (Project No. 201829),
303
China Scholarship Council and South Australian Research and Development Institute
304
(SARDI). We thank Mr Andy Dyer of Kinkawooka Mussels for the provision of mussel
305
broodstock. We also thank master student Zhongling Lin of Dalian Ocean University for
306
technical assistance.
307 308
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K., 2007. The permeability to water and cryoprotectants of immature and mature
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36. Simon, N.A., Yang, H., 2018. Cryopreservation of trochophore larvae from the hard
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37. Suneja, S., Alfaro, A.C., Rusk, A.B., Morrish, J.R., Tervit, H.R., McGowan, L.T.,
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New. Zeal., J. Mar. Fresh. 48, 335-349.
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38. Titterington, J.L., Robinson, J., 1996. The protective action of polyvinylpyrrolidone-
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subsequent developmental potential post-thaw in vitro and in vivo. Hum. Reprod. 11,
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2697-2702.
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39. Ushijima, H., Yamakawa, H., Nagashima, H., 1999. Cryopreservation of bovine pre-
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morula-stage in vitro matured/in vitro fertilized embryos after delipidation and before
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40. Usuki, H., Hamaguchi, M., Ishioka, H., 2005. Effects of developmental stage,
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seawater concentration and rearing temperature on cryopreservation of Pacific oyster
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Crassostrea gigas larvae. Fisheries. Sci. 68, 757-762.
419
41. Veleva, Z., Orava, M., Nuojua-Huttunen, S., Tapanainen, J.S., Martikainen, H., 2013.
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Factors affecting the outcome of frozen-thawed embryo transfer. Hum. Reprod. 28,
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422
42. Wang, H., Li, X., Wang, M., Clarke, S., Gluis, M., Zhang, Z., 2011. Effects of larval
423
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424
galloprovincialis Lamarck. Aquac. Res. 42, 1816-1823.
425 426 427 428
43. Woods, E.J., Benson, J.D., Agca, Y., Critser, K., 2004. Fundamental cryobiology of reproductive cells and tissues. Cryobiology 48, 146-156. 44. Youngs, C.R., 2011. Cryopreservation of preimplantation embryos of cattle, sheep, and goats. J. Vis. Exp. 54, e2764.
429
45. Zhang, T.T., 2004. Cryopreservation of gametes and embryos of aquatic species. In:
430
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431
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432
46. Zhu, W., Li, X., Qu, D., Liu, Y., Clarke, S., 2014. Cryopreservation of sperm from
433
wild greenlip abalone (Haliotis laevigata) in Australia. Aquaculture 432, 60-66.
434
Table Legend
435
Table 1. Comparison of survival rates and relative mortality rate between fresh and
436
cryopreserved larvae during periods post-fertilization, n = 3. Different letter indicates
437
significant difference (P < 0.05).
438
Table 1.
Survival rate (%)
Relative mortality rate (%)
Days post fertilization
fresh larvae
cryopreserved larvae
day 2 (D larvae)
91.7 ± 3.0 A
71.6 ± 0.9 B
day 8
72.0 ± 3.9 A′
44.5 ± 1.3 B′
21.3 ± 6.4 b
37.8 ± 1.9 a
day 14
59.3 ± 5.1 A″
37.3 ± 5.4 B″
17.2 ± 11.8 a′
16.3 ± 9.5 a′
fresh larvae
cryopreserved larvae
day 20
48.7 ± 3.5 A‴
30.3 ± 1.2 B‴
17.8 ± 6.1 a″
17.9 ± 9.5 a″
day 26 (eyed larvae)
46.7 ± 2.7 A″″
28.5 ± 2.3 B″″
3.8 ± 6.7 a‴
6.1 ± 5.3 a‴
day 32 (spat)
37.5 ± 1.5 A″‴
21.3 ± 0.6 B″‴
19.7 ± 1.5 a″″
25.0 ± 4.4 a″″
439
Figure Legends
440
Fig. 1. Post-thaw D-larval rates (%) when larvae were cryopreserved at different
441
developmental stages, n = 3. Different letter indicates significant difference (P < 0.05). All
442
the data has been normalized to the controls.
443
Fig. 2. Post-thaw D-larval rates (%) when larvae were cryopreserved in different CPA
444
combination(s), n = 3. Different letter indicates significant difference (P < 0.05). All the data
445
has been normalized to the controls.
446
Fig. 3. Post-thaw D-larval rates (%) when larvae were cryopreserved in 10% EG + 7.5% FIC
447
with addition of different concentrations of PVP, n = 3. Different letter indicates significant
448
difference (P < 0.05). All the data has been normalized to the controls.
449
Fig. 4. Post-thaw D-larval rates (%) of larvae thawed at various thawing temperatures after
450
cryopreservation, n = 3. Different letter indicates significant difference (P < 0.05). All the
451
data has been normalized to the controls.
