**Effects of Slow Cooling Methods and Vitrification Methods on Red Seabream (***Pagrus major***) Embryos**

Q. H. Liu, J. Li, Z.Z. Xiao, S.H. Xu, D.Y. Ma and Y.S. Xiao

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58830

#### **1. Introduction**

[22] Taylor MJ, Campbell LH, Rutledge RN, Brockbank KGM. Comparison of Unisol with Euro-Collins Solution as a Vehicle Solution for Cryoprotectants. Transplantation Pro‐

[23] Baust JM, Van Buskirk RG, Baust JG. Improved Cryopreservation Outcome is Facili‐ tated by an Intracellular-Type medium and Inhibition of Apoptosis. Journal of the Bi‐

[24] Baust JM, Van Buskirk RG, Baust JG. Cell Viability Improves Following Inhibition of Cryopreservation-Induced Apoptosis. In Vitro Cellular and Developmental Biology

[25] Campbell LH, Brockbank KGM. Serum-Free Solutions for Cryopreservation of Cells.

[26] Campbell LH, Brockbank KGM. Cryopreservation of Porcine Aortic Heart Valve Leaflet-derived Myofibroblasts. Bioprocessing and Biobanking 2010; 8(4) 211-217. [27] Campbell LH, Brockbank KGM. Culturing with Trehalose Produces Viable Endothe‐

[28] Brockbank KGM, Spyropoulos DD, Baatz JE, Chen Z, Greene ED, Campbell LH. Stor‐ age and Distribution Methods for Products Based upon Engineered Cell and Tissue Models. Abstract, 18th Annual Hilton Head Workshop & Annual Engineering Tissues

In Vitro Cellular & Developmental Biology-Animal 2007; 43 269–275.

lial Cells after Cryopreservation. Cryobiology 2012; 64(3) 240-4.

ceedings 2001; 33 677-79.

30 Recent Advances in Cryopreservation

2000; 36(4) 262-70.

omedical Engineering Society 1999; 1 206-7.

Workshop, Hilton Head Island, SC., 2014.

Fish gametes cryopreservation would benefit global germplasm shipping and supply, aquaculture, aquatic resources conservation and scientific research [1]. In fish sperm cryopre‐ servation, more than 200 species have been successfully cryopreserved [2]. However, the cryopreservation of fish embryo has not been successful due to its complex multi-compart‐ mental system, large content of water, high sensitivity to chilling, large amount of egg-yolk and low membrane permeability [3,4]. In recent years, many researches have been carried out on cryopreservation protocols [5] and mechanism of cryoinjuries in fish embryos [6].

Conventional slow cooling and vitrification are commonly used methods for long term storage of mammalian embryos. Conventional slow cooling method has been widely used in various species, but it suffers from several limitations such as chilling injury, ice formation damage, expensive equipment and tedious cooling protocols [7]. Vitrification, a solidification of a liquid without crystallization, seemed to be a promising approach. It is an extreme increase of viscosity and requires either rapid cooling rates or high concentration cryoprotectants [8], and can greatly simplify the process of cooling, avoids physical damage, and lessens the chilling injury to embryo [7]. However, the embryo cryopreserved by vitrification may still be injured by toxicity and osmotic effects of cryoprotectants [9].

Despite the fact that cryopreservation of embryos for some fish species have been attempted, successful results were not achieved [10]. Slow-cooling method had been approved to be not suitable for cryopreservation of zebrafish embryos, starfish oocytes and *Xenopus laevis* oocytes [11,12]. Therefore, some researchers suggested that vitrification would be a good option for its absence from ice crystal formation. In olive flounder, some researchers reported survival embryos were obtained after vitrification [13], however, it could not be replicated by others

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

[14]. In spite of this, as a promising method, attempts have been made on various fish species by vitrification.

**2.2. Chemicals and solutions**

percentage of volume (v/v).

**2.3. Effect of cryoprotectant solutions on the embryos**

S1\* 5%DMSO+5%PG

S2\* 8%MeOH V1\*\* 40%DMSO

V2\*\* 40%PG

\* For conventional slow cooling methods

\*\* For vitrification methods

number of each group.

(18 ± 1 °C). Three replicates were taken for each experiment group.

**Table 1.** Cryprotectant concentrations and treatment time used in cryopreservation of red seabream.

**2.4. Morphological changes during exposure to cryoprotectant solutions**

embryos were observed for each cryoprotectant solution.

After immersion the embryos were first removed from the cryoprotectant solution using a nylon mesh, and carefully washed three times with fresh seawater. Then the embryos were transferred to a 100 mL beaker containing 80 mL fresh seawater for incubation with a change of seawater an hour later. Control groups were incubated in the filtered seawater at room temperature. The toxicity of cryoprotectant was assessed by the hatching rate which was calculated as the percentage of hatched larvae (48 h after fertilization) in relation to the total

One embryo with seawater was loaded on a concave slide under a light microscope (Nikon-YS100). We removed the seawater with filter paper and added 100 µl cryoprotectant solution. Morphological changes were observed by taking pictures using a digital camera (Nikon CoolPix 4500) under microscope with 40× magnification and the interval was about 1 min. 5

Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, methanol (MeOH), 1,2 propylene glycol (PG) and the other chemicals were purchased from Beijing Chemical Agents Ltd. All the chemicals were analytical grade. The cryoprotectant solutions used in the following experiments were made with Hank's solution [16] (8 g/L NaCl, 0.4 g/ L KCl, 0.14 g/ L CaCl2, 0.1 g/ L l MgSO4 7H2O, 0.1 g/ L MgCl2 6H2O, 0.06 g/ L Na2HPO4 12H2O, 1g/ L glucose, 0.35 g/ L NaHCO3) as the extender. The concentration of cryoprotectant was expressed as the

Effects of Slow Cooling Methods and Vitrification Methods on Red Seabream (*Pagrus major*) Embryos

For each exposure test, approximately 50 embryos were exposed to 10 mL cryoprotectant solutions with various concentrations for different exposure time (Tab 1) at room temperature

**Cryoprotectants extender Exposure time(min)**

10 30 60

http://dx.doi.org/10.5772/58830

33

5 10 15

Hank's solution

Red seabream (*Pagrus major*) is one of the most important cultured fish species in China, Japan and South Korea. We have carried out some researches on red seabream embryos in cryopre‐ servation protocols [15] and mechanism of cryoinjuries [6]. Potential protocols of slowing cooling and vitrification (sorts of cryoprotectant, programs of cooling and thrawing) have been screened [15]. However, little was known upon the morphological changes issue during cooling and thawing process by the two methods. The objective of the present study was to investigate the effects of two conventional slow cooling methods (S1 and S2) and two vitrifi‐ cation methods (V1 and V2) on red seabream embyos. The main topics are as follows:1) the effect of different cryoprotectant solutions on hatching rate of red seabream embryos; 2) the morphological changes during exposure to different cryoprotectant solutions; 3) and the changes of embryos during cooling and thawing process under cryomicroscope. 1 World Aquaculture Society, pp. 179–187. 2 [24] Billard R., Zhang T., 2001, Techniques of genetic resource banking in fish. In: 3 Watson P.F., Holt W.V., (Eds.) Cryobanking the genetic resource. Wildlife

4 conservation for the future. London, Taylor and Francis Press, pp. 145–170. 5 [25] Baudot A., Alger L., Boutron P., Glass-forming tendency in the system

8 Six-Somite Stage Zebrafish (*Brachydanio rerio*) Embryos to Water and Methanol.

#### **2. Materials and methods** 6 water–dimethyl sulfoxide. Cryobiology 2000, 40, 151–158. 7 [26] Zhang T., Rawson D.M., 1998, Permeability of Dechorionated One-Cell and

8

24 heart-beat stage embryo.

