**4. Discussion**

298 Current Frontiers in Cryopreservation

egg yolk as 54.2±3.46 s. Differences between the means of motility durations were significant

20.3±2.57Ad 24.5±2.47Acd 28.6±3.46Ac 40.2±1.29Aa 42.6±4.57Aa 46.4±2.38Ba 15.23±4.39Abf 19.6±4.39Abef 23.4±3.47Abe 37.2±3.45Ac 45.3±5.39Ab 54.2±3.46Aa 12.3±4.17Bd 15.7±1.28Bd 20.4±8.25Bcd 20.4±4.59Bcd 25.3±2.48Bc 32.5±5.27Cb

Means followed by different superscripts (lowercase for lines and uppercase for columns within the

Sperm motility rate (Figure 1) and longevity of motility (Figure 2) values following cryopreservation in the ionic extender containing 15% egg yolk were determined. Sperm was thawed at 25ºC, 35ºC or 45ºC for 5s, 15s or 25s and activated in 0.3% NaCl and 1%

Fig. 1. Post-thaw motility (%) of brown trout sperm thawed at different degrees, periods and

**45ºC/5s**

**45ºC/15s**

**45ºC/25s**

Post-thaw sperm motility rates were affected by thawing rates and activation agents and ranged from 25% to 50%. Also, the activating agents affected the duration of motility. All sperm samples triggered in 1% NaHCO3 were motile for a longer period (32-57 s) compared with samples triggered in 0.3% NaCl (24-53 s). Differences between the post-thaw motility

Table 2. Post-thaw longevity (s) of brown trout sperm cryopreserved with different

Egg Yolk (5%)

Egg Yolk (10%)

Egg Yolk (15%)

**0.3%NaCl**

**1%NaHCO3**

Methanol (15%)

(P<0.05). (Table 2).

Methanol (5%)

cryoprotectants.

activating agents.

**0**

**25ºC/5s**

**25ºC/15s**

**25ºC/25s**

**35ºC/5s**

**35ºC/15s**

**35ºC/25s**

**Thawing Temperatures (C) and Periods (s)**

**10**

**20**

**30**

**Post-thaw motility (%)**

**40**

**50**

**60**

and longevity values were significant (P<0.05).

NaHCO3.

Methanol (10%)

same sperm feature) are different (p<0.05). (mean±SE, n=3).

Successful cryopreservation of fish spermatozoa depends on a range of factors including the collection of high quality sperm, equilibration conditions, choice of cryoprotectant medium, cooling/thawing regimes, and conditions for fertilization. Even though some general rules can be applied to any fish species, optimization of the protocol is needed for each individual species (Kopeika et al. 2007). Several factors have affected post-thaw quality of cryopreserved sperm from both brown trout (*Salmo trutta macrostigma*) and ornamental koi carp (*Cyprinus carpio*). The results obtained in the present study contribute significantly

Cryopreservation of Brown Trout

1% NaHCO3 was used as an activating agent.

**4.2 Ornamental koi carp (***Cyprinus carpio***)**

2009).

al. 1998).

(*Salmo trutta macrostigma*) and Ornamental Koi Carp (*Cyprinus carpio*) Sperm 301

On the other hand, thawing temperature also play an important role in the post-freeze semen quality of fish (Wayman et al., 1998). Generally, thawing rates should be high to avoid recrystallization (Lahnsteiner, 2000). Significant post-thaw motility was determined when brown trout sperm was thawed at temperature of 35ºC in the present study. According to the results of the present study, it was shown that higher temperatures are necessary to recover membrane stability or metabolism of spermatozoa. Also it appears that either recrystallization and ice crystal formation during thawing were reduced or avoided by this thawing procedure, or enzymatic activities were the best reactivated (Lahnsteiner, 2000). Although thawing from -196ºC to 4ºC is generally considered as critical phase because of potential recrystallization, the process was similar for all species. Furthermore, the two activating agents (0.3% NaCl and 1% NaHCO3) tested did not affect post-thaw motility rates or quality motility score, although, in general, higher scores were observed when 1% NaHCO3 was used in the present study. Duration of motility was significantly higher when

The main purpose of the current experiment was to develop an appropriate protocol for ornamental koi carp (*Cyprinus carpio*) sperm cryopreservation to increase sperm availability outside the breeding season. By banking male gametes when they are abundant, most efforts can be devoted to raising healthy female broodstock and obtaining good quality eggs within a short captivity culture period. Through cryopreservation, a sperm repository can also be established for all males in captivity or from the wild. Such repository is important to maintain the genetic diversity to avoid inbreeding or loss of heterozygosity for captive breeding programs as well as possible future stock enhancement in the wild (Cabrita et al.,

In the present experiment, koi carp males gave sperm characterized by good spermatozoa density and percent of motility. Such sperm should be used for cryopreservation experiments when considering minimization of artificial selection and sperm competition during hatchery operations in order to maintain the greatest biodiversity (Campton, 2004). During the cryopreservation process, one of the important issue is the use of cryoprotectants, which role is to prevent cell damage during the freezing and thawing steps. Several cryoprotectants have been used for fish sperm cryopreservation, including methanol, ethylene glycol and dimethyl sulphoxide (DMSO); however, DMSO is reported to be the most efficient to cryopreserve fish spermatozoa (Anel & Cabrita, 2000) due mainly to its small molecular size, which allows it to enter and exit the spermatic cell easily (Tiersch et