452
Fig. 5. Post-thaw D-larval rates (%) of larvae diluted with different concentrations of sucrose
453
after thawing, n = 3. Different letter indicates significant difference (P < 0.05). All the data
454
has been normalized to the controls.
100.0
D-larval rate (%)
80.0 A 60.0 B 40.0
C
C
20.0
0.0 10 h
455 456
Fig.1.
20 h 25 h Periods post-fertilization
30 h
457 100.0 A
D-larval rate (%)
80.0 B
60.0 B
B
40.0
20.0
0.0 10% EG
458 459 460
Fig.2.
10% EG + 7.5% FIC
10% EG + 0.2% PVP 10% EG + 7.5% FIC + 0.2% PVP
100.0 A B
D-larval rate (%)
80.0
B
B 60.0
C
40.0 20.0 0.0 10% EG + 7.5% 10% EG + 7.5% 10% EG + 7.5% 10% EG + 7.5% 10% EG + 7.5% FIC FIC + 0.05% PVP FIC + 0.1% PVP FIC + 0.2% PVP FIC + 0.4% PVP Cryoprotectant agents
461 462
Fig.3.
100 A
D-larval rate (%)
80
AB
B
AB
AB
60 40 20 0 18°C
463 464
Fig.4.
28°C
38°C 48°C Thawing temperatures
58°C
100.0 A D-larval rate (%)
80.0 B
B
B
60.0 40.0 20.0 0.0 6% sucrose
465 466
Fig.5.
9% sucrose 12% sucrose Sucrose concentrations
15% sucrose
Highlights
This study developed a programmable freezing technique to cryopreserve larvae in Mytilus galloprovinvialis. The post-thaw survival rate has been improved significantly by the application of Ficoll and polyvinylpyrrolidone together. The technique can facilitate breeding programs and hatchery management in the mussel aquaculture industry.
S0044-8486(19)31599-6
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734554
Reference:
AQUA 734554
To appear in:
Aquaculture
Received Date: 27 June 2019 Revised Date:
28 September 2019
Accepted Date: 29 September 2019
Please cite this article as: Liu, Y., Gluis, M., Miller-Ezzy, P., Qin, J., Han, J., Zhan, X., Li, X., Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734554. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis Yibing Liu a, Mark Gluis b, Penny Miller-Ezzy b, Jianguang Qin c, Jiabo Han a, Xin Zhan d,*, Xiaoxu Li b,*
a
Dalian Key Laboratory of Conservation Biology for Endangered Marine mammals,
Liaoning Ocean and Fisheries Science Research Institute, Dalian, 116023, China b
South Australian Research and Development Institute - Aquatic Sciences Centre, Adelaide,
5024, Australia c
College of Science and Engineering, Flinders University, Adelaide, 5042, Australia
d
The Ocean College, Hainan University, Haikou, 570228, China
* Corresponding author: Xin Zhan Email: [email protected]
* Corresponding author: Xiaoxu Li Tel: ++ 61 8 8429 0504 Fax: ++ 61 8 8207 5481 Email: [email protected]
Highlights
This study developed a programmable freezing technique to cryopreserve larvae in Mytilus galloprovinvialis. The post-thaw survival rate has been improved significantly by the application of Ficoll and polyvinylpyrrolidone together. The technique can facilitate breeding programs and hatchery management in the mussel aquaculture industry.
Abstract This study investigated the factors important to the development of a programmable larval cryopreservation technique in Mytilus galloprovinvialis, including (1) larval developmental stages; (2) cryoprotectant agents (CPAs); (3) thawing temperatures; (4) sucrose concentrations to remove CPA after thawing and (5) straw volumes, using D-larval rate as the post-thaw survival indicator. The results showed that larvae at 25 h post-fertilization (PF) had the highest resistance to cryopreservation. A post-thaw D-larval rate higher than 80% was achieved when the larvae were cryopreserved with 10% ethylene glycol + 7.5% Ficoll + 0.2% polyvinylpyrrolidone, thawed at 28 °C and used 9% sucrose solution as the medium to remove CPA after thawing. No significant difference in post-thaw D-larval rates (P > 0.05) was found between larvae cryopreserved in 0.25 mL and 0.5 mL straws. The performance comparison experiment showed that although a significantly lower survival rate (P < 0.05) was found in cryopreserved larvae than that in fresh larvae at day 2 PF (D larvae), no significant difference (P > 0.05) was shown on relative mortality rate from day 8 PF (early umbonal larvae) to day 32 PF (spat) and on shell length at day 8 PF and day 32 PF between fresh and cryopreserved larvae. Therefore, the larval cryopreservation technique developed in this study would enhance the breeding program and all year-round hatchery production in this species.