19

25 26

27

28

29

#### **2.1. Fish breeding and embryo collection** 9 Cryobiology 37, 13–21.

Sexually mature red seabream (8 female, 12 male; body weight, 3–4 kg) were maintained in a 12 m3 concrete rearing pond (temperature: 16-18 °C) with filtered seawater changed two times a day and pumped air supply. The photoperiod was fixed at L: D=16 h: 8 h. They were fed twice a day with cooked meat of mussel. Naturally fertilized embryos were collected each morning before feeding and then incubated in filtered seawater with pumped air supply at 18 ± 1 °C in a small plastic barrel. Embryos developed to heart-beat stage (heart rate: 60–90 beats/ min (fig.1); approximately 36 h after fertilization) were used for experiments. The develop‐ mental stages of the embryo were determined morphologically using a light microscope (Nikon-YS100). 10 [27] Robles V., Cabrita E., Fletcher G.L., Shears M.A., King M.J., Herraéz M.P., 2005, 11 Vitrification assays with embryos from a cold tolerant sub-arctic fish species. 12 Theriogenology 64, 1633–1646. 13 14 15 16 17 Figures and Figures captions 18

20 Fig 1. The collected fresh embryos of red seabream. Naturally fertilized embryos were 21 collected each morning before feeding and then incubated in filtered seawater with pumped air supply at 18 ± 1<sup>o</sup> 22 C in a small plastic barrel. Embryos developed to 23 heart-beat stage were used for experiments. a. The collected fertilized embryos. b. The **Figure 1.** The collected fresh embryos of red seabream. Naturally fertilized embryos were collected each morning be‐ fore feeding and then incubated in filtered seawater with pumped air supply at 18 ± 1°C in a small plastic barrel. Em‐ bryos developed to heart-beat stage were used for experiments. a. The collected fertilized embryos. b. The heart-beat stage embryo.

#### **2.2. Chemicals and solutions**

[14]. In spite of this, as a promising method, attempts have been made on various fish species

Red seabream (*Pagrus major*) is one of the most important cultured fish species in China, Japan and South Korea. We have carried out some researches on red seabream embryos in cryopre‐ servation protocols [15] and mechanism of cryoinjuries [6]. Potential protocols of slowing cooling and vitrification (sorts of cryoprotectant, programs of cooling and thrawing) have been screened [15]. However, little was known upon the morphological changes issue during cooling and thawing process by the two methods. The objective of the present study was to investigate the effects of two conventional slow cooling methods (S1 and S2) and two vitrifi‐ cation methods (V1 and V2) on red seabream embyos. The main topics are as follows:1) the effect of different cryoprotectant solutions on hatching rate of red seabream embryos; 2) the morphological changes during exposure to different cryoprotectant solutions; 3) and the

Sexually mature red seabream (8 female, 12 male; body weight, 3–4 kg) were maintained in a

 concrete rearing pond (temperature: 16-18 °C) with filtered seawater changed two times a day and pumped air supply. The photoperiod was fixed at L: D=16 h: 8 h. They were fed twice a day with cooked meat of mussel. Naturally fertilized embryos were collected each morning before feeding and then incubated in filtered seawater with pumped air supply at 18 ± 1 °C in a small plastic barrel. Embryos developed to heart-beat stage (heart rate: 60–90 beats/ min (fig.1); approximately 36 h after fertilization) were used for experiments. The develop‐ mental stages of the embryo were determined morphologically using a light microscope

changes of embryos during cooling and thawing process under cryomicroscope.

6 water–dimethyl sulfoxide. Cryobiology 2000, 40, 151–158.

17 Figures and Figures captions

a b

2 [24] Billard R., Zhang T., 2001, Techniques of genetic resource banking in fish. In: 3 Watson P.F., Holt W.V., (Eds.) Cryobanking the genetic resource. Wildlife 4 conservation for the future. London, Taylor and Francis Press, pp. 145–170. 5 [25] Baudot A., Alger L., Boutron P., Glass-forming tendency in the system

7 [26] Zhang T., Rawson D.M., 1998, Permeability of Dechorionated One-Cell and 8 Six-Somite Stage Zebrafish (*Brachydanio rerio*) Embryos to Water and Methanol.

10 [27] Robles V., Cabrita E., Fletcher G.L., Shears M.A., King M.J., Herraéz M.P., 2005, 11 Vitrification assays with embryos from a cold tolerant sub-arctic fish species.

20 Fig 1. The collected fresh embryos of red seabream. Naturally fertilized embryos were 21 collected each morning before feeding and then incubated in filtered seawater with pumped air supply at 18 ± 1<sup>o</sup> 22 C in a small plastic barrel. Embryos developed to 23 heart-beat stage were used for experiments. a. The collected fertilized embryos. b. The

**Figure 1.** The collected fresh embryos of red seabream. Naturally fertilized embryos were collected each morning be‐ fore feeding and then incubated in filtered seawater with pumped air supply at 18 ± 1°C in a small plastic barrel. Em‐ bryos developed to heart-beat stage were used for experiments. a. The collected fertilized embryos. b. The heart-beat

by vitrification.

32 Recent Advances in Cryopreservation

**2. Materials and methods**

9 Cryobiology 37, 13–21.

12 m3

18

19

25 26

stage embryo.

27

28

29

(Nikon-YS100).

8

24 heart-beat stage embryo.

**2.1. Fish breeding and embryo collection**

12 Theriogenology 64, 1633–1646.

1 World Aquaculture Society, pp. 179–187.

Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, methanol (MeOH), 1,2 propylene glycol (PG) and the other chemicals were purchased from Beijing Chemical Agents Ltd. All the chemicals were analytical grade. The cryoprotectant solutions used in the following experiments were made with Hank's solution [16] (8 g/L NaCl, 0.4 g/ L KCl, 0.14 g/ L CaCl2, 0.1 g/ L l MgSO4 7H2O, 0.1 g/ L MgCl2 6H2O, 0.06 g/ L Na2HPO4 12H2O, 1g/ L glucose, 0.35 g/ L NaHCO3) as the extender. The concentration of cryoprotectant was expressed as the percentage of volume (v/v).

#### **2.3. Effect of cryoprotectant solutions on the embryos**

For each exposure test, approximately 50 embryos were exposed to 10 mL cryoprotectant solutions with various concentrations for different exposure time (Tab 1) at room temperature (18 ± 1 °C). Three replicates were taken for each experiment group.


