**3. Methodology employed for semen cryopreservation of freshwater fishes in Malaysia**

As in cryopreservation of other fish species, the cryopreservation of the semen of freshwater fishes from Malaysia to be discussed later falls under the following general procedures (i) collection of semen, (ii) dilution of semen sample, (iii) semen sample packaging, (iv) equilibration, (v) freezing, (vi) cryo-storage, and (vii) thawing. Successful cryopreservation of fish sperm very much depends on a range of factors in each step of the cryopreservation procedures as highlighted by Kopeika et al. (2007).

#### **3.1 Collection of fish semen**

Mature and healthy males broodfish were selected and anaesthesized using MS222 or clove oil according to the dosage recommended by the manufacturer. Semen sample was expelled from the male fish by gentle abdominal pressure and collected into a clean and dry tube. Extra precaution should be taken while collecting semen sample. Contamination of sample with blood, water, urine or the feces should be avoided as these contaminants significantly reduced the semen quality and caused poor post-thaw sperm motility. The semen samples were then transferred back to laboratory for quantification of the fresh sperm quality and proceed with further dilution before freezing procedure. The sperm motility rates and sperm concentration of the freshly collected semen sample was evaluated prior to cryopreservation. In order to obtain good post-thaw motility, only semen samples showing sperm motility >70% were used for cryopreservation.

#### **3.2 Dilution of semen sample**

274 Current Frontiers in Cryopreservation

a b

c d

e f Fig. 1. Photos of the Malaysian fishes involved in the cryopreservation studies, (a) Isok barb (*P. jullieni*), (b) Malaysian Mahseer (*T. tambroides*), (c) Malaysian Mahseer (*T. deauronensis*), (d) Tropical bagrid catfish (*H. nemurus*), (e) Local pangasiid catfish (*P. nasutus*), and (f) Kerai

**3. Methodology employed for semen cryopreservation of freshwater fishes in** 

As in cryopreservation of other fish species, the cryopreservation of the semen of freshwater fishes from Malaysia to be discussed later falls under the following general procedures (i) collection of semen, (ii) dilution of semen sample, (iii) semen sample packaging, (iv) equilibration, (v) freezing, (vi) cryo-storage, and (vii) thawing. Successful cryopreservation of fish sperm very much depends on a range of factors in each step of the cryopreservation

Mature and healthy males broodfish were selected and anaesthesized using MS222 or clove oil according to the dosage recommended by the manufacturer. Semen sample was expelled from the male fish by gentle abdominal pressure and collected into a clean and dry tube. Extra precaution should be taken while collecting semen sample. Contamination of sample with blood, water, urine or the feces should be avoided as these contaminants significantly

Kunyit (*H. wetmorei*).

**3.1 Collection of fish semen** 

procedures as highlighted by Kopeika et al. (2007).

**Malaysia** 

The semen samples with good quality were subsequently diluted at an appropriate sperm to diluent ratio, with suitable extender solution and cryo-protectant. Sperm to diluent ratios ranged between 1:3 to 1:9 were reported to produce best results in fish sperm cryopreservation studies (Scott & Baynes, 1980; Lahnsteiner et al., 1996). Gwo (2000) reported the fish sperm could loss its viability in high dilution ratio especially in marine species. The type of extender solution, cryoprotectant and dilution ratio that were optimum for each Malaysian fish species studied were summarized in Table 1. The type of diluents and dilution ratios workable to preserve sperm motility appear to vary among different fish species. Thus each of these parameters needs to be optimized accordingly via a series of experimentations.