Keywords: Mytilus galloprovinvialis, larval cryopreservation, Ficoll, polyvinylpyrrolidone
1
1. Introduction
2
Larval cryopreservation in aquaculture has been acknowledged as an effective and reliable
3
technique to preserve superior genetic resources, facilitate breeding design flexibility, provide
4
a reference family for selective breeding and supply larvae without seasonal limitation
5
(Zhang, 2004; Paredes et al., 2013; Labbé et al., 2018). However, larval cryopreservation in
6
marine bivalves is very difficult with few competent pediveliger larvae produced, < 3% in
7
oysters, clams and mussels (Chao et al., 1997; Lin et al., 1999; Usuki et al., 2005; Wang et al.,
8
2011; Paredes et al., 2012, 2013; Suneja et al., 2014; Labbé et al., 2018; Simon and Yang,
9
2018). The reasons causing this phenomenon are still unclear, may be due to that larvae in
10
marine bivalves are large in size and have large propositions of yolk, thus a low surface area
11
to volume ratio, leading to high sensitivity to cryopreservation (Ushijima et al., 1999; Seki et
12
al., 2007; Gosling, 2015).
13
Major steps in marine bivalve larval cryopreservation include (1) larval collection and
14
preparation; (2) cryoprotectant agent (CPA) stock solution preparations; (3) preparation of
15
CPA and larval suspension, and equilibration; (4) transfer CPA and larval suspension into
16
cryo-containers; (5) cooling by liquid nitrogen (LN) vapour; (6) storage in LN; (7) thawing
17
and CPA removal; and (8) post-thaw larval quality evaluation. In the development of a larval
18
cryopreservation technique, controlling the formation of intracellular ice is of importance.
19
One of the most effective strategies is to optimize CPAs or their combination to improve the
20
post-thaw survival rate (Massip et al., 2001; Jain and Paulson, 2006; Paredes et al., 2012;
21
Veleva et al., 2013; Liu and Li, 2015). Normally, CPA is divided into permeable and non-
22
permeable types depending on their ability to penetrate the cell membrane, a combination of
23
these two types is commonly used for the embryo cryopreservation in humans and livestock
24
species (Michelmann and Nayudu, 2006; Pereira and Marques, 2008; Youngs, 2011). In
25
Mytilus galloprovincialis larval cryopreservation, ethylene glycol has been shown as a
26
suitable permeable CPA, whereas a suitable non-permeable CPA is yet to be determined,
27
although sugars, such as polyvinylpyrrolidone and trehalose, have been evaluated (Wang et
28
al., 2011; Paredes et al., 2013). Recently, Ficoll has been found as an effective non-permeable
29
CPA for the oocyte cryopreservation in M. galloprovincialis (Liu and Li, 2015) which also
30
has shown beneficial effects in embryo cryopreservation of other species (Kuleshova et al.,
31
2001; Pereira and Marques, 2008). Nevertheless, Ficoll has not been evaluated on larval
32
cryopreservation in marine bivalves.
33
Methods to remove CPA after thawing are important for the success of embryo
34
cryopreservation, as inappropriate CPA removal could compromise embryo quality due to
35
osmotic pressure imbalance (Michelmann and Nayudu, 2006; Pereira and Marques, 2008). In
36
humans and livestock species, these adverse osmotic effects have been mitigated by using
37
sucrose solution to remove the CPA after thawing (Michelmann and Nayudu, 2006; Pereira
38
and Marques, 2008; Youngs, 2011). However, this method has not been evaluated in marine
39
bivalves.
40
M. galloprovincialis is one of the most important bivalve species farmed in the world
41
(Pettersen et al., 2010; Paredes et al., 2013). In addition, their larvae have also been
42
extensively utilized as a biological indicator in environmental monitoring programs (Jha et al.,
43
2000; Geffard et al., 2002; Beiras et al., 2003). Therefore, development of a M.
44
galloprovincialis larval cryopreservation technique could be beneficial for providing
45
progenies without seasonal limitations not only for the aquaculture productions, but also for
46
environmental monitoring programs (Pettersen et al., 2010; Sánchez-Lazo and Martínez-Pita,
47
2012a, b). In our previous study in this species, we have found that the combination of Ficoll
48
and ethylene glycol as CPA and using 9% sucrose solution to remove CPA after thawing
49
were suitable for oocyte cryopreservation (Liu and Li, 2015). Therefore, in order to achieve
50
the post-thaw larvae survival level that could potentially be used in M. galloprovincialis
51
commercial production or other applications, such as, selective breeding, environment
52
monitoring, etc., the protocol developed from oocyte cryopreservation was investigated in
53
this study with the purpose to develop a programmable freezing technique for larval
54
cryopreservation in M. galloprovincialis.