\* For conventional slow cooling methods

\*\* For vitrification methods

**Table 1.** Cryprotectant concentrations and treatment time used in cryopreservation of red seabream.

After immersion the embryos were first removed from the cryoprotectant solution using a nylon mesh, and carefully washed three times with fresh seawater. Then the embryos were transferred to a 100 mL beaker containing 80 mL fresh seawater for incubation with a change of seawater an hour later. Control groups were incubated in the filtered seawater at room temperature. The toxicity of cryoprotectant was assessed by the hatching rate which was calculated as the percentage of hatched larvae (48 h after fertilization) in relation to the total number of each group.

#### **2.4. Morphological changes during exposure to cryoprotectant solutions**

One embryo with seawater was loaded on a concave slide under a light microscope (Nikon-YS100). We removed the seawater with filter paper and added 100 µl cryoprotectant solution. Morphological changes were observed by taking pictures using a digital camera (Nikon CoolPix 4500) under microscope with 40× magnification and the interval was about 1 min. 5 embryos were observed for each cryoprotectant solution.

#### **2.5. Changes of embryos during cooling and thawing process**

The embryos were immersed in the four cryoprotectant solutions for different time (Tab 2), respectively. After immersion, the embryos suspended in 20 µll of cryoprotectant solution were loaded into a small quartz holding vessel and placed onto a Linkam Cryostage (Linkam-THMS600, UK). The embryos were cooled with different methods (Tab 2). After thawing, the embryos were transferred to a 100 mL beaker containing 80 mL filtered seawater for incuba‐ tion. Each experiment was repeated three times. The morphological changes during the cooling-thawing process were recorded using a microscope (Olympus BX-51, Japan) with a video attachment and monitor (Nikon-E200, Japan). In addition, two temperature values, *TEIF* and *TIIF* were recorded. They were identified as the temperature when a flash appears in the field of view and the temperature when the embryo suddenly blackens, respectively. The temperatures reported were obtained by the Linkam cryostage thermocouple.

**3. Results**

V1 and V2, respectively.

S1 5%DMSO+5%PG

S2 8%MeOH

V1 40%DMSO

V2 40%PG

Values with different letters are significantly different (*P*<0.05) (means ± SD)

**Table 3.** Hatching rates of embryos treated with the four selected solutions

**3.2. Morphological changes during exposure to cryoprotectant solutions**

The morphological changes of the embryos in S1 and V1 are shown in Figure 2. The morpho‐ logical changes in S2 and V2 were similar with those in S1 and V1, respectively. No obvious change was found after immersed in S solutions (after exposure for 30 min (Fig. 2-S-2). But there was an obvious dark strip around the yolk sac in V groups as soon as embryo was exposed in the cryoprotectants (Fig. 2-V-2), and the dark strip became narrower until disappeared gradually at the time of 10 min (V1, Fig. 2-V-4)/13 min (V2). At the end of immersion, the

**3.1. Effect of cryoprotectant solutions on the hatching rate of red seabream embryos**

Control ― ― 99.67±0.82a

The hatching rates of embryos treated with cryoprotectant solutions are shown in Table 3. After exposure to S1 and S2 solutions, the hatching rates of embryos showed no significant decrease compared to control except for S2 solution with 60 min exposure. However, the hatching rates of embryos exposed to V1 and V2 solutions decreased sharply, only the embryos immersed in V2 solution for 5 min and 10 min had higher hatching rates (>80%). So in the later cooling experiment, we choose 60 min, 30 min, 5 min and 10 min as exposure time for S1, S2,

**Cryoprotectants Exposure time Hatching rate (%)**

Effects of Slow Cooling Methods and Vitrification Methods on Red Seabream (*Pagrus major*) Embryos

30 min

60 min

10 min 98.00±2.00a 30 min 100.00±0.00a 60 min 100.00±0.00a

10 min 98.67±2.31a

5 min 59.33±14.19c 10 min 15.33±5.03d 15 min 15.00±15.52d

5 min 96.67±5.77a 10 min 81.33±22.74ab 15 min 65.00±27.73bc

96.67±2.31a 65.67±5.13bc

http://dx.doi.org/10.5772/58830

35


**Table 2.** Cooling methods used in cryopreservation of red seabream.

After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. The proportion of morphologically intact embryos was calculated as the percentage of embryos with normal morphology in relation to the total number of each group.

#### **2.6. Statistical analysis**

Percentage data were normalized through arcsine transformation and analyzed by one-way ANOVA with SPSS software (SPSS Inc., USA). The results were expressed as means ± SD. The significant differences between treatments in the different experiments were detected using an SNK (Student Newman Keuls) statistical test (*P* < 0.05). The percentages of embryos with intact morphology were arithmetical means.

## **3. Results**

**2.5. Changes of embryos during cooling and thawing process**

Immersed for 50

Immersed for 30

Immersed for 10

**Table 2.** Cooling methods used in cryopreservation of red seabream.

V1 40%DMSO Immersed for 5 min cooling to-150°C with -130°C /min

min

min

min

intact morphology were arithmetical means.

the total number of each group.

**2.6. Statistical analysis**

S1 5%DMSO+5%PG

34 Recent Advances in Cryopreservation

S2 8% Methanol

V2 40%PG

The embryos were immersed in the four cryoprotectant solutions for different time (Tab 2), respectively. After immersion, the embryos suspended in 20 µll of cryoprotectant solution were loaded into a small quartz holding vessel and placed onto a Linkam Cryostage (Linkam-THMS600, UK). The embryos were cooled with different methods (Tab 2). After thawing, the embryos were transferred to a 100 mL beaker containing 80 mL filtered seawater for incuba‐ tion. Each experiment was repeated three times. The morphological changes during the cooling-thawing process were recorded using a microscope (Olympus BX-51, Japan) with a video attachment and monitor (Nikon-E200, Japan). In addition, two temperature values, *TEIF* and *TIIF* were recorded. They were identified as the temperature when a flash appears in the field of view and the temperature when the embryo suddenly blackens, respectively. The

temperatures reported were obtained by the Linkam cryostage thermocouple.

**Cryoprotectants Cooling methods Thawing methods**

and for 1 min;

1 min;

and for 1 min;

and for 1 min;

After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. The proportion of morphologically intact embryos was calculated as the percentage of embryos with normal morphology in relation to

Percentage data were normalized through arcsine transformation and analyzed by one-way ANOVA with SPSS software (SPSS Inc., USA). The results were expressed as means ± SD. The significant differences between treatments in the different experiments were detected using an SNK (Student Newman Keuls) statistical test (*P* < 0.05). The percentages of embryos with

0°C for 1 min, to -15.5°C with -3.5 °C /min, to -30°C with 3 °C /min, then to -150°C with -130°C /min

0°C for 1 min, to -20°C with -2°C /min and for 5 min, to -60°C with -2°C/min and for 5 min, then to -150°C with -10°C/min and for

cooling to-150°C with -130°C /min

to -80°C with 30°C/min, to 20°C

to -70°C with 30°C/min, to 20°C

with 130°C/min

with 130°C/min

to 20°C with 130°C/min

to 20°C with 130°C/min

#### **3.1. Effect of cryoprotectant solutions on the hatching rate of red seabream embryos**

The hatching rates of embryos treated with cryoprotectant solutions are shown in Table 3. After exposure to S1 and S2 solutions, the hatching rates of embryos showed no significant decrease compared to control except for S2 solution with 60 min exposure. However, the hatching rates of embryos exposed to V1 and V2 solutions decreased sharply, only the embryos immersed in V2 solution for 5 min and 10 min had higher hatching rates (>80%). So in the later cooling experiment, we choose 60 min, 30 min, 5 min and 10 min as exposure time for S1, S2, V1 and V2, respectively.


