Cryopreservation of Mirror Carp (Cyprinus carpio L. 1758) Semen: With Emphasis on Post-Thaw Motility Ergun Akçay1*, Yusuf Bozkurt2, Sezer Kayam3 1*

Ankara University, Veterinary Faculty, Department of Reproduction and Artificial Insemination 06110 Dıskapı, Ankara, Turkey. E-mail: [email protected]

2

Ankara University, Faculty of Agriculture, Department of Fisheries and Aquaculture 06110 Dıskapı, Ankara, Turkey. E-mail: [email protected] 3

S.H.W. Fish Production Station, Bolu, Turkey.

Abstract In this experiment, the main spermatological properties of the mirror carp (Cyprinus carpio L. 1758) semen and the effects of various extenders and cryoprotectants on the post-thaw motility were investigated. Semen was collected from the anaesthetized males by the hand stripping method. Having determined the main spermatological properties (volume, motility, movement duration, concentration, total spermatozoa number and pH), the pooled samples were diluted at a 1:3 ratio with two extenders (described by Kurokora et al., 1984 and Zhang & Liu, 1991) containing different cryoprotectants (15 % DMSO,15% DMA, 15 % Glycerol) individually. The diluted semen was packaged in 0.5 ml straws and left to the equilibration for 45 min at 4 °C. Following the equilibration, the straws were exposed for 10 min to liquid nitrogen vapour and plunged into the liquid nitrogen. Afterwards, cryopreserved semen in the straws were thawed in a water bath at 30 °C for 30 s to determine the motility (%) and movement duration (s) as regards to the postthaw period. The success of freezing was assessed from sperm motility. According to the results of the experiment, the highest post-thaw motility (%) and movement duration (s) were determined by using extender II (described by Zhang & Liu, 1991) containing 15 % DMSO as 60 % and 4 min 10 s respectively.

Key Words: Mirror carp, semen, spermatological properties, extender, cryoprotectant, cryopreservation

Introduction The successful cryopreservation of fish spermatozoa might be used to increase the number of offspring from genetically superior males, aid in the transport of semen and provide a year round supply of male gametes. Furthermore, semen cryopreservation can increase the economic utilization of males and it is a prerequisite for the establishment of gene banks. There has been considerable research on semen preservation in teleosts, salmonids and tilapias. Cryopreservation of the sperm of carp, has been attempted by many authors (Kurokura et al., 1984; Linhart et al., 1988; Cognie et al., 1989; Zhang & Lio, 1991). Besides the influence of the variability in biological material on fertilizing ability of cryopreserved semen, multiplicity of cryopreservation procedures affects the concistency of fertilization results. Differences in diluents, cryoprotectants and freezing techniques make it difficult to get variable estimations of the efficiency of various procedures (Akçay et al., 1995; Lubzens et al., 1997). Most of the experiments in this field have focussed rol and DMSO-glycerol mixture are also efficient (Linhart et al. 1988; Drokin et al., 1989; Cognie et al., 1989). Especially, using an appropriate cryoprotectant solution prevents cells from cellular disruption and membrane damage during freezing and thawing (Brown & Brown, 2000). However, there is a little data on milt quality in connection with cryopreservation. The motility is the simplest and most reliable indicator of the semen quality. The criterion is commonly used in the selection of milt for insemination and preservation. The sperm motility varies in vigor and duration not only among males but also within an individual male depending on his ripeness. In numerous fish species with external fertilization, duration of sperm motility is very short (Billard & Cosson, 1989). The highest motility of the sperm is observed at the height of the breeding season (Terner, C. 1986). Studies on most of the fish species reported the duration and motility of semen may show seasonal variation (Benau & Terner, 1980). Furthermore, sperm motility is an important component of a cryopreservation program in order to prevent poor quality semen samples prior to freezing and to estimate the fertility of the stored sperm after thawing. In cyprinid fishes, sperm motility is often used to estimate semen quality and viability when spermatozoa exposed to cryopreservation; a relationship between motility and fertilizing capacity has been assumed by several authors (Billard & Cosson, 1992; Lahnsteiner et al., 1996). Especially post-thaw motility is one of the important indicators of an successfull cryopreservation. From this point of view, the main purpose of this study was to

investigate the effects of different extenders containing different cryoprotectants on post-thaw motility and movement duration of cryopreserved mirror carp (Cyprinus carpio L. 1758) spermatozoa.