The extender solution helps to maintain sperm viability prior to and during the freezing process. Extender solution is a balanced salt buffer of specific pH and osmotic strength. Apart from salts, sometimes extender solution is prepared with addition of organic compounds such as glucose. The nature of the effect of extenders is based on the control of pH and salt concentration as well as the supply of energy, and can extend the functional life and fertilizing capability of the sperm (Tiersch, 2000). Cryoprotectants function to protect cells from cryodamage or cryo-injury during freezing and thawing process. The permeating cryoprotectants, namely dimethyl sulfoxide, methanol, ethylene glycol and propylene glycol are among the most frequently used cryoprotective agents among the aquatic organisms (Lahnsteiner et al., 1997; Tiersch, 2000; Tiersch, 2006). However, the permeating cryoprotectants are often toxic to cells, and thus the choice of the types of cryoprotectant and their optimal concentration should be at a balance between protection and toxicity. On the other hand, the non-permeating cryoprotectants such as sucrose, glucose and polymers (e.g. alginate) were often used in combination with the extender solution in the diluents. Sometimes, a combination of different cryoprotectants in certain ratios could help improving the post-thaw motility. The studies of African catfish in our laboratory has shown that a combination of methanol and N, Ndimethylacetamide at ratio 70%:30% produced significant higher post-thaw motility compared to the use of a single cryoprotectant in sample dilution. Tiersch (2000) has also reported that the application of cryoprotectant at concentration between 5% to 20% ususally provides good protection in most fish species. The protective effect and optimal concentration of cryoprotectants could be species specific (Gwo et al., 1991; Suquet et al., 2000; Rideout et al., 2003). Therefore, the optimal concentration needs to be determined individually in each species studied through experimentations.

For Isok barb (*P. jullieni*), Malaysian Mahseer (*Tor* spp.), local pangasiid catfish (*P. nasutus*) and Kerai Kunyit (*H. wetmorei*), a total of 14 extender formulations, five types of cryoprotectants (dimethyl sulfoxide, ethylene glycol, glycerol, methanol and N,N dimethylacetamide) with concentrations between 5-20% (v/v), semen to diluent ratios (1:1 to 1: 14) were examined as described by Chew et al. (2010a). This may be compared with Muchlisin et al. (2004) who used three extender solutions (the Ringer, physiological saline

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 277

sperm cryopreservation and was found to be successful in maintaining a good post-thaw sperm motility (Chew et al., 2010b). In most circumstances, the equilibration duration is set at 15 to 30 min, but it can be varied depending on the type and concentration of

Cryopreservation involves the removal of excess water from the inside of the cell to the exterior where it can form ice (Tiersch, 2000). A two-step rapid freezing method was used for semen cryopreservation in Isok barb, Malaysian Mahseer, local pangasiid catfish and Kerai Kunyit. After the equilibration procedure, semen samples of these species were subjected to liquid nitrogen vapour exposure for 5-10 min in an insulated styrofoam cooler box filled with liquid nitrogen, with the samples placed between 3 to 4 cm above the liquid nitrogen, after which the samples were directly plunged into liquid nitrogen. For tropical bagrid catfish (Muchlisin et al., 2004), the semen samples were equilibrated on crushed ice (0°C) for 5 min. After the equilibration procedure, the samples were then placed in an ice box containing dry ice (-120°C) for 5 min and subsequently plunged into liquid nitrogen. Besides the styrofoam cooler and ice box, the vapour shipper is another choice of method could be used for the freezing of fish semen samples. To cryopreserve semen samples by using dry shipper, the extended and packed semen sample was transferred into the fully charged vapour shipper and subsequently transferred and stored in liquid nitogen. The use of the vapour shipper method in freezing as reported in *Tor* spp. (Chew et al., 2010b) was convenient to be used in the field. Such method is simple to use, the cooling rate is more consistent and controllable and it consumes less liquid nitrogen and requires less space

In our opinion, the use of the sophisticated bench top type of controlled rate programmable freezer is not practical in cryopreservation of fish semen in some laboratories because of difficulty to move this equipment from one location to another. Unfortunately portable type of controlled rate programmable freezer is not user friendly, time consuming and limited number of samples could be processed at a single run. Compared with programmable freezer, the two-step freezing method employing insulated styrofoam cooler box or ice box is simpler, rapid and more cost effective. Nevertheless, the main shortcomings of this simple freezing method is the inconsistency in cooling rates and non-reproducible cryopreservation

Thawing is a process to recover the sperm cells from the dormant stage in ultra low temperature. Frozen semen is usually thawed at 40°C, with different optimal durations applied according to the type of packaging and the storage volume as shown in Table 2.