**Table 3.** Hatching rates of embryos treated with the four selected solutions

#### **3.2. Morphological changes during exposure to cryoprotectant solutions**

The morphological changes of the embryos in S1 and V1 are shown in Figure 2. The morpho‐ logical changes in S2 and V2 were similar with those in S1 and V1, respectively. No obvious change was found after immersed in S solutions (after exposure for 30 min (Fig. 2-S-2). But there was an obvious dark strip around the yolk sac in V groups as soon as embryo was exposed in the cryoprotectants (Fig. 2-V-2), and the dark strip became narrower until disappeared gradually at the time of 10 min (V1, Fig. 2-V-4)/13 min (V2). At the end of immersion, the 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

25

26

18 solutions

24 Scale bar = 300.00 μm.

9

embryos presented different extent of degeneration (Fig. 2-S-6), and the degeneration in S2 was more serious (Fig. 2-S-7).

**3.3. Changes of embryos during cooling and thawing process**

and *TIIF* in the four groups are shown in Table 4.

1

2

3

4

5

6

7

8

9

10

11

13 conventional slow cooling groups.

10

21 = 400.00 μm.

in V groups.

The representative changes of embryos during cooling and thawing process under cryomi‐ croscope are shown in Figure 3 and Figure 4. Intra-cellular ice formation occured in both group (Fig. 3-S-4,5; Fig. 4-V-2,3) when the embryos suddently blacken. According to the extent of blackening, we separated the intra-cellular ice formation temprations (*TIIF*) into two categories, high temperature blackeners (*TIIF1*) and low temperature blackeners (*TIIF2*). The values of *TEIF*

Effects of Slow Cooling Methods and Vitrification Methods on Red Seabream (*Pagrus major*) Embryos

http://dx.doi.org/10.5772/58830

37

**S-1 S-2 S-3** 

**S-4 S-5 S-6** 

**S-7 S-8 S-9** 

12 Fig 3. The representative changes of embryos during cooling and thawing in

**Figure 3.** The representative changes of embryos during cooling and thawing in conventional slow cooling groups. This figure shows the representative changes of embryos during cooling and thawing in conventional slow cooling groups. (S-1) Embryos before cooling. (S-2) Extra-cellular ice formation: a flash appears and dendritic spears of ice (ar‐ row) project in the field of view. (S-3) Extra-cellular ice formation. (S-4) Intra-cellular ice formation in partly embryos. (S-5) Intra-cellular ice formation in all embryos (S-6) Completely frozen embryos. (S-7) Embryo with extra-cellular ice thawing. (S-8) Embryo with intra-cellular ice thawing. (S-9) Completely thawed embryos. Scale bar=400.00 μm.

Both *TEIF*s and *TIIF*s in S groups were significantly higher than those in V groups. In V groups, *TIIF* was higher than *TEIF*, indicating intra-cellular ice formed earlier than extra-cellular ice (Fig. 4-V-2), opposite to S groups (Fig. 3-S-2). And the intra-cellular ice firstly formed in the yolk and developing embryo (Fig. 4-V-3), then the perivitelline space (Fig. 4-V-4). Besides, dendritic ice crystals were observed in S groups (Fig. 3-S-2, arrow), and no big ice crystals were observed

14 This figure shows the representative changes of embryos during cooling and 15 thawing in conventional slow cooling groups. (S-1) Embryos before cooling. (S-2) 16 Extra-cellular ice formation: a flash appears and dendritic spears of ice (arrow) project 17 in the field of view. (S-3) Extra-cellular ice formation. (S-4) Intra-cellular ice 18 formation in partly embryos. (S-5) Intra-cellular ice formation in all embryos (S-6) 19 Completely frozen embryos. (S-7) Embryo with extra-cellular ice thawing. (S-8) 20 Embryo with intra-cellular ice thawing. (S-9) Completely thawed embryos. Scale bar

19 This figure shows the morphological changes of red seabream embryo immersed 20 in cryoprotectant solutions. (S-1) Untreated embryo for S1 group. (S-2~6) 21 Morphological changes of embryo immersed in S1 for different times. (S-7) 22 Degeneration of embryo immersed in S2 for 60 min. (V-1) Untreated embryo for V1 **Figure 2.** Representative morphological changes of embryo immersed in cryoprotectant solutions. This figure shows the morphological changes of red seabream embryo immersed in cryoprotectant solutions. (S-1) Untreated embryo for S1 group. (S-2~6) Morphological changes of embryo immersed in S1 for different times. (S-7) Degeneration of em‐ bryo immersed in S2 for 60 min. (V-1) Untreated embryo for V1 group. (V-2~5) Morphological changes of embryo im‐ mersed in V1 for different time. Scale bar=300.00 μm.

17 Fig 2. Representative morphological changes of embryo immersed in cryoprotectant

23 group. (V-2~5) Morphological changes of embryo immersed in V1 for different time.

#### **3.3. Changes of embryos during cooling and thawing process**

embryos presented different extent of degeneration (Fig. 2-S-6), and the degeneration in S2

Untreated 1 min 10 min

**S-1 S-2**

30 min 53 min 60 min

**S-4 S-5**

**S-6**

**V-2**

**V-5**

**S-3**

17 Fig 2. Representative morphological changes of embryo immersed in cryoprotectant

**V-3 V-4**

5 min 10 min 15 min

**Figure 2.** Representative morphological changes of embryo immersed in cryoprotectant solutions. This figure shows the morphological changes of red seabream embryo immersed in cryoprotectant solutions. (S-1) Untreated embryo for S1 group. (S-2~6) Morphological changes of embryo immersed in S1 for different times. (S-7) Degeneration of em‐ bryo immersed in S2 for 60 min. (V-1) Untreated embryo for V1 group. (V-2~5) Morphological changes of embryo im‐

60 min Untreated 1 min

**S-7 V-1**

19 This figure shows the morphological changes of red seabream embryo immersed 20 in cryoprotectant solutions. (S-1) Untreated embryo for S1 group. (S-2~6) 21 Morphological changes of embryo immersed in S1 for different times. (S-7) 22 Degeneration of embryo immersed in S2 for 60 min. (V-1) Untreated embryo for V1 23 group. (V-2~5) Morphological changes of embryo immersed in V1 for different time.

was more serious (Fig. 2-S-7).