Materials and Methods Adult Fish and Care: In this experiment, 15 adult males of mirror carp aging from 3 to 7 cultured at S.H.W. Fish Production Station in Bolu, Turkey were used. In the pre-spawning period the parenteral brood fishes were kept seperately in small pods and fasted 48 hour prior to semen collection. Milt Collection: Milt was collected from anesthetized (0.1 g/l MS 222) males by manual abdominal stripping 12 h after a single injection 2 mg/kg carp pituitary extract, CPE (Saad & Billard, 1987) at 20-22 °C water temperature. Their abdomens and urogenital papilla were dried before stripping. Milt was collected into 50 ml calibrated glass mezures. Samples contaminated with fecal material or urine were discarded. Evaluation of Motility, Movement Duration, Concentration and pH: Motility was evaluated using a light microscope at x200 magnification and was expressed as a percentage of motile spermatozoa. An activating solution 0.3 % NaCl was used for estimating motility. Only samples showing high motility (>80 %) were used for freezing. Movement duration of was estimated using a sensitive chronometer (1/100 s). Motility and movement duration were evaluated according to the following criterias: 1) Mass progressive motility when most of the spermatozoa were still actively swimming with progressive movement 2) Total duration of movement until most spermatozoa stopped swimming. The sperm concentration was estimated using the hemocytometric method and expressed as spermatozoa x109 / ml. pH was measured by using indicator papers (Merck 5.5-9.0). Dilution: Semen from 15 males were pooled in equal amounts. The pooled semen was diluted at a ratio of 1:3 (one part semen/three part extender) with two extenders containing three different cryoprotectants (15 % DMSO, 15 % DMA, 15 % Glycerol) resulting six extenders. Extender I containing 0.75 g NaCl, 0.02 g KCl, 0.02 g CaCl2 and 0.02 g NaHCO3 supplemented to 100 ml with distilled water described by Kurokura et al., 1984. Extender II containing 6 g

glucose, 0.3 g NaCl and 0.05 g NaHCO3 supplemented to 100 ml with distilled water described by Zhang et al., 1991. The sperm and diluent were kept at 4 °C prior to dilution. Packaging and Equilibration: The diluted samples were drawn into 0.5 ml plastic straws (IMV, France) and were sealed with polyvinile alcohol (PVA) . Having diluted, the samples were equilibrated for 45 min at 4 °C. Freezing: After equilibration, the straws were placed on a styfoam rack that floated on the surface of liquid nitrogen in a styfoam box. The straws were frozen in liquid nitrogen vapour 3 cm above the surface of liquid nitrogen for 10 min. After 10 min the straws were plunged into the liquid nitrogen and stored for one day. Thawing: Frozen straws were thawed by plunging them into water at 30 °C for 30 s. Thawed sperm were activated using 0.3 % NaCl and motility and duration of motility were tested and recorded again. Statistical Analysis:

The results were analysed by variance analysis (ANOVA) and

significant differences were detected using Duncan test.

Acknowledgments The authors wish to thank Dr. N. Tekin and Dr. S. Secer for help and advice about this study and S.H.W. Fish Production Station workers for technical assistance.

References Akcay, E., Tekin, N. & Seçer, S. (1995) Preservation of fish semen. Ege University Journal of Fisheries, 12 (3-4) 367-373. Babiak, I., Glogowski, J., Goryczko, K., Dobosz, S., Kuzminski, H, Strzezek, J. & Demianowicz, W. (2001) Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa. Theriogenology, 56 (1) 177-192. Benau, D. & Terner, C. (1980) Initiation, prolongation and reactivation of the motility of salmonid spermatozoa. Gamete Research, 3, 247-257. Billard, R. & Cosson M. P. (1989) Measurement of sperm motility in trout and carp. Aquaculture (A Biotechnology in Progress), 499-503.

