**1. Introduction**

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Cryopreservation is considered as one component in an effective strategy to save endangered species by facilitating the storage of their gametes in gene banks (Gausen, 1993; Akcay et al. 2004). Cryopreservation offers several benefits that in this way stocks can be protected from being totally eliminated due to sudden disease outbreak, natural utilization in hatcheries' production and laboratory experiments can be ensured. Stocks can be maintained more economically and experimental materials for advanced studies, such as gene transfer, can be made more accessible (Chao & Liao, 2001; Tekin et al. 2003).

Cryopreservation techniques involve addition of cryoprotectants, freezing and thawing of sperm samples, all of which may result in some damage to the spermatozoa and may decrease egg fertilization rate (Kopeika et al. 2003). Therefore, before cryopreservation of sperm, a through evaluation of different extender solutions, cryoprotectants, straw sizes and thawing rates is essential to develop optimum cryopreservation protocols for various species (Yavas & Bozkurt, 2011).

The species-specific cryopreservation procedure needs a suitable extender, as undiluted semen is not suitable for long-term preservation. Similarly, addition of optimum amount of cryoprotectant reduces cell damages associated with dehydration, cellular injuries and ice crystal formation (Leung, 1991). Although cryoprotectants help to prevent cryoinjuries during freezing and thawing, they can become toxic to cells when the exposure time and concentration are more (Tekin et al. 2007). In addition, type of cryoprotectants is also very specific to many species. There are no universal extenders and cryoprotectants available that can be used across species.

On the other hand, motility is the most commonly used parameter to evaluate sperm quality in fishes (Billard et al. 1995). This parameter is acceptable so that spermatozoa must be motile to achieve fertilization. Furthermore, sperm motility is an important component of a cryopreservation program in order to prevent poor sperm quality semen samples prior to freezing and to estimate the fertility of the stored sperm after thawing (Akçay et al., 2004;

Cryopreservation of Brown Trout

samples.

30 min of sampling.

(*Salmo trutta macrostigma*) and Ornamental Koi Carp (*Cyprinus carpio*) Sperm 295

anesthetized (0.1 g/l MS 222) males by manual abdominal stripping 12 h after a single injection of 2 mg/kg of carp pituitary extract (CPE) at 20-22 ºC water temperature. Eggs were collected by hand stripping 10-12 h after a double injection of 3.5 mg/kg of CPE. The

For sperm collection, the urogenital papilla's of mature male fishes were carefully dried and sperm was hand-stripped directly into test tubes. Following sperm collection, the tubes containing sperm were placed in a styrofoambox containing crushed ice (4ºC). Contamination of sperm with water, urine or faeces was carefully avoided. Sperm was transported to the laboratory within 15 min. For collection of eggs from koi carps, females were wiped dry, stripped by gentle abdominal massage and the eggs from each female were collected in a dry metal bowl. Eggs were checked visually and only those lots of

Motility was estimated subjectively using light microscope (Olympus, Japan) with a x400 magnification. Samples were activated by mixing 1 μl of sperm with 20 μl activation solution (0.3% NaCl) on a glass slide. The percentage of motility was defined as the percentage of spermatozoa moving in a forward motion every 20% motile increment (i.e., 0, 20%, 40%, 60%, 80%, and 100%) (Vuthiphandchai & Zohar, 1999). Motility measurements were performed within 15 s. after activation. Sperm cells that vibrated in place were not considered to be motile. Sperm motility was estimated with three replicates of samples. For cryopreservation experiments, samples below 80% motile spermatozoa were discarded. Duration of sperm motility was determined using a sensitive chronometer (sensitivity: 1/100 s) by recording the time following addition of the activation solution to the sperm

Spermatozoa density was determined according to the haemacytometric method. Sperm was diluted at ratio of 1:1000 with Hayem solution (5g Na2SO4, 1g NaCl, 0.5g HgCl2, 200 mL bicine) and density was determined using a 100 μm deep Thoma haemocytometer (TH-100, Hecht-Assistent, Sondheim, Germany) at 400x magnification with Olympus BX50 phase contrast microscope (Olympus, Japan) and expressed as spermatozoa x109 mL-1 (three replicates). Counting chambers were always kept in a moist atmosphere for at least 10 min before cell counting. Sperm pH was measured using indicator papers (Merck, 5.5-9) within

Collected sperm from 10 males that showing >80 motility was pooled into equal aliquots according to the required semen volume and sperm density to eliminate effects of individual variability of gamete donors. Semen and extenders were kept at 4°C prior to dilution. Pooled semen was diluted at 1:3 ratio (semen/extender) with extender containing 4.68 g l- NaCl, 2.98 g l- KCl, 0.11 g l- CaCl2 and Trizma-HCl 3.15 g l- in distilled water; pH 9.0 (Billard & Cosson, 1992). The extender contained methanol and egg yolk at ratios of 5%, 10% and 15% separately. Dilution of semen with extender resulted in sperm concentrations of around 2.5x109 cells/ml extender that was enough to avoid damage due to sperm

first injection, 10% (0.35 mg/kg) CPE was given 10 h before the second (3.15 mg/kg).

homogenous shape, colour and size were used in the fertilization experiments.

**2.3 Determination of fresh sperm quality parameters** 

**2.4 Experiment 1 - Brown trout (***Salmo trutta macrostigma***)**

Bozkurt, 2008). Thawing temperature and duration are also critical factors in the survival of cryopreserved sperm cells (Morris, 1981). Optimal freezig/thawing procedures have not been reported for *Salmo trutta macrostigma* sperm. So, in the present study three different thawing temperatures and thawing durations were also tested related to motility.

For this reason, there is a need to improve techniques on gamete storage and evaluation of sperm quality to facilitate optimization of controlled reproduction in fish (Alavi & Cosson, 2005). Important parameters for cryopreservation include type of extenders and cryoprotectants, dilution ratios, freezing/thawing rates and fertilization rates (Bozkurt et al. 2005).

*Salmo trutta macrostigma* is a salmonid species occurring in inland water habitats of Southern Europe, Western Asia, Northern Africa and Anatolia (Geldiay & Balik, 1988). It is also critically endangered fish species in inland waters because of illegal shing, overshing, and other environmental changes, including hydroelectric plants and pollution. For this reason a biological conservation program has been considered for *Salmo trutta macrostigma* in Turkey. On the other hand, ornamental koi carp is evaluated by its colour and have been used in the selecive propagation programs. These brightly colored koi carps are the result of selective breeding of wild carp. Over centuries a range of pleasing colors, patterns and shapes have been developed for this valuable species. Therefore, reliable methods for brown trout and koi carp sperm cryopreservation could benefit both aquaculture application and conservation of biodiversity.

Therefore, the present study was conducted in order to examine the effect of ionic extenders combined with different cryoprotectants at different ratios and to test the effect of different thawing temperatures and thawing periods on the post-thaw sperm quality of brown trout (*Salmo trutta macrostigma*) and koi carp (*Cyprinus carpio*) and development of a cryopreservation protocol for sperm of this commercially valuable two species.