36 Recent Advances in Cryopreservation

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

25

26

18 solutions

24 Scale bar = 300.00 μm.

mersed in V1 for different time. Scale bar=300.00 μm.

9

The representative changes of embryos during cooling and thawing process under cryomi‐ croscope are shown in Figure 3 and Figure 4. Intra-cellular ice formation occured in both group (Fig. 3-S-4,5; Fig. 4-V-2,3) when the embryos suddently blacken. According to the extent of blackening, we separated the intra-cellular ice formation temprations (*TIIF*) into two categories, high temperature blackeners (*TIIF1*) and low temperature blackeners (*TIIF2*). The values of *TEIF* and *TIIF* in the four groups are shown in Table 4.

12 Fig 3. The representative changes of embryos during cooling and thawing in 13 conventional slow cooling groups. 14 This figure shows the representative changes of embryos during cooling and 15 thawing in conventional slow cooling groups. (S-1) Embryos before cooling. (S-2) 16 Extra-cellular ice formation: a flash appears and dendritic spears of ice (arrow) project **Figure 3.** The representative changes of embryos during cooling and thawing in conventional slow cooling groups. This figure shows the representative changes of embryos during cooling and thawing in conventional slow cooling groups. (S-1) Embryos before cooling. (S-2) Extra-cellular ice formation: a flash appears and dendritic spears of ice (ar‐ row) project in the field of view. (S-3) Extra-cellular ice formation. (S-4) Intra-cellular ice formation in partly embryos. (S-5) Intra-cellular ice formation in all embryos (S-6) Completely frozen embryos. (S-7) Embryo with extra-cellular ice thawing. (S-8) Embryo with intra-cellular ice thawing. (S-9) Completely thawed embryos. Scale bar=400.00 μm.

17 in the field of view. (S-3) Extra-cellular ice formation. (S-4) Intra-cellular ice 18 formation in partly embryos. (S-5) Intra-cellular ice formation in all embryos (S-6) 19 Completely frozen embryos. (S-7) Embryo with extra-cellular ice thawing. (S-8) 20 Embryo with intra-cellular ice thawing. (S-9) Completely thawed embryos. Scale bar 21 = 400.00 μm. Both *TEIF*s and *TIIF*s in S groups were significantly higher than those in V groups. In V groups, *TIIF* was higher than *TEIF*, indicating intra-cellular ice formed earlier than extra-cellular ice (Fig. 4-V-2), opposite to S groups (Fig. 3-S-2). And the intra-cellular ice firstly formed in the yolk and developing embryo (Fig. 4-V-3), then the perivitelline space (Fig. 4-V-4). Besides, dendritic ice crystals were observed in S groups (Fig. 3-S-2, arrow), and no big ice crystals were observed in V groups.

10

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When the embryos cooled to-150 °C, the phenomena was similar in S groups and V1, the whole field of view was obscured by the extra-cellular ice (Fig.3-S-6, Fig.4-V-5). In V2, the embryos were obscure, while the solutions outside the embryos were transparent, just like solid glass (Fig.4-V-9). The transparent spaces among embryos were obscured during the thawing process (Fig.4-V-10).

During the thawing process, the ice of the outside solution thawed firstly and then thawed the ice inside the embryo with the embryo brightening (Fig. 3-S-8, Fig. 4-V-7) in all four groups. After thawing, the percentage of embryos with intact morphology in the four groups were S1, 10.26%; S2, 8.19%; V1, 55.26%; and V2, 70.00%, respectively. In total, the percentage of embryos with intact morphology in V groups (62.82%) was significantly higher than that in S groups

Effects of Slow Cooling Methods and Vitrification Methods on Red Seabream (*Pagrus major*) Embryos

http://dx.doi.org/10.5772/58830

39

**Cryoprotectants** *TEIF* (℃) *TIIF1* (℃) *TIIF2* (℃) *TEIF – TIIF2* (℃)

S1 5%DMSO+5%PG -5.00±0.17a -17.74±0.59a -21.40±0.31a 16.40±0.47a S2 8%MeOH -4.93±0.35a -16.34±0.08a -17.51±0.48b 12.58±0.74b V1 40%DMSO -70.30±2.72b -56.83±1.48b -64.33±0.51c -5.97±2.21c

V2 40%PG ― -46.23±0.31c -53.70±1.00d ―

Fish embryo is composed of three membrane-limited compartments: a large yolk surrounded by the yolk syncytial layer, a developing embryo limited by its own cell plasma membranes, and perivitelline space surrounded by chorion [17]. The different layers and membranes represent permeability barriers hindering the movement of water and cryoprotectants, and consequently, making the balance of cryoprotectant in the whole embryo difficult [18].

In the observation of morphological changes, we found a dark strip around the yolk sac after immersion in the V solutions. This may ascribe to the different refractive indexes between outer solution and perivitelline space. With the time going on, the strip became narrower and eventually disappeared. It indicated that the chorion of red seabream embryo was permeable to the cryoprotectant solutions. Similar results were found in studies on zebrafish embryos and medaka oocytes [18,19]. In V groups, the dark strip was obvious, while in S groups, the dark strip was not observed. This may be due to higher concentrations of V solutions than S

In our cooling experiment, we have observed some different phenomena between the two methods. The first representative phenomenon in our experiment was the large dendritic ice crystals observed in S groups, but not in V groups. High cooling rate and cryoprotectant

Values with different letters within the same row are significantly different (*P*<0.05) (means ± SD)

**4.1. Morphological changes during exposure to cryoprotectant solutions**

solutions, which caused higer refractive index difference.

**4.2. Changes of embryos during cooling and thawing process**

(9.21%).

**Table 4.** The values of *TEIF* and *TIIF*

**4. Discussion**

16 vitrification groups. 17 This figure shows the representative changes of embryos during cooling and 18 thawing in vitrification groups. (V-1) Embryos before cooling. (V-2) Intra-cellular ice 19 formation in partly embryos. (V-3,4) Intra-cellular ice formation: the intra-cellular ice 20 firstly forms in the yolk and developing embryo (arrow), then the perivitelline space 21 (arrow). (V-5) Completely frozen embryos in V1 group. (V-6) Embryo with **Figure 4.** The representative changes of embryos during cooling and thawing in vitrification groups. This figure shows the representative changes of embryos during cooling and thawing in vitrification groups. (V-1) Embryos before cool‐ ing. (V-2) Intra-cellular ice formation in partly embryos. (V-3,4) Intra-cellular ice formation: the intra-cellular ice firstly forms in the yolk and developing embryo (arrow), then the perivitelline space (arrow). (V-5) Completely frozen em‐ bryos in V1 group. (V-6) Embryo with extra-cellular ice thawing. (V-7) Embryo with intra-cellular ice thawing. (V-8) Completely thawed embryos. (V-9) Vitrification in the solutions outside embryos in V2 group. (V-10) Devitrification during the thawing process in V2 gourp. Scale bar=400.00 μm.

