Effect of Different Extenders and Storage Periods on Motility and Fertilization Rate of Rainbow Trout (Oncorhynchus Mykiss) Semen

The effects of different extenders and storage periods on the motility and fertilization rate of rainbow trout (Oncorhynchus mykiss) semen were evaluated after short-term storage. Semen was collected from anesthetized males by the abdominal massage. After determination of main semen characteristics, the pooled ejaculates were diluted with 4 different extenders at a ratio of 1:3, and stored at 4oC for 72 h. During preservation, spermatozoa motility (%) were determined every 12 h. Fertilization was carried out using the dry fertilization technique. The sperm-egg ratio was approximately 0.5x106 sperm/egg for optimum fertilization success. The highest motility (64.4±5.27%) and fertilization rate (94.3 ± 0.58 %) were obtained from semen stored with glucose based extender after 72 h storage. These results indicate that glucose based extender is a better preservative than the other solutions used in the study for the short term preservation of rainbow trout semen.


Introduction
Preservation of fish sperm for short-term duration is generally useful from the commercial point of view and facilitates various hatchery operations. The short-term storage of sperm at low temperature (4ºC) is mostly applied in short-distance transport of gametes collected in different locations, in synchronizing the timing of obtaining good quality of gamete collection from males and females during artificial insemination, in avoiding the aging of sperm, in facilitating hatchery operations, also in experimental programs for genetic studies [1][2][3].
The fish sperm could be preserved by storage in undiluted and diluted form. Undiluted sperm stored at low temperature has been reported to cause a reduction in fertilization capacity [4]. Storage of diluted sperm with extender provides better control compared to undiluted storage [5]. Extender solutions can be mixed with semen to increase the volume of the semen samples and to prevent it from deteriorating while it is being held or shipped for insemination.
The quality of the stored sperm can be assessed by taking into consideration motility, motility duration, and by its insemination ability [3]. Buyukhatipoglu and Holtz [6] reported that the spermatozoa of rainbow trout were maintained fertilization capacity at 4°C under oxygen and air medium for 15 and 9 days, respectively. Sperm of rainbow trout diluted with ovarian fluid and borax-boric acid buffer and the highest insemination achievement (81.76%) was obtained when ovarian fluid was used as a sperm extender but the spermatozoa demonstrated maximum (34 seconds) forward motility aftcr dilution with borax-boric acid buffer [7]. Bencic et al. [8] suggested that atmospheric air served as a suitable gas for the short-term preservation of salmonid semen. Canyurt et al. [9] indicated that the best results in fertilization and motility were obtained from the sperm samples of rainbow trout diluted with artificial seminal plasma with the rate of 1:1 and stored for 7 days [9]. Hatipoğlu and Akçay [10] found that glucose based extender was a better preservative than Ringer solution for the short term preservation of Abant trout (Salmo trutta abanticus) semen. Niksirat et al. [11] reported that semen samples stored in the absence of antibiotics completely lost fertilizing capacity within 19 days of storage. Şahin et al. [12] clarified that Salmo coruhensis sperm stored in extenders containing glucose, calcium chloride or magnesium chloride remained motile for 24, 14 and 18 days, respectively.
As seen in previous studies, despite the use of various diluents to preserve fish sperm without reducing fertilizing capacity, results showed that sperm motility, motility duration, viability and fertilizing capacity vary widely. Individual variation, collecting method and storage conditions affect the success in fish sperm dilution [13]. The goal of this study was to identify the effect of cold storage of 66 Effect of Different Extenders and Storage Periods on Motility and Fertilization Rate of Rainbow Trout (Oncorhynchus Mykiss) Semen sperm from rainbow trout by assessing sperm motility and its insemination ability using different extenders.

Broodstock Care and Collection of Sperm and Eggs
The experiment was carried out at the Recep Tayyip Erdoğan University, Iyidere Fisheries Research Centre (IFRC), Rize, Turkey. The broodstock were held in a circular fiberglass tank under a natural illumination and fed with a commercial trout diet at 2% of their body weight per day. The average water temperature was measured as 12.9±0.58 °C (8.0-18.5 °C) during spawning season. A total of 6 mature rainbow trout males (total weight 2536 ±316.3g, total length 56.7 ± 2.82 cm) were randomly selected from broodstock and were used as semen donors in the middle of the spawning season. Fish were fasted for two days prior to collection of semen, anaesthetized in 30 ppm of benzocaine and their abdomens were dried before stripping in order to avoid contamination of semen with urine, mucus and blood cells. The semen was collected into 10 ml graduated glass tubes by gentle abdominal massage. Each male was stripped once only and the total amount of expressible milt was collected individually and the sperm volume was expressed as ml. The semen samples were held on crushed ice (4ºC) before analysis, which was undertaken within 4 h of stripping. Eggs were gathered from 5 mature females without anesthesia.