55
2. Materials and methods
56
2.1. Larval collection
57
Mature M. galloprovincialis were supplied by Kinkawooka Mussels in Port Lincoln, South
58
Australia and transported in a refrigerated container overnight to South Australian Research
59
and Development Institute (SARDI) - Aquatic Sciences Centre. Upon arrival, the animals
60
were washed with 1 µm filtered seawater (FSW) prior to spawning induction. Mussels were
61
spawned individually by thermal shock (increasing water temperature from 17 to 20 °C for
62
30 min) and the gametes were collected as described by Liu and Li (2015). In each
63
experiment, fresh eggs and sperm were collected from at least 10 and 5 individuals,
64
respectively. Fresh eggs were fertilized in a 2 L beaker at a sperm to egg ratio of 20:1. At 10
65
min post-fertilization, the fertilized eggs were gently washed on a 35 µm screen and then
66
cultured in 50 L tanks at a concentration of approximate 20 individuals mL-1 at 17 °C. When
67
the fertilized eggs were cultured for a predetermined time post-fertilization (PF), the larvae
68
were collected from the top of tanks on a 35 µm screen. The larvae were then transferred into
69
10 mL tubes and stored on ice, which would minimize the potential temperature shock on
70
larvae when mixed with cold CPAs. The larvae density was counted and diluted to 4 x 105
71
individuals mL-1 for the subsequent experiments. The larvae stored on ice were used within
72
30 min in each experiment.
73
2.2. Chemicals and equipment
74
All chemicals, ethylene glycol (EG), sucrose, Ficoll PM 70 (FIC) and polyvinylpyrrolidone
75
(PVP) were purchased from Sigma-Aldrich Pty Ltd (St. Louis, MO, USA). The
76
cryoprotective stock solution was prepared in Milli-Q water at a concentration two times as
77
high as that required in the experiments. Therefore, when the same volume of stock solution
78
and larvae suspension were mixed, the required final chemical concentration was produced.
79
The programmable controller applied in Liu and Li (2015) was used in this study. The
80
required temperatures in the thawing bath were produced by mixing ambient and boiled
81
seawater. The recovery bath (18 ºC) was produced by mixing ambient and cold seawater.
82
2.3. Experiments
83 84
2.3.1. Effects of different larval developmental stages on post-thaw D-larval rates
85
In this study, 10% EG + 7.5% FIC was selected as CPA because this combination has
86
produced the highest post-thaw D-larval rates in oocyte cryopreservation for this species (Liu
87
and Li, 2015). The larvae were collected at 10, 20, 25 and 30 h post-fertilization and were
88
mixed with CPA for 10 min on ice. The mixtures were then transferred into 0.25 mL straws
89
and maintained at 0 °C for 5 min in the programmable freezer. The straws were then cooled
90
at a rate of -1 °C/min from 0 to -10 °C and at -0.3 °C/min from -10 to -34 °C before being
91
plunged into LN. After at least 12 h storage in LN, the straws were thawed individually in a
92
28°C water bath until the ice melted. They were then moved into an 18 °C seawater bath for
93
recovery for approximately 5 min. The content in each straw was then expelled into a 4 mL
94
tube and diluted for 10 min using 0.25 mL medium consisting of 9% sucrose. The CPA
95
concentration was further diluted twice by adding a same volume of FSW as that in the tube
96
at 10 min intervals. The larvae were then cultured in 500 mL containers until reaching D-
97
larval stage (48 h post-fertilization). The D-larval rate was calculated as the percentage of
98
larvae that develop into D-larvae. Controls were established and cultured in the same way as
99
those cryopreserved. Each treatment was replicated three times.
100
2.3.2. Comparison of different CPAs on post-thaw D-larval rates
101
The highest post-thaw D-larval rates were achieved when the larvae were cryopreserved at 25
102
h post-fertilization in the previous experiment. Therefore, this stage of larvae was used for
103
this and subsequent experiments. In this experiment, different CPAs, 10% EG, 10% EG + 7.5%
104
FIC, 10% EG + 0.2% PVP and 10% EG + 7.5% FIC + 0.2% PVP were evaluated for the
105
post-thaw D-larval rates. The other procedures were the same as Experiment 2.3.1
106 107 108 109
2.3.3. Effects of polyvinylpyrrolidone (PVP) concentrations on post-thaw Dlarval rates The CPA combination, 10% EG + 7.5% FIC + 0.2% PVP produced the highest post-thaw D-
110
larval rates in the previous experiment. In this experiment, different PVP concentrations (0.05,
111
0.1, 0.2 or 0.4%) were compared for post-thaw D-larval rates. The other procedures were the
112
same as Experiment 2.3.2.
113 114 115
2.3.4. Effects of thawing temperatures on post-thaw D-larval rates The highest post-thaw D-larval rates were achieved when the 0.2% PVP was added in 10%
116
EG + 7.5% FIC in the previous experiment. Therefore, this CPA combination was used for
117
this and subsequent experiments. In this experiment, thawing temperatures at 18, 28, 38, 48
118
and 58 °C were evaluated on post-thaw D-larval rates. The other procedures were the same as
119
Experiment 2.3.3.