15 Fig 4. The representative changes of embryos during cooling and thawing in

22 extra-cellular ice thawing. (V-7) Embryo with intra-cellular ice thawing. (V-8) 23 Completely thawed embryos. (V-9) Vitrification in the solutions outside embryos in 24 V2 group. (V-10) Devitrification during the thawing process in V2 gourp. Scale bar = During the thawing process, the ice of the outside solution thawed firstly and then thawed the ice inside the embryo with the embryo brightening (Fig. 3-S-8, Fig. 4-V-7) in all four groups. After thawing, the percentage of embryos with intact morphology in the four groups were S1, 10.26%; S2, 8.19%; V1, 55.26%; and V2, 70.00%, respectively. In total, the percentage of embryos with intact morphology in V groups (62.82%) was significantly higher than that in S groups (9.21%).


Values with different letters within the same row are significantly different (*P*<0.05) (means ± SD)

**Table 4.** The values of *TEIF* and *TIIF*

#### **4. Discussion**

When the embryos cooled to-150 °C, the phenomena was similar in S groups and V1, the whole field of view was obscured by the extra-cellular ice (Fig.3-S-6, Fig.4-V-5). In V2, the embryos were obscure, while the solutions outside the embryos were transparent, just like solid glass (Fig.4-V-9). The transparent spaces among embryos were obscured during the thawing process

**V-1 V-2 V-3**

**V-4 V-5 V-6**

**V-7 V-8 V-9**

15 Fig 4. The representative changes of embryos during cooling and thawing in

**V-10**

during the thawing process in V2 gourp. Scale bar=400.00 μm.

17 This figure shows the representative changes of embryos during cooling and 18 thawing in vitrification groups. (V-1) Embryos before cooling. (V-2) Intra-cellular ice 19 formation in partly embryos. (V-3,4) Intra-cellular ice formation: the intra-cellular ice 20 firstly forms in the yolk and developing embryo (arrow), then the perivitelline space 21 (arrow). (V-5) Completely frozen embryos in V1 group. (V-6) Embryo with 22 extra-cellular ice thawing. (V-7) Embryo with intra-cellular ice thawing. (V-8) 23 Completely thawed embryos. (V-9) Vitrification in the solutions outside embryos in 24 V2 group. (V-10) Devitrification during the thawing process in V2 gourp. Scale bar =

**Figure 4.** The representative changes of embryos during cooling and thawing in vitrification groups. This figure shows the representative changes of embryos during cooling and thawing in vitrification groups. (V-1) Embryos before cool‐ ing. (V-2) Intra-cellular ice formation in partly embryos. (V-3,4) Intra-cellular ice formation: the intra-cellular ice firstly forms in the yolk and developing embryo (arrow), then the perivitelline space (arrow). (V-5) Completely frozen em‐ bryos in V1 group. (V-6) Embryo with extra-cellular ice thawing. (V-7) Embryo with intra-cellular ice thawing. (V-8) Completely thawed embryos. (V-9) Vitrification in the solutions outside embryos in V2 group. (V-10) Devitrification

(Fig.4-V-10).

38 Recent Advances in Cryopreservation

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16 vitrification groups.

11

25 400.00 μm.

#### **4.1. Morphological changes during exposure to cryoprotectant solutions**

Fish embryo is composed of three membrane-limited compartments: a large yolk surrounded by the yolk syncytial layer, a developing embryo limited by its own cell plasma membranes, and perivitelline space surrounded by chorion [17]. The different layers and membranes represent permeability barriers hindering the movement of water and cryoprotectants, and consequently, making the balance of cryoprotectant in the whole embryo difficult [18].

In the observation of morphological changes, we found a dark strip around the yolk sac after immersion in the V solutions. This may ascribe to the different refractive indexes between outer solution and perivitelline space. With the time going on, the strip became narrower and eventually disappeared. It indicated that the chorion of red seabream embryo was permeable to the cryoprotectant solutions. Similar results were found in studies on zebrafish embryos and medaka oocytes [18,19]. In V groups, the dark strip was obvious, while in S groups, the dark strip was not observed. This may be due to higher concentrations of V solutions than S solutions, which caused higer refractive index difference.

#### **4.2. Changes of embryos during cooling and thawing process**

In our cooling experiment, we have observed some different phenomena between the two methods. The first representative phenomenon in our experiment was the large dendritic ice crystals observed in S groups, but not in V groups. High cooling rate and cryoprotectant concentration were reported to be prone to vitrify and avoid dendritic ice crystal formation [20], which may explain the phenomenon in this study.

**5. Conclusion**

**Acknowledgements**

Resources.

**Author details**

Qingdao, P.R. China

**References**

tectant concertration in red seabream embryos.

In the present study, we compared conventional slow cooling method and vitrification method in red seabream embryos cryopreservation. In V groups, the *TEIF*s and *TIIF*s were significantly lower than those in S groups, and no big ice crystals formed. Besides, after thawing, the percentage of embryos with intact morphology in V groups was significantly higher than that in S groups. All the results indicated that vitrification method would be a good option for red seabream embryos cryopreservation, though the embryos were better tolerant of S solutions than V solutions. Therefore, further study is still required to optimize the cryopreservation protocol for reducing toxicity of cryopreservation as well as improving the internal cryopro‐

Effects of Slow Cooling Methods and Vitrification Methods on Red Seabream (*Pagrus major*) Embryos

http://dx.doi.org/10.5772/58830

41

This work is supported by Regional Demonstration of Marine Economy Innovative Develop‐ ment Project (No. 12PYY001SF08)" and National Infrastructure of Fishery Germplasm

1 Center of Biotechnology R&D, Institute of Oceanology, Chinese Academy of Sciences,

2 National &, Local Joint Engineering Laboratory of Ecological Mariculture, Chinese Acade‐

[1] Cabrita E., Chereguini O., Luna M., Paz de.P., Herráez M.P., 2003, Effect of different treatments on the chorion permeability to DMSO of turbot embryos. Aquaculture

[2] Gwo J.C., 2000, Cryopreservation of sperm of some marine fishes. In: Tiersch T.R., Mazik P.M. (Eds.) Advances in World Aquaculture. Baton Rouge, Louisiana, USA,

Q. H. Liu1,2, J. Li1,2, Z.Z. Xiao1,2, S.H. Xu1,2, D.Y. Ma1,2 and Y.S. Xiao2,2

\*Address all correspondence to: junli@qdio.ac.cn

World Aquaculture Society, pp. 138–160.

my of Sciences, Qingdao, P.R. China

221, 593–604.

Secondly, in our result, *TIIF*s in S groups were significantly higher than those in V groups. This may be because the nucleation temperature of red seabream embryos is too high to achieve sufficient cell dehydration to avoid IIF [21]. *TIIF* is a main factor many researchers focus on in the study of cryoproservation. Slow cooling usually causes dehydration and decreases *TIIF* largely in mammal oocytes or embryos, and contributes to successful cryopreservation [22]. However, it was not the case in red seabream embryos, zebrafish embryos [23] and starfish oocytes [24] mainly due to the high water content and multi-membrane structures which inhibit sufficient dehydration of fish embryos.