Billard, R. & Cosson, M.R. (1992) Some problems related to the assesment of sperm motility in freshwater fish. Journal of Experimental Zoology, 261, 122-131. Brown, G.G. & Brown, L. D. (2000) Cryopreservation of sperm of stripped bass and white bass, In: Cryopreservation in Aquatic Species.Tiersch, T.R. & Mazik, P.M., Editors. World Aquculture Soociety, Baton Rouge, Louisiana, pp. 130-137. Cognie, F., Billard, R. & Choao, N.H. (1989) Freezing of the milt of the common carp, Cyprinus carpio. Journal of Applied Ichthyology, 5 (4) 165-176. Drokin, S., Kopeika, E.F. & Grishchenko, V.I. (1989) Differences in the resistance to cryopreservation and specifics of lipid compositions of spermatozoa of marine and freshwater fish species. Doklady Biochemistry, 304 (1-6) 65-68. Kurokura, H., Hirano, R., Toita, M. & Iwahashi, M. (1984) Cryopreservation of carp sperm. Aquaculture,37, 267-273. Lahnsteiner, F., Berger, B., Weismann, T. & Patzner, R.A. (1996) Motility of spermatozoa of Alburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism. Fish Pathology and Biochemistry, 15 (2) 197-179. Linhart, O., Liehman, P., & Rab, P. (1988) The first results in freezing of carp semen. Bulletin VURH, Vodnany, 24 (2), 3-13. Lubzens, E., Daube, N., Pekarsky, I., Magnus, Y., Cohen, A., Yusefovich, F. & Feigin, P. (1997). Carp (Cyprinus carpio L.) spermatozoa cryobanks-strategies in research and application. Aquaculture, 155, 13-30. Saad, A. & Billard, R. (1987) Spermatozoa production and volume of semen collected after hormonal stimulation in the carp. Aquaculture, 65, 67-77. Stoss, J. & Holtz, W. (1983) Cryopreservation of rainbow trout sperm. IV. The effect of DMSO concentration and equilibration time on sperm survival, sucrose and KCl as extender components and the osmolality of the thawing solution. Aquaculture, 32 , 321-330. Terner C. (1986) Evaluation of salmonid sperm motility for cryopreservation. The Progressive Fish Culturist 48, 230-232. Zhang, X. & Liu, Y. (1991) Study of cryopreservation of fish spermatozoa. Acta Scientiarum Naturalium Universitatis Normalis Hunanensis, 14 (3) 255-259.

Table 1. Spermatological properties of collected samples Total Concentration spermatzoa (x109/ml) number (x109) 24.625 591

Sample no

Volume (ml)

Motility (%)

Motility Duration (min:s)

1

24

90

10:35

2

40

95

11:26

12.375

495

8.0

3

18

80

7:24

16.925

304.6

7.5

4

11

90

9:05

18.750

206.2

8.0

5

2

70

5:18

22.800

45.6

8.0

6

1

90

14:18

18.325

18.3

7.5

7

9

90

5:15

23.500

211.5

8.0

8

14

95

10:30

16.100

225.4

8.0

9

8

90

8:16

22.700

181.6

8.0

10

12

90

15:30

21.250

255.0

8.0

11

20

75

8:28

12.400

248.0

8.5

12

9

90

13:40

11.800

106.2

8.5

13

7

90

5:50

13.850

96.9

8.5

14

16

95

12:10

12.900

206.4

8.0

15

8

95

12:10

11.700

93.6

8.5

9.31±0.90

17.33±1.22

X±sx n=15

13.26±2.51 88.33±1.68

pH 8.0

219.02±40.4 8.06±0.08

Table 2. Postthaw motility and movement duration of cryopreserved samples -

Extender*

Cryoprotectant %15 DMSO

Extender 1 b

%15 DMA %15 Glycerol

%15 DMSO Extender 2 a

%15 DMA %15 Glycerol

Motility (%) 35 a 10 a 30 a

Motility Duration** (min:s) 1:18 c 0:10 b 2:00 a

60 a 50 a 40 a

4:10 c 3:03 b 3:10 b

*: P