120 121 122 123
2.3.5. Effects of sucrose CPA dilution medium concentrations on post-thaw D-larval rates The highest post-thaw D-larval rates were achieved when a 28 °C thawing temperature was
124
used in the previous experiment. Thus, this temperature was used for this and subsequent
125
experiments. In this experiment, 6, 9, 12 and 15% sucrose solution were evaluated to dilute
126
the CPA after thawing. The other procedures were the same as Experiment 2.3.4.
127 128 129
2.3.6. Effects of straw volumes on post-thaw D-larval rates Sucrose at concentration of 9% produced the highest post-thaw D-larval rates in the previous
130
experiment and this medium was used for this and subsequent experiments. In this
131
experiment, straw volume of 0.25 mL (Minitube, Germany) and 0.5 mL (IMV, France) was
132
evaluated on post-thaw D-larval rates. The other procedures were the same as Experiment
133
2.3.5.
134 135 136 137
2.3.7. Performance comparison between progenies produced with cryopreserved and fresh larvae The cryopreservation protocol (larvae: 25 h PF; CPA: 10% EG + 7.5% FIC + 0.2% PVP;
138
post-thaw CPA removal medium: 9% sucrose; thawing temperature: 28 °C; straw volume: 0.5
139
mL) developed in this study was applied to compare the performance between progenies
140
produced with fresh and cryopreserved larvae. After the assessment of D-larval rates, D
141
larvae were transferred into 10 L tanks and stocked at a density of ～10 individuals mL-1 for
142
cryopreserved larvae (3 tanks) and fresh larvae (3 tanks). Methods for larval culture and
143
settlement were the same as those used by Wang et al. (2011) and Pettersen et al. (2010). The
144
survival rate (%) was calculated in terms of dividing the number of alive on the sample
145
collection date (PF) by the number of larvae initially stocked in the tank. The relative
146
mortality rate (%) was calculated by dividing the difference in survival rate between adjacent
147
sampling collection dates with the survival rate at the start of this period. At day 8 and day 32
148
PF, 30 larvae from each tank were randomly selected to measure shell length.
149 150
2.4. Statistical analysis
151
The D-larval rate was normalized against the controls before data analysis. Data was
152
normalized to the control mean percentage of larval abnormality using Abbot’s formula: P =
153
(Pe-Pc)/(100-Pc) x 100, where Pc and Pe are control and experimental percentages of response,
154
respectively (Paredes et al., 2013). The data were presented as mean ± standard deviation (SD)
155
and was arcsine transformed for statistical analyses using SPSS 22. One-way analysis of
156
variance (ANOVA) was applied to analyze the data on the effects of different larval stages,
157
different CPAs, different PVP concentrations, sucrose medium concentrations and thawing
158
temperatures on post-thaw D-larval rate. The Least-Significant Difference (LSD) comparison
159
test was used when significance was observed. A t-test was applied to compare the straw
160
volumes on D-larval rate after cryopreservation and a paired sample t-test was applied to
161
compare the performances (survival rate, relative mortality rate and spat size) between
162
progenies produced with fresh and cryopreserved larvae. Differences were considered
163
statistically significant at P < 0.05.
164
3. Results
165
3.1. Effects of different larval developmental stages on post-thaw D-larval rate
166 167
The highest post-thaw D-larval rate of 62.6 ± 4.1% was achieved when the 25 h PF larvae
168
were cryopreserved, which was significantly higher than those collected at other periods (P <
169
0.05; Fig. 1).
170
3.2. Comparison of different CPAs on post-thaw D-larval rate
171 172
The addition of 7.5% FIC + 0.2% PVP in 10% EG significantly improved the post-thaw D-
173
larval rate to 78.0 ±7.7% in comparison with 10% EG, 10% EG +7.5% FIC and 10% EG +
174
0.2% PVP treatments (P < 0.05; Fig. 2). The addition of 7.5% FIC or 0.2% PVP in 10% EG
175
did not affect the post-thaw D-larval rate in comparison with 10% EG alone (P > 0.05).
176
3.3 Effects of polyvinylpyrrolidone (PVP) concentrations on post-thaw D-larval
177
rate
178 179
The addition of 0.2% PVP in 10% EG + 7.5% FIC resulted in the highest post-thaw D-larval
180
rate of 84.0 ± 1.5% in comparison with 10% EG + 7.5% FIC and the addition of PVP at
181
other concentrations evaluated (P < 0.05; Fig. 3).
182 183 184
3.4. Effects of thawing temperatures on post-thaw D-larval rate The highest post-thaw D-larval rate of 80.5 ± 6.0% was produced when a 28 °C thawing
185
temperature was used (Fig. 4). This thawing temperature produced significantly higher post-
186
thaw D-larval rate than an 18 °C thawing temperature (P < 0.05), whereas no significant
187
difference was found in comparison with other thawing temperatures (P > 0.05).