Type of packaging Volume of semen Duration of thawing

4s – 6s 6s - 8s 12s – 15s

0.25 mL 0.5 mL 1.2 mL

Cryo-vial 5 mL 5 min Table 2. The optimal durations used to thaw the frozen semen samples of freshwater fish

species in Malaysia in different types of packaging and storage volume.

cryoprotectant used (Tiersch, 2000).

compared to the styrofoam cooler box or ice box method.

experiments if performed by different operators.

**3.5 Thawing** 

PE straw

**3.4 Sperm cryopreservation** 


and saline solution); four types of cryoprotectants (DMSO, ethanol, glycerol and methanol) at three concentrations (5%, 10% and 15%) and three sperm to diluent ratios (1:20, 1:30 and 1:40) in tropical bagrid catfish sperm cryopresevation.

Table 1. A list of extender solution and its composition, type of cryoprotectant and its optimal concentration and sperm to diluent ratio for successful semen cryopreservation of various species of freshwater fishes in Malaysia.

#### **3.3 Packaging and equilibration of diluted semen sample**

In this procedure which is performed after the addition of extender solution and cryoprotectant, the diluted semen sample is packed into polyethylene (PE) straws (Chew et al., 2010a; Chew et al., 2010b) or cryo-vials (Muchlisin et al. ,2004). Extended semen sample is subjected to equilibration at temperature <10°C prior to freezing procedure. The duration taken for equilibration is the time required for the cryoprotectant to permeate the cells. Equilibration duration between 15 min to 3 h has been practised for Malaysian fish species sperm cryopreservation and was found to be successful in maintaining a good post-thaw sperm motility (Chew et al., 2010b). In most circumstances, the equilibration duration is set at 15 to 30 min, but it can be varied depending on the type and concentration of cryoprotectant used (Tiersch, 2000).

### **3.4 Sperm cryopreservation**

276 Current Frontiers in Cryopreservation

and saline solution); four types of cryoprotectants (DMSO, ethanol, glycerol and methanol) at three concentrations (5%, 10% and 15%) and three sperm to diluent ratios (1:20, 1:30 and

*P. nasutus* CF-HBSS (Tiersch et al., 1994) Methanol 9 - 10 % 1:7 Unpublished

Table 1. A list of extender solution and its composition, type of cryoprotectant and its optimal concentration and sperm to diluent ratio for successful semen cryopreservation of

In this procedure which is performed after the addition of extender solution and cryoprotectant, the diluted semen sample is packed into polyethylene (PE) straws (Chew et al., 2010a; Chew et al., 2010b) or cryo-vials (Muchlisin et al. ,2004). Extended semen sample is subjected to equilibration at temperature <10°C prior to freezing procedure. The duration taken for equilibration is the time required for the cryoprotectant to permeate the cells. Equilibration duration between 15 min to 3 h has been practised for Malaysian fish species

Type of cryoprotectant & concentration (v/v)

Methanol + DMA

Sperm to diluent ratio

Methanol 10% 1:4 – 1:6 Unpublished

(70:30) 8% 1:6 Unpublished

Methanol 9 - 10% 1:3 – 1:5 Chew et al.,

DMSO 10% 1:7 Chew et al.,

Methanol 9 - 10 % 1:4 – 1:7 Unpublished

Methanol 10% 1:20 Muchlisin et

Reference

data

data

2010a

2010b

data

data

al., 2004

1:40) in tropical bagrid catfish sperm cryopresevation.

solution

Modified from Kurokura et al. 1984 - 62 mM NaCl, 134 mM KCl, 1.5 mM CaCl2, 0.4 mM MgCl2, 2.4 mM NaHCO3 (Horvath et al., 2003)