Thirdly, intra-cellular ice formed earlier than extra-cellular ice in V groups, which is opposite to the phenomenon in S groups. And the intra-cellular ice in V groups firstly formed in the yolk and developing embryo, then in the perivitelline space. Based on the result of section 3.2 and previous research [19], the cryoprotectant could penetrate into the chorion but hardly penetrate into the yolk syncytial layer and the developing embryo. So the concentration of cryoprotectant outside the chorion was the highest, followed by the perivitelline space, and the yolk and developing embryo was the lowest. We speculated that the sequence of ice formation was determined by the different cryoprotectant concentration in the different parts.

At the point of-150 °C, the phenomena in the four groups were different. In the group of S groups and V1, the whole field of view was obscured by the extra-cellular ice. However, in V2 group, the solutions outside embryos were transparent, like solid glass. We inferred that the outer solution vitrified. The difference of cryoprotectants caused the different phenomena between V1 and V2, because PG (used in V2) vitrifies more easily than DMSO [25]. The embryos in V group did not vitrify, which indicated that the cryoprotectant concentration of inner embryo was not high enough to form vitrification at this cooling rate. It indicated that limited or no cryoprotectant penetrated into the yolk and developing embryo. Yolk syncytial layer (YSL) was the major obstacle for the penetration of the cryoprotectant into the embryo. This was coincide with many researches [26].

In the thawing process, devitrification or/and recrystallization usually take place. Devitrifica‐ tion is the transition of glassy to crystalline state and recrystallization is the growth of existing ice crystals. In our experiment, the transparent space in V2 group was obscured during thawing, which indicated that devitrification had occurred in extra-cellular ice. Recrystalliza‐ tion had also occurred within the embryos in all four groups, for the embryos were all turned bright in the thawing process. Devitrification and recrystallization were thought to be the major reason for cellular damage associated with the thawing process of cryopreserved cells, for they could cause ice crystals formation and larger ice crystals [27]. To avoid devitrification and recrystallization, the specimens must be warming at higher rate than the critical warming rate [23]. However, there are no effective means to avoid them at the present time because of the technique limitation.

## **5. Conclusion**

concentration were reported to be prone to vitrify and avoid dendritic ice crystal formation

Secondly, in our result, *TIIF*s in S groups were significantly higher than those in V groups. This may be because the nucleation temperature of red seabream embryos is too high to achieve sufficient cell dehydration to avoid IIF [21]. *TIIF* is a main factor many researchers focus on in the study of cryoproservation. Slow cooling usually causes dehydration and decreases *TIIF* largely in mammal oocytes or embryos, and contributes to successful cryopreservation [22]. However, it was not the case in red seabream embryos, zebrafish embryos [23] and starfish oocytes [24] mainly due to the high water content and multi-membrane structures which

Thirdly, intra-cellular ice formed earlier than extra-cellular ice in V groups, which is opposite to the phenomenon in S groups. And the intra-cellular ice in V groups firstly formed in the yolk and developing embryo, then in the perivitelline space. Based on the result of section 3.2 and previous research [19], the cryoprotectant could penetrate into the chorion but hardly penetrate into the yolk syncytial layer and the developing embryo. So the concentration of cryoprotectant outside the chorion was the highest, followed by the perivitelline space, and the yolk and developing embryo was the lowest. We speculated that the sequence of ice formation was determined by the different cryoprotectant concentration in the different parts.

At the point of-150 °C, the phenomena in the four groups were different. In the group of S groups and V1, the whole field of view was obscured by the extra-cellular ice. However, in V2 group, the solutions outside embryos were transparent, like solid glass. We inferred that the outer solution vitrified. The difference of cryoprotectants caused the different phenomena between V1 and V2, because PG (used in V2) vitrifies more easily than DMSO [25]. The embryos in V group did not vitrify, which indicated that the cryoprotectant concentration of inner embryo was not high enough to form vitrification at this cooling rate. It indicated that limited or no cryoprotectant penetrated into the yolk and developing embryo. Yolk syncytial layer (YSL) was the major obstacle for the penetration of the cryoprotectant into the embryo. This

In the thawing process, devitrification or/and recrystallization usually take place. Devitrifica‐ tion is the transition of glassy to crystalline state and recrystallization is the growth of existing ice crystals. In our experiment, the transparent space in V2 group was obscured during thawing, which indicated that devitrification had occurred in extra-cellular ice. Recrystalliza‐ tion had also occurred within the embryos in all four groups, for the embryos were all turned bright in the thawing process. Devitrification and recrystallization were thought to be the major reason for cellular damage associated with the thawing process of cryopreserved cells, for they could cause ice crystals formation and larger ice crystals [27]. To avoid devitrification and recrystallization, the specimens must be warming at higher rate than the critical warming rate [23]. However, there are no effective means to avoid them at the present time because of

[20], which may explain the phenomenon in this study.

40 Recent Advances in Cryopreservation

inhibit sufficient dehydration of fish embryos.

was coincide with many researches [26].

the technique limitation.

In the present study, we compared conventional slow cooling method and vitrification method in red seabream embryos cryopreservation. In V groups, the *TEIF*s and *TIIF*s were significantly lower than those in S groups, and no big ice crystals formed. Besides, after thawing, the percentage of embryos with intact morphology in V groups was significantly higher than that in S groups. All the results indicated that vitrification method would be a good option for red seabream embryos cryopreservation, though the embryos were better tolerant of S solutions than V solutions. Therefore, further study is still required to optimize the cryopreservation protocol for reducing toxicity of cryopreservation as well as improving the internal cryopro‐ tectant concertration in red seabream embryos.

#### **Acknowledgements**

This work is supported by Regional Demonstration of Marine Economy Innovative Develop‐ ment Project (No. 12PYY001SF08)" and National Infrastructure of Fishery Germplasm Resources.

### **Author details**

Q. H. Liu1,2, J. Li1,2, Z.Z. Xiao1,2, S.H. Xu1,2, D.Y. Ma1,2 and Y.S. Xiao2,2

\*Address all correspondence to: junli@qdio.ac.cn

1 Center of Biotechnology R&D, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, P.R. China

2 National &, Local Joint Engineering Laboratory of Ecological Mariculture, Chinese Acade‐ my of Sciences, Qingdao, P.R. China

#### **References**


[3] Chen S.L., 2002, Progress and prospect of cryopreservation of fish gametes and em‐ bryos. J. Fish. Chi. 26, 161–168.