188
3.5. Effects of sucrose CPA dilution medium concentrations on post-thaw D-
189
larval rate
190 191
Significantly higher post-thaw D-larval rate was produced when the 9% sucrose was used as
192
a medium to dilute the CPA after thawing in comparison with either the lower or higher
193
sucrose concentrations evaluated (P < 0.05; Fig. 5).
194 195 196
3.6. Effects of straw volumes on post-thaw D-larval rate No significant difference was found when the larvae were cryopreserved in 0.25 and 0.5 mL
197
straws (P > 0.05), resulting in 78.4 ± 6.3% and 78.0 ± 5.8% post-thaw D-larval rates,
198
respectively.
199 200
3.7. Performance comparison between progenies produced with cryopreserved and fresh larvae
201 202
Table 1 demonstrated that the survival rate of fresh larvae was significantly higher than that
203
of cryopreserved larvae at all the periods PF evaluated (P < 0.05). However, after day 8 PF,
204
there was no significant difference in relative mortality rate between fresh and cryopreserved
205
larvae. Moreover, no significant difference (P > 0.05) in shell length was found between
206
fresh and cryopreserved progenies at day 8 PF (fresh larvae: 146.7 ± 5.8 µm; cryopreserved
207
larvae: 143.9 ± 2.5 µm, n = 90) and day 32 PF (fresh larvae: 380.0 ± 8.7 µm; cryopreserved
208
larvae 371.7 ± 9.1 µm, n = 90).
209 210
4. Discussion
211
This study has developed a programmable larval cryopreservation technique for M.
212
galloprovincialis. The post-thaw larval survival rate was improved to >80% when the 25 h
213
post-fertilization larvae were cryopreserved with 10% EG + 7.5% FIC + 0.2% PVP, thawed
214
at 28 °C and using 9% sucrose solution as medium to remove CPA after thawing.
215
It has been generally acknowledged that larval age is of importance for cryosurvival, as
216
larvae at different developmental stages have various ability to be cryopresered (Gwo 1995;
217
Nascimento et al., 2005; Paredes et al., 2012, 2013). In this study, M. galloprovincialis larvae
218
collected at 25 h post-fertilization had the highest resistance to cryopreservation, with 62%
219
post-thaw D-larval rates achieved. This developmental stage was later than Paredes et al.
220
(2013) study (20 h) on the same species with about 50% post-thaw D-larval rates produced.
221
This difference may be related to early larvae having more lipid, which is stored as an energy
222
reserve and used for larval development (Bayne et al., 1978; Gosling, 2015). An embryo with
223
high lipid content would be more sensitive to cryopreservation and this phenomenon has been
224
shown in livestock species, such as pigs (Nagashima et al., 1995; Dobrinsky 2002), cattle
225
(Massip 2001; Pryor et al., 2011) and sheep (Massip 2001).
226
Ethylene glycol has been shown as a suitable permeable CPA in larval cryopreservation in M.
227
galloprovincialis and Perna canaliculus (Paredes et al., 2012, 2013). For the non-permeable
228
CPAs, on the other hand, although some sugars such as trehalose and PVP have been
229
investigated in mussels and oysters, no spat has been produced in the published results
230
(Paredes et al., 2012, 2013). In our previous study, the non-permeable CPA, FIC,
231
demonstrated the ability to significantly improve the post-thaw oocyte quality in M.
232
galloprovincialis (Liu and Li, 2015). Although the PVP was not used for the larval
233
cryopreservation in this species (Paredes et al., 2013), it has been applied in mice and cattle
234
cryopreservation (Leibo and Oda, 1993; Saha et al., 1996; Titterington and Robinson, 1996;
235
Kim et al., 2008). Furthermore, some studies have shown that a combination of different non-
236
permeable CPAs could be more effective for improving the cryosurvival rate of larvae (Saha
237
et al., 1996; Titterington and Robinson, 1996). In this study, 10% EG + 7.5% FIC and 10%
238
EG + 0.2% PVP had the similar cryoprotective ability as 10% EG used alone (Fig. 2). This is
239
in agreement with the study by Paredes et al. (2013) on the same species where 10% EG with
240
or without trehalose produced a similar post-thaw D-larval rate. However, in the present
241
study, when the 7.5% FIC was applied with 0.2% PVP together, the post-thaw D-larval rate
242
was significantly improved (Fig 2, 3). This result indicates that the combined effects of FIC
243
and PVP could play an important role in M. galloprovincialis larval cryopreservation.
244
Reasons for this improvement are not clear, although it might be because the intercellular ice
245
formation is limited by the application of suitable non-permeable CPAs as suggested by Gao
246
and Critser (2000) in their review on cryoinjury mechanisms. In addition, further study would
247
be needed to determine if any correlation between the improvement in post-thaw survival rate
248
achieved in this study and the toxicity of CPAs used. Result in this study agrees with the
249
study reported by Saha et al. (1996) that the hatching rate was improved significantly when
250
cattle embryos were cryopreserved in EG (40%) plus two sugars (11.3% trehalose + 20%
251
PVP) in comparison with EG used alone or in combination with either sugars.