Ringer solution – 128 mM NaCl, 2.7 mM KCl, 1.4 mM CaCl2, 2.4 mM NaHCO3 (Kurokura et al., 1984)

Calcium free Hank's Balance Salt Solution (CF-HBSS) – 152 mM NaCl, 5.9 mM KCl, 0.9 mM MgSO4, 0.36 mM Na2HPO4, 0.5 mM KH2PO4, 4.6 mM NaHCO3, 6.16 mM Fructose (Tiersch et al., 1994)

202 mM D(+)-glucose monohydrate, 51.5 mM sodium chloride and 6 mM sodium bicarbonate, with pH 7.1 to 7.8 and osmolality 309 + 30 mOsmol/kg

Modified Fish Ringer – 111 mM NaCl, 40.2 mM KCl, 2.1 mM CaCl2 , 2.4 mM NaHCO3 (Wolf, 1963)

Modified Fish Ringer – 128 mM NaCl, 2.7 mM KCl, 1.4 mM CaCl2, 2.4 mM NaHCO3, 25.3 mM glucose

**3.3 Packaging and equilibration of diluted semen sample** 

various species of freshwater fishes in Malaysia.

Species Chemical composition of extender

*B. gonionotus*

*C. gariepinus*

*P. jullieni* 

*Tor* spp.

*H. wetmorei* 

*H. nemurus*  Cryopreservation involves the removal of excess water from the inside of the cell to the exterior where it can form ice (Tiersch, 2000). A two-step rapid freezing method was used for semen cryopreservation in Isok barb, Malaysian Mahseer, local pangasiid catfish and Kerai Kunyit. After the equilibration procedure, semen samples of these species were subjected to liquid nitrogen vapour exposure for 5-10 min in an insulated styrofoam cooler box filled with liquid nitrogen, with the samples placed between 3 to 4 cm above the liquid nitrogen, after which the samples were directly plunged into liquid nitrogen. For tropical bagrid catfish (Muchlisin et al., 2004), the semen samples were equilibrated on crushed ice (0°C) for 5 min. After the equilibration procedure, the samples were then placed in an ice box containing dry ice (-120°C) for 5 min and subsequently plunged into liquid nitrogen.

Besides the styrofoam cooler and ice box, the vapour shipper is another choice of method could be used for the freezing of fish semen samples. To cryopreserve semen samples by using dry shipper, the extended and packed semen sample was transferred into the fully charged vapour shipper and subsequently transferred and stored in liquid nitogen. The use of the vapour shipper method in freezing as reported in *Tor* spp. (Chew et al., 2010b) was convenient to be used in the field. Such method is simple to use, the cooling rate is more consistent and controllable and it consumes less liquid nitrogen and requires less space compared to the styrofoam cooler box or ice box method.

In our opinion, the use of the sophisticated bench top type of controlled rate programmable freezer is not practical in cryopreservation of fish semen in some laboratories because of difficulty to move this equipment from one location to another. Unfortunately portable type of controlled rate programmable freezer is not user friendly, time consuming and limited number of samples could be processed at a single run. Compared with programmable freezer, the two-step freezing method employing insulated styrofoam cooler box or ice box is simpler, rapid and more cost effective. Nevertheless, the main shortcomings of this simple freezing method is the inconsistency in cooling rates and non-reproducible cryopreservation experiments if performed by different operators.

#### **3.5 Thawing**

Thawing is a process to recover the sperm cells from the dormant stage in ultra low temperature. Frozen semen is usually thawed at 40°C, with different optimal durations applied according to the type of packaging and the storage volume as shown in Table 2.


Table 2. The optimal durations used to thaw the frozen semen samples of freshwater fish species in Malaysia in different types of packaging and storage volume.