[17] Cabrita E., Robles V., Chereguini O., Paz de. P., Anel L., Herraéz M.P., 2003, Dimeth‐ yl sulfoxide influx in turbot embryos exposed to a vitrification protocol. Theriogenol‐

Effects of Slow Cooling Methods and Vitrification Methods on Red Seabream (*Pagrus major*) Embryos

http://dx.doi.org/10.5772/58830

43

[18] Rawson D.M., Zhang T., Kalicharan D., Joegebloed W.L., 2000, Field emission scan‐ ning microscopy transmission electron microscopy studies of the chorion plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish *Brachy‐ danio rerio*: a consideration of the structural and functional relationship with respect

[19] Valdez D.M., Miyamoto A., Hara T., Seki S., Kasai M., Edashige K., 2005, Water-and cryoprotectant-permeability of mature and immature oocytes in the medaka (*Oryzias*

[20] Kobayashi A., Shirai Y., Nakanishi K., Matsuno R., 1996, A method for making large

[21] Liu X.H., Zhang T.T., Rawson D.M., 1998, Feasibility of vitrification of zebrafish

[22] Bernard A., Fuller B.J., 1996, Cryopreservation of human oocytes: a review of current

[23] Bart A., 2000, New approaches in cryopreservation of fish embryos. In: Tierch T.R., Mazik P.M., (Eds.) Cryopreservation in aquatic species. Baton Rouge, LA, World

[24] Billard R., Zhang T., 2001, Techniques of genetic resource banking in fish. In: Watson P.F., Holt W.V., (Eds.) Cryobanking the genetic resource. Wildlife conservation for

[25] Baudot A., Alger L., Boutron P., Glass-forming tendency in the system water–di‐

[26] Zhang T., Rawson D.M., 1998, Permeability of Dechorionated One-Cell and Six-So‐ mite Stage Zebrafish (*Brachydanio rerio*) Embryos to Water and Methanol. Cryobiolo‐

[27] Robles V., Cabrita E., Fletcher G.L., Shears M.A., King M.J., Herraéz M.P., 2005, Vitri‐ fication assays with embryos from a cold tolerant sub-arctic fish species. Therioge‐

agglomerated ice crystals for freeze concentration. J. Food Eng. 27, 1–15.

(*Danio rerio*) embryos using methanol. Cryo-Letters 19, 309–318.

problems and perspectives. Hum. Reprod. Update 2, 193–207.

the future. London, Taylor and Francis Press, pp. 145–170.

methyl sulfoxide. Cryobiology 2000, 40, 151–158.

to cryoprotectant penetration. Aquac. Res. 31, 325–336.

ogy 60, 463–473.

*latipes*). Cryobiology 50, 93–102.

Aquaculture Society, pp. 179–187.

gy 37, 13–21.

nology 64, 1633–1646.


[17] Cabrita E., Robles V., Chereguini O., Paz de. P., Anel L., Herraéz M.P., 2003, Dimeth‐ yl sulfoxide influx in turbot embryos exposed to a vitrification protocol. Theriogenol‐ ogy 60, 463–473.

[3] Chen S.L., 2002, Progress and prospect of cryopreservation of fish gametes and em‐

[4] Robles V., Cabrita E., Paz de. P., Cuñado S., Anel L., Herráez M.P., 2004, Effect of a vitrification protocol on the lactate dehydrogenase and glucose-6-phosphate dehy‐ drogenase activities and the hatching rates of Zebrafish (*Danio rerio*) and Turbot

[5] Ding F.H., Xiao Z.Z., Li J., 2007, Preliminary studies on the vitrification of red sea

[6] Li J., Zhang L.L., Liu Q.H., Xu X.Z., Xiao Z.Z., Chen Y.K., Ma D.Y., Xu S.H., Xue Q.Z., 2009, Extra-and intra-cellular ice formation of red seabream (*Pagrus major*) embryos

[7] Naik B.R., Rao B.S., Vagdevi R., Gnanprakash M., Amarnath D., Rao V.H., 2005, Con‐ ventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rab‐

[8] Kuleshova L.L., Lopata A. 2002, Vitrification can be more favorable than slow cool‐

[9] Kasai M., Zhu S.E., Pedro P.B., Nakamura K., Sakurai T., Edashige K., 1996, Fracture damage of embryos and its prevention during vitrification and warming. Cryobiolo‐

[10] Robles V., Cabrita E., Real M., Álvarez R., Herráez M.P., 2003, Vitrification of turbot

[11] Hagedorn M., Peterson A., Mazur P., Kleinhans F.W., 2004, High ice nucleation tem‐ perature of zebrafish embryos: slow-cooling is not an option. Cryobiology 49, 181–

[12] Guenther J.F., Seki S, Kleinhans F.W., Edashige K., Roberts D.M., Mazur P., 2006, Ex‐ tra-and intra-cellular ice formation in Stage I and II *Xenopus laevis* oocytes. Cryobiolo‐

[13] Chen S.L., Tian Y.S., 2005, Cryopreservation of flounder (*Paralichthys olivaceus*) em‐

[14] Edashige K., Delgado M., Valdez Jr., Hara T., Saida N., Seki S., Kasai M., 2006, Japa‐ nese flounder (Paralichthys olivaceus) embryos are difficult to cryopreserve vitrifica‐

[15] Ding F.H., 2005, Sperm and Embyo Cryopreservation of Red Sea Bream (*Pagrosomus Major*). M.S. Thesis. Graduate University of Chinese Academy of Sciences, Beijing

[16] Chambers J.A.A., Rickwood D., 1993,.Biochemistry LabFaX. Oxford, UK, Bios scien‐

(*Scophthalmus maximus*) embryos. Theriogenology 61, 1367–1379.

bream (*Pagrus major*) embryos. Theriogenology 68, 702–708.

at different cooling rates. Cryobiology 59, 48-53.

embryos: preliminary assays. Cryobiology 47, 30–39.

bryos by vitrification. Theriogenology 63, 1207–1219.

bit embryos. Ani. Reprod. Sci. 86, 329–338.

ing. Fertil. Steril. 78, 449-454.

gy 33, 459–464.

gy 52, 401–416.

City, P. R. China.

tion. Cryobiology 53, 96-106.

tific Publishers, Academic Press.

189.

bryos. J. Fish. Chi. 26, 161–168.

42 Recent Advances in Cryopreservation


**Chapter 3**

**Cryopreservation of Embryos and Oocytes of South**

Danilo Pedro Streit Jr., Leandro Cesar de Godoy, Ricardo Pereira Ribeiro,

The importance of animal genetic resources for wildlife maintenance as well as farming production has become more and more evident in recent years. Fish stocks are globally threatened mainly due to overfishing and environmental pollution. Cryopreservation of aquatic germplasm brings the possibility of preserving the genome of endangered species, increasing the representation of genetically valuable animals for farming purposes and

In fish, successful cryopreservation of semen from many species has been well documented and cryopreserved semen has been used for reproduction of many wild and farmed species. Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive.

In this chapter we cover the cryobiology applications on assisted reproduction for fish farming, focused on embryo and oocyte cryopreservation. Our research group has been working in this area for more than 10 years and the chapter shows the main results that we have achieved from researches on embryos cryopreservation. The barriers that have been identified as a hamper for successful cryopreservation and the sensitiveness of tropical fish embryos to low temper‐

Recently, researches have reported that the use of oocytes may offer some advantages when compared to fish embryos, improving the chances of a successful cryopreservation. On this point, the chapter presents the most recent achievements in oocytes cryopreservation from

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Darci Carlos Fornari, Melanie Digmayer and Tiantian Zhang

Additional information is available at the end of the chapter

avoiding genetic losses through diseases and catastrophes.

South American fish, new trends and future works on this area.

ature exposure are also detailed here.

**American Fish Species**

http://dx.doi.org/10.5772/58703

**1. Introduction**