The best fertilization rate obtained with extender II with 99.7% eyeing rate in koi carp. These results can be explained by the presence of 10% DMSO as a cryoprotectant in this extender. It can be concluded that DMSO has higher permeability by permeating into cell, causing reduced ice crystal formation for koi carp sperm. On the other hand, Lahnsteiner et al., (1996) used 10% methanol, 10% DMSO, 10% DMA, 5% glycerol and mixture of 5% DMSO and 1% glycerol for semen cryopreservation of the grayling (*Thymallus thymallus*) and the Danube salmon (*Hucho hucho*), which methanol showed the highest fertilization rates in

Cryopreservation protocol carried out in the present study with a 1:100,000 egg: spermatozoa ratio, almost the same fertilization efficiency was obtained whether frozen or fresh semen was

relation to control 95.3% and 91.1% for grayling and Danube salmon, respectively.

improve the development protocol of sperm cryopreservation in brown trout and ornamental koi carp at large scale.

#### **4.1 Brown trout (***Salmo trutta macrostigma***)**

The results of the present study demonstrate for the first time cryopreservation of brown trout (*Salmo trutta macrostigma*) sperm. In the present study, post-thaw sperm quality was initially evaluated on the basis of sperm motility score and duration of motility for brown trout. For this aim, the effect of two cryoprotectants and three thawing temperatures on the post-thaw sperm quality of brown trout were assessed.

Motility is induced after the spermatozoa released into the aquatic environment during natural reproduction or after transfer to an activation medium during controlled reproduction (Alavi & Cosson, 2006). When salmonid spermatozoa are released into water they have a brief period of sperm activity between 20 and 40s (Morisawa & Morisawa, 1986). A better knowledge of the characteristics of fresh sperm motility is necessary to evaluate sperm quality in commercial hatcheries before artificial reproduction and in laboratories before experiments. Preliminary examination of fresh sperm was carried out in order to determine the relationship between sperm motility and seminal plazma composition of *Salmo trutta macrostigma* sperm (Bozkurt et al. 2011a).

Comparison of different cryoprotectant recipes and freeze-thaw protocols are difficult when each treatment tested for the ability of sperm to fertilise eggs. Cryoprotectants can suppress most cryoinjuries when used higher concentrations but at the same time it can become toxic to the cells. Therefore, a suitable concentration was needed for the development of a cryopreservation protocol. Methanol has been used successfully for sperm cryopreservation in African catfish (*Clarias gariepinus*) (Burchell) (Steyn & Van Vuren, 1987), tilapia (*Sarotherodon mossambicus*) (Peters) (Rana & McAndrew, 1989), bagrid catfish (*Mystus nemurus*) (Muchlisin et al., 2004) and salmonid fish (Lahnsteiner et al., 1996). Mansour et al. (2006) showed that 10% methanol was more effective as a cryoprotectant for Arctic char spermatozoa than 10% DMA or 10% DMSO when used with a glucose diluent. However, the effects of higher levels of methanol cryoprotectant were not investigated. In the present study, brown trout semen in an extender containing 15% egg yolk resulted in the highest overall percentage of sperm motility.

In addition, penetrating cryoprotectants could affect the percentage of motile sperm. In salmonids, some authors reported that higher post-thaw motility from methanol than from DMSO and other cryoprotectants (Mansour et al. 2006). In the present study, methanol and egg yolk have statistically significant effect on the percentage of sperm motility. On the other hand, it should be noted that egg yolk achieved better results than methanol for cryopreservation of brown trout sperm. Extender containing glucose, egg yolk and DMSO described by Alderson and McNeil (1984) gave good results in cryopreservation experiments with large straws. Baynes and Scott (1987) also reported that egg yolk is a valuable component in extenders for salmonid sperm cryopreservation. Furthermore, the addition of egg yolk to the medium interferes with the good visualization of spermatozoa during the motility rate analysis. With this in mind, we have tested several extender/cryoprotectant combinations with the addition of egg yolk that preserve sperm during storage and yet allow good visualization during motility analysis.

On the other hand, thawing temperature also play an important role in the post-freeze semen quality of fish (Wayman et al., 1998). Generally, thawing rates should be high to avoid recrystallization (Lahnsteiner, 2000). Significant post-thaw motility was determined when brown trout sperm was thawed at temperature of 35ºC in the present study. According to the results of the present study, it was shown that higher temperatures are necessary to recover membrane stability or metabolism of spermatozoa. Also it appears that either recrystallization and ice crystal formation during thawing were reduced or avoided by this thawing procedure, or enzymatic activities were the best reactivated (Lahnsteiner, 2000). Although thawing from -196ºC to 4ºC is generally considered as critical phase because of potential recrystallization, the process was similar for all species. Furthermore, the two activating agents (0.3% NaCl and 1% NaHCO3) tested did not affect post-thaw motility rates or quality motility score, although, in general, higher scores were observed when 1% NaHCO3 was used in the present study. Duration of motility was significantly higher when 1% NaHCO3 was used as an activating agent.