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 279

(*B. gonionotus*) 2.52 x 109 – 1.03 x 1010 <15 sec (10 s)

(*C. gariepinus*) 5.44 x 109 – 1.14 x 1010 15 – 30 s, (13 s)

(*P. jullieni*) 4.00 x 108 – 6.24 x 1010 20 s, (10 s)

(*Tor* spp.) 2.20 x 108 – 5.98 x 109 40 -50 s, (20 s)

(*P. nasutus*) 6.60 x 108 – 1.36 x 1010 25 – 50 s, (15 s)

(*H. wetmorei*) 7.30 x 109 – 1.33 x 1010 20 -70 s, (20 s)

Table 3. Sperm concentration and motility duration of each freshwater fish species studied

Osmolality is another critical variable in sperm quality (Honeyfield & Krise, 2000). As seen in many studies, seminal plasma osmolality among males fish is highly variable (Aas et al., 1991). According to Babiak et al. (2001), the extender solution that worked well to cryopreserve spermatozoa of a species should posses the ability to maintain the sperm cell viability by inhibiting sperm motility. The key of success is via the use of an extender solution which is almost isotonic or mimicking the seminal plasma of that particular species. Therefore, it is crucial to know the seminal fluid osmolality of a species before media and

In all species of Malaysian fishes studied, sperm motility generally reduced after freezing and thawing process compared to the sperm motility before any freezing procedure. In *P. jullieni*, the motility of cryopreserved semen has reduced by approximately 45% compared to the fresh semen. A reduction of sperm motility by an average of 15% and 30% in *T. tambroides* and *T. deuronensis* respectively was observed. In general, sperm motility reduced between 10 - 70% on average in the species studied (Table 4)*.* These observations are similar to studies in several other species such as *Cyprinus carpio* L. (Wamecke & Pluta, 2003), *Oncorhynchus mykiss* 

> Javanese barb 88 - 97% 15 – 65% African catfish 60 – 100% 15 – 70%

Kerai Kunyit 90 - 100% 35-80%

Table 4. Sperm motility (%) of the freshwater fish species studied before freezing and after

Isok barb 85 - 100% 30-84% (Mean: 49%) Malaysian Mahseer 85 -100% 35-89% (Mean: 55%)

Before freezing Post-thaw

per mL) Duration of motility (\*)

Species Sperm count (Number of cells

\*Duration of progressive movement before slowed down and finally stopped

(Lahnsteiner et al., 1996) and *Scophthalmus maximus* (Dreanno et al., 1997).

Species Motility %

Local pangasiid catfish (Patin Buah) 85 -100% 35-70%

Tropical bagrid catfish (Muchlicin et al. 2004) 80 – 94% 58%

diluents for that particular species can be formulated.

Javanese barb

African catfish

Isok barb

Malaysian Mahseer

Local pangasiid catfish

Kerai Kunyit

at FRI Glami Lemi.

thawing procedures

### **3.6 Egg fertilization**

A cryopreservation protocol developed for a species is considered success if the semen that cryopreserved according to the certain protocol could successfully fertilize eggs and produced offspring. Fresh semen is usually included in the control treatment. The optimal sperm to egg ratio used should be determined prior to fertilization. The sperm: egg ratio at approximately 250,000:1 is usually sufficient and worked well in most species in our laboratory. However, other sperm to egg ratios from 1000:1 to 500000: 1 were tested by Butts et al. (2009) and 100 000: 1 was found to yield the best fertilization performance in Atlantic cod. The dry fertilization method is favorable in the fertilization test for freshwater fish and thus was applied for all Malaysian fish species studied in our laboratory. In the dry fertilization method, both eggs and sperms were mixed well before hatchery water was later added into the sperm/egg mixture to water-harden the fertilized eggs. After rinsing with hatchery water, fertilized eggs were then incubated in aquarium (with or without using a hatching trough depended on species). Water temperature was kept at room temperature between 23 - 28°C throughout the period of incubation. The duration of embryo development varied between species. Therefore, the duration required for the fertilized egg to hatch is also varied among different species.