252
Thawing temperature is a critical factor affecting the success of larval cryopreservation. High
253
thawing temperature could inhibit recrystallization of the internal ice, while a low
254
temperature might prevent osmotic pressure on the larvae as extracellular medium melts fast.
255
The reduced pressure could result in a rapid shift of free water into the larvae if the
256
intracellular CPA cannot diffuse out quickly, leading to extreme larval swelling, and possible
257
rupture (Jain and Paulson, 2006). Therefore, controlling thawing temperature is necessary,
258
although studies in this area have been lacking in marine bivalves. In this study, 28 °C was
259
the optimal thawing temperature for M. galloprovincialis larval cryopreservation. This
260
temperature has also been applied for the oocyte and larval cryopreservation in the same
261
species (Paredes et al., 2013; Liu and Li, 2015), Crassostrea gigas (Lin et al., 1999; Paredes
262
et al., 2013; Suneja et al., 2014) and P. canaliculus (Paredes et al., 2012). This temperature is
263
higher than that applied in Pinctada fucata martensii (25 °C; Choi and Chang, 2003).
264
Besides thawing temperature, application of proper medium to remove CPA from the larvae
265
after thawing could also improve the success of cryopreservation (Woods et al., 2004; Jain
266
and Paulson, 2006). Medium containing sucrose has been widely used to remove CPA in
267
embryo cryopreservation in cattle (Mahmoudzadeh et al., 1993; Youngs, 2011), dogs
268
(Guaitolini et al., 2012), sheep (Youngs, 2011), mice (Fathi et al., 2012) and pigs (Castillo-
269
Martín et al., 2013). In the larvae cryopreservation of marine mollusc species, seawater and
270
bovine serum albumin are commonly used to remove CPA in oysters (Lin et al., 1999; Choi
271
and Chang, 2003; Paredes et al., 2013; Suneja et al., 2014) and mussels (Wang et al., 2011;
272
Paredes et al., 2012, 2013), although low or no spat productions were reported in these
273
studies. In the current study, 9% sucrose solution achieved the highest post-thaw D-larval
274
rates (Fig. 5) which has also been used on the oocyte cryopreservation in the same species
275
(Liu and Li, 2015).
276
Different straw volumes have different surface to volume ratio which is important for
277
cryosurvival. Higher surface to volume ratio could enable a uniform cooling rate and proper
278
heat exchange properties which would be advantageous for maintaining cell quality (Zhu et
279
al., 2014). In this study, 0.25 mL and 0.5 mL straws produced similar post-thaw D-larval
280
rates. This is in agreement with a study on P. canaliculus larvae cryopreservation where no
281
significant difference on post-thaw D-larval rate was found when larvae were cryopreserved
282
in 0.25 mL and 0.5 mL straws (Paredes et al., 2012).
283
After cryopreservation, the developmental capacity of larvae is reduced, resulting in a
284
significantly lower survival rate compared to fresh larvae (Table 1). This phenomenon has
285
been observed in other studies, as the mechanism of cell cleavage might be compromised
286
during cryopreservation (Wang et al., 2011; Paredes et al., 2012). Nevertheless, in the present
287
study, the relative mortality rates after day 8 PF remained the same between fresh and
288
cryopreserved larvae (Table 1). Moreover, the shell length of larvae at day 8 and spat at day
289
32 PF was similar (P > 0.05) between the fresh and cryopreserved larvae. Therefore, the
290
results of this study indicate that cryopreservation affects the larvae at an early developmental
291
stage in M. galloprovincialis. Similar phenomenon has also been observed in other studies on
292
the same species (Wang et al., 2011; Paredes et al., 2012, 2013; Liu and Li, 2015).
293
In conclusion, the larval cryopreservation technique developed in this study has significantly
294
improved the post-thaw D-larval rate to >80% in M. galloprovincialis with cryodamage being
295
expressed only at early development stages. Therefore, this technique could be applied to
296
guarantee a reliable year round supply of progenies for hatchery production and
297
environmental monitoring programs, and enhance the management efficiency of breeding
298
programs in the M. galloprovincialis aquaculture industry.
299
300
Acknowledgments
301
This research was funded by the Talent Project of Revitalizing Liaoning (Project No.
302
XLYC1807087), Department of Ocean and Fisheries of Liaoning (Project No. 201829),
303
China Scholarship Council and South Australian Research and Development Institute
304
(SARDI). We thank Mr Andy Dyer of Kinkawooka Mussels for the provision of mussel
305
broodstock. We also thank master student Zhongling Lin of Dalian Ocean University for
306
technical assistance.
