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Milt Properties and Sperm Cryopreservation in Filefish ( Thamnaconus modestus )

Thesis for the Degree of Master of Science UR Interdisciplinary Program of Fisheries and Oceanography Graduate School. Milt Properties and Sperm Cryopreservation in Filefish ( Thamnaconus modestus ). Presenter: Le Minh Hoang Advisor: Young Jin Chang. Introduction.

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Milt Properties and Sperm Cryopreservation in Filefish ( Thamnaconus modestus )

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  1. Thesis for the Degree of Master of Science UR Interdisciplinary Program of Fisheries and Oceanography Graduate School Milt Properties and Sperm Cryopreservation in Filefish (Thamnaconus modestus) Presenter: Le Minh Hoang Advisor: Young Jin Chang

  2. Introduction • Filefish, Thamnaconus modestus: • Small member of family Monacanthidae • Inhabiting in the bottom of seawater • Distribution throughout Northwest Pacific Ocean • Annual yield of filefish has gradually decreased • Milt properties provide a basic knowledge for understanding: • Physiology of spermatozoa and determination of diluents constituent • To make artificial seminal plasma for fish sperm preservation

  3. Fine structure of spermatozoa provides information for understanding: • Reproductive biology, taxonomic & evolutionary relationships at family level • Artificial reproduction, prevention of polyspermy • Development of sperm cryopreservation techniques • Profits of sperm cryopreservation: • Solving the unbalanced sex ratio and asynchronous spawning season between male and female • Easy transportation of sperm to other place • Easy selective breeding or hybridization and preservation of decreasing indigenous species

  4. Contents • Milt properties: • Physical properties of milt • Biochemical properties of seminal fluid • Fine structure of fresh spermatozoa • Sperm cryopreservation: • Fine structure of cryopreserved spermatozoa • Sperm cryopreservation with different diluents and cryoprotectants

  5. Materials and Methods • Fish and milt collection 5 males (BW:321.0±9.0 g) Spermiation was induce by a single intramuscular injection of hCG 100 IU·kg-1 fish weigh Volume: 2 m3 Temperature: 15.0-16.5℃ Salinity: 32 psu Oxygen: 6-7 mg·L-1 Water flow rate: 0.2 L·s-1 Feed: commercial feed Male filefish Spawning tank Serial waves of abdominal pressure Milt properties analysis Sperm cryopreservation Milt collection and stored with ice

  6. X200 magnification Under microscope CCD camera, video-recorder and image analyzer Sperm concentration • Milt properties analysis Motilities and swimming speed Biochemical property Hemocytometer chamber Samples In 1.5 mL Eppendorf tubes VITRO DTII chemistry system Centrifugation at 15,000 rpm for 10 min Sperm volume pH and Osmolality In 1.5 mL Eppendorf tubes In 75 mm capillary tubes Electronic pH meter and osmometer Spermatocrit

  7. Fine structure of spermatozoa (spz): Immersing Fixing Samples 2.5% glutaraldehyde solution in PBS 0.1M (4oC) 1.0% osmium tetroxide solution in PBS 0.1M (20oC) Dehydrating 20% uranylacetate in 50% ethanol and lead citrate solution Transmission Electronic Microscopy (TEM) Photographed Ethanol from 50% to 100% Post-stained Embedding Copper grids In Epon812 Hardened blocks (0.5-0.7 µm-thick) Mounted Sectioned

  8. Straw method LN2 vapor • Sperm cryopreservation Milt collection Dilution 1:3 with diluent containing cryoprotectant Liquid nitrogen • Diluents: Two kinds of artificial • seminal plasma (ASP1, ASP2), • 0.3M glucose, marine fish • Ringer’s solution (MFRS) • Cryoprotectants: Dimethyl • sulfoxide (DMSO) and • methanol with concentration: • 5%, 10%, 15% and 20% Sperm activity index (SAI) Thawing Motility Swimming speed

  9. Constituents of diluents • Statistical analysis • Data were as means ± SE. Statistical evaluation was performed by one-factor ANOVA (SPSS 16.0). Means were separated using Tukey’s range test and differences were considered to be significant at P<0.05.