Evaluation of Sperm Density and Spermatocrit
Sperm density was determined according to the haemacytometric method [14]. Semen was diluted by pipetting 10 μl semen into 990 μl 0.7% NaCl solution. One droplet of diluted semen was placed on a hemocytometer slide (depth 0.1 mm) with a coverslip, the sperm was allowed to settle for 3-5 min, sperm cells were counted using light microscopy (x 40), and spermatozoa density was expressed as x 10 9 cells/ml. Spermatocrit was defined as the ratio of white packed material volume to the total volume of semen multiplied by 100 [15]. Heparinized microhematocrit capillary tubes (75 x 1.1-1.2 mm) were filled with semen and one end was sealed with clay. The capillary tubes were centrifuged at 10,000 rpm for 10 min.

Evaluation of Sperm Motility, Motility Duration and Ph
The motility of fresh spermatozoa from each male was determined immediately after semen was collected. The percent of spermatozoa exhibiting rapid, vigorous, forward movement was determined subjectively under a microscope (x 400 magnification) by diluting the semen in activation solution (0.3% NaCl) at a ratio of 1:100 (1 μl sperm to 99 μl activation solution). Motility duration was assessed using a sensitive chronometer (1/100) that was started simultaneously with the addition of activation solution into the samples. Sperm motility observations were done using three replicates per sample. All of the experiments were performed at 17-20°C and all of measurements were carried out by the same investigator under the same conditionsfor avoiding subjective errors. pH was measured by using indicator papers (Merck 6.4-8).

Selection of the Sperm Extenders
Four extenders containing calcium chloride (E1), magnesium chloride (E2), glucose (E3) and calcium+magnesium chloride (E4) [16] were selected for evaluation as potential extenders for rainbow trout sperm. The chemical composition of each extender is shown in Table 1. The semen and extenders were maintained in a refrigerator before dilution. For selecting the most suitable extender, the semen was mixed with extender solution at a ratio of 1:3 and placed into 2 ml small plastic vials that were three replicates per treatment. After a rapid shaking, the vials were stored in a refrigerator at 4°C. The samples were analyzed by 12 hours intervals.

Fertilization
Eggs were pooled from 5 females. Fertilization took place in dry plastic dishes using dry fertilization technique and a portion of ca. 100 eggs (10 g) was placed into each dish. The egg groups were fertilized with cold preserved semen for 72 h storage. Eggs and sperm cells were gently mixed for 10 s. The proportion of sperm-egg was approximately 0.5x10 6 sperm/egg for optimum fertilization success [17]. After fertilization, 25 ml of 0.3% sodium chloride was added to the sperm-egg mixture as fertilization solution and left for 45 min for swelling of eggs. Subsequently, the fertilized eggs were rinsed with hatchery water (10ºC) and placed into vertical incubation trays. Unfertilized and dead eggs were counted and removed continuously. The fertilization rate was determined with the percent of eyed-egg 21 days after insemination.

Statistical Analysis
All measurements were done in triplicate for each sample and the average of three measurements was used in subsequent statistical analyses. Data were expressed as means±SD. Motility data were normalized through arcsine transformation. Pearson correlation analysis was used to estimation with spermatologic parameters. Differences between parameters were analyzed by one-way analysis of variance (ANOVA). Significant means were subjected to a multiple comparison test (Tukey HSD) for post-hoc comparisons at a level of α = 0.05. All analyses were carried out using SPSS 15.0 for Windows statistical software package.

Results
Spermatologic parameters of fresh semen from rainbow trout are shown in Table 2. The volume of semen collected ranged from 10.4 to 21.8 mL. The sperm concentration in the seminal plasma varied between individuals and ranged from 1.6 x 10 9 to 15.4 x 10 9 sperm cells per milliliter of semen. The semen pH ranged from 7.0 to 7.7, and motility ranged from 80 to 100 per cent.
The effects of extenders and storage periods up to 72 h at 4ºC on motility are shown in Table 3.All semen samples were stored in the refrigerator (4ºC) for 72 h and the motility drastically declined during storage period. The mixing of fresh semen with the glucose based extender solution (E3) significantly (P < 0.05) increased the motility of spermatozoa. In contrast, the semen mixed with calcium chloride (E1), magnesium chloride (E2), calcium+magnesium chloride (E4) and the semen stored without the extender (control) had a greatly reduced sperm motility. The effect of extenders on fertilization rates are shown in Table 4. The differences between the groups were statistically significant (p < 0.05). The highest fertilization rate (94.3±0.58%) was obtained with glucose-based extender.
The fertilization results were quite high except for extender E1. Although extenders E2 and E4 were suitable for short-term preservation of rainbow trout sperm, the fertilization rates were all lower than those obtained with extender E3 which gave the best fertilization rates. The reason for the differences among groups in fertilization rate can be explained with decrease in spermatozoa motility. Furthermore, the fertilization can be affected by factors such as dilution ratio of sperm, extender composition, and egg quality [27].
In this investigation, it has been shown that the effect of storage time and extender on motility and fertilization rate was significant (p < 0.05).The fertilization success obtained when extender E3 used as a sperm diluent was significantly better (p < O. 05) than al1 the other diluents tested. Extender E3 was the most suitable diluent for cold storage of rainbow trout semen at 4°C.