307 308
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43. Woods, E.J., Benson, J.D., Agca, Y., Critser, K., 2004. Fundamental cryobiology of reproductive cells and tissues. Cryobiology 48, 146-156. 44. Youngs, C.R., 2011. Cryopreservation of preimplantation embryos of cattle, sheep, and goats. J. Vis. Exp. 54, e2764.
429
45. Zhang, T.T., 2004. Cryopreservation of gametes and embryos of aquatic species. In:
430
Fuller, B.J., Lane, N., Benson, E.E. (Eds.), Life in the Frozen State. CRC Press, Boca
431
Raton, pp. 415-435.
432
46. Zhu, W., Li, X., Qu, D., Liu, Y., Clarke, S., 2014. Cryopreservation of sperm from
433
wild greenlip abalone (Haliotis laevigata) in Australia. Aquaculture 432, 60-66.
434
Table Legend
435
Table 1. Comparison of survival rates and relative mortality rate between fresh and
436
cryopreserved larvae during periods post-fertilization, n = 3. Different letter indicates
437
significant difference (P < 0.05).
438
Table 1.
Survival rate (%)
Relative mortality rate (%)
Days post fertilization
fresh larvae
cryopreserved larvae
day 2 (D larvae)
91.7 ± 3.0 A
71.6 ± 0.9 B
day 8
72.0 ± 3.9 A′
44.5 ± 1.3 B′
21.3 ± 6.4 b
37.8 ± 1.9 a
day 14
59.3 ± 5.1 A″
37.3 ± 5.4 B″
17.2 ± 11.8 a′
16.3 ± 9.5 a′
fresh larvae
cryopreserved larvae
day 20
48.7 ± 3.5 A‴
30.3 ± 1.2 B‴
17.8 ± 6.1 a″
17.9 ± 9.5 a″
day 26 (eyed larvae)
46.7 ± 2.7 A″″
28.5 ± 2.3 B″″
3.8 ± 6.7 a‴
6.1 ± 5.3 a‴
day 32 (spat)
37.5 ± 1.5 A″‴
21.3 ± 0.6 B″‴
19.7 ± 1.5 a″″
25.0 ± 4.4 a″″
439
Figure Legends
440
Fig. 1. Post-thaw D-larval rates (%) when larvae were cryopreserved at different
441
developmental stages, n = 3. Different letter indicates significant difference (P < 0.05). All
442
the data has been normalized to the controls.
443
Fig. 2. Post-thaw D-larval rates (%) when larvae were cryopreserved in different CPA
444
combination(s), n = 3. Different letter indicates significant difference (P < 0.05). All the data
445
has been normalized to the controls.
446
Fig. 3. Post-thaw D-larval rates (%) when larvae were cryopreserved in 10% EG + 7.5% FIC
447
with addition of different concentrations of PVP, n = 3. Different letter indicates significant
448
difference (P < 0.05). All the data has been normalized to the controls.
449
Fig. 4. Post-thaw D-larval rates (%) of larvae thawed at various thawing temperatures after
450
cryopreservation, n = 3. Different letter indicates significant difference (P < 0.05). All the
451
data has been normalized to the controls.
452
Fig. 5. Post-thaw D-larval rates (%) of larvae diluted with different concentrations of sucrose
453
after thawing, n = 3. Different letter indicates significant difference (P < 0.05). All the data
454
has been normalized to the controls.
100.0
D-larval rate (%)
80.0 A 60.0 B 40.0
C
C
20.0
0.0 10 h
455 456
Fig.1.
20 h 25 h Periods post-fertilization
30 h
457 100.0 A
D-larval rate (%)
80.0 B
60.0 B
B
40.0
20.0
0.0 10% EG
458 459 460
Fig.2.
10% EG + 7.5% FIC
10% EG + 0.2% PVP 10% EG + 7.5% FIC + 0.2% PVP
100.0 A B
D-larval rate (%)
80.0
B
B 60.0
C
40.0 20.0 0.0 10% EG + 7.5% 10% EG + 7.5% 10% EG + 7.5% 10% EG + 7.5% 10% EG + 7.5% FIC FIC + 0.05% PVP FIC + 0.1% PVP FIC + 0.2% PVP FIC + 0.4% PVP Cryoprotectant agents
461 462
Fig.3.
100 A
D-larval rate (%)
80
AB
B
AB
AB
60 40 20 0 18°C
463 464
Fig.4.
28°C
38°C 48°C Thawing temperatures
58°C
100.0 A D-larval rate (%)
80.0 B
B
B
60.0 40.0 20.0 0.0 6% sucrose
465 466
Fig.5.
9% sucrose 12% sucrose Sucrose concentrations
15% sucrose
Highlights
This study developed a programmable freezing technique to cryopreserve larvae in Mytilus galloprovinvialis. The post-thaw survival rate has been improved significantly by the application of Ficoll and polyvinylpyrrolidone together. The technique can facilitate breeding programs and hatchery management in the mussel aquaculture industry.