  10. 1 2 3 4 Spawning tank Hormone injection Milt collection Milt tube 5 Sperm cryopreservation Milt properties analysis Stored milt with ice 9 7 6 10 11 Physical properties analysis Liquid nitrogen tank Straws method LN2 vapor 8 14 13 12 Biochemical properties analysis Straws thawing and cutting Frozen/thawed sperm Evaluation

  11. Results • Physical properties of milt

  12. Biochemical properties of seminal plasma

  13. C B A n n p m d d m f f m D • Fine structure of spermatozoa Cryopre. spermatozoa Fresh spermatozoa Photographs of spermatozoa of filefish by TEM. A: fresh spermatozoon. B: cross section of mid-piece with five mitochondria. C: cross section of flagellum with 9+2 pattern. D: cryopreserved spermatozoon with ASP1 and 10% DMSO as cryoprotectant. d: distal centriole, f: flagellum, m: mitochondrion, n: nucleus, p: proximal centriole, (scale bar=0.2 µm).

  14. 5 5 10 10 15 15 20 20 5 5 10 10 15 15 20 20 DMSO (%) DMSO(%) Methanol (%) Methanol(%) ASP1 ASP2 • Sperm cryopreservation in ASP1 and ASP2

  15. 5 5 10 10 15 15 20 20 5 5 10 10 15 15 20 20 DMSO (%) DMSO (%) Methanol (%) Methanol (%) MFRS Glucose • Sperm cryopreservation in MFRS and 0.3 M glucose

  16. Discussion • Milt properties in filefish were different to compare with other fish species. These could be related to many factors: • Age, weight, sampling period and method (Suquet et al., 1994) • Ecology and spawning behavior (Piironen and Hyvärien, 1983) • Milt properties and composition of their plasma were subject to change with (Chang, 1997): • Season and frequency of milt collection • Age, size and species as well as condition of objective fish • Several studies have indicated that seminal plasma composition (Alavi and Cosson, 2006): • Not only shows inter-specific variation but also varies among different groups of fish of same species

  17. Fine structure of fish spermatozoa: • Filefish spz is similar to spz of teleost fishes: usually without acrosome (Afzelius, 1978) • Head of filefish spz was small (1-1.3 µm). Other teleost fishes spz (2-4 µm) (Jamiesion, 1991) • Different shapes of spz head: northern pike (ball-shaped), cardinal fish (ovoid-shaped), Atlantic eel (banana-shaped) and filefish (horseshoe-shaped) • Flagellum of spz in filefish “9+2” pattern but “9+0” pattern in spz of Atlantic eel (Jamiesion, 1991) • There was a small alteration of shape in cryopreserved spermatozoa compared with fresh spermatozoa

  18. Successful fish sperm cryopreservation depends on: • Quality of sperm before cryopreservation • Diluent, cryoprotectant, equilibrium time, freezing rate, storage procedure and thawing rate • Diluents: • Play an important role as the regulator on osmotic pressure, pH and ion component etc • Must maintain sperm alive but immotile prior to freezing • Various components and efficacy from species to species • For example: Atlantic salmon (0.3Mglucose) (Stoss and Refstie, 1983), grey mullet (MFRS) (Chao et al., 1975) • In the case of filefish: ASP1 and ASP2 were better than MFRS and 0.3M glucose

  19. Cryoprotectants: • Protect sperm cells from damage during to freezing and thawing • Characteristic: water soluble and low toxic • DMSO, methanol, glycerol and propylene glycol are commonly used • Effectiveness of it varies with different species • For example: tilapia (Chao et al., 1987) and zebra fish (Harvey et al., 1982) (methanol); striped bass (Kerby, 1983), rainbow trout (Stoss and Holtz, 1981) and grouper (Wither and Lim, 1982) (DMSO) • In the case of filefish: DMSO was better than methanol

  20. Conclusion • Physical properties of milt in filefish had 0.3±0.1 mL∙individual-1 in sperm volume, 2.6±0.1x107 spermatozoa∙mL-1 in sperm concentration and 73.3±6.7% in spermatocrit • Biochemical properties of seminal fluid in filefish contained 9.8±0.9 mEq∙L-1 potassium, 164.0±4.0 mEq∙L-1 sodium, 151.0±1.2 mEq∙L-1 chloride, 14.9±0.6 mEq∙L-1 calcium, 7.2±0.1 magnesium, 1.0 mg∙dL-1 glucose, 0.1 g∙dL-1 total protein, 1.0 mg∙dL-1 total lipid, 322.8±2.8 mOsm∙kg-1 osmolality and 7.7±0.1 pH

  21. Spermatozoa of filefish consisted head without acrosome, mid-piece and tail. The shape of spermatozoa head was horseshoe shaped in longitudinal section with 1.3-1.6 µm long and 1.0-1.3 µm wide • The highest motility (40.5±2.8%) & swimming speed (81.5±4.1 µm∙s-1) in frozen/thawed sperm were observed when sperm was frozen in ASP1 containing 10% DMSO and in ASP2 containing 15% DMSO, respectively

  22. Thank for your attention!

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