Sper m quality and cr yopr eser vation in Pe r c a fluviatilis O - PowerPoint PPT Presentation

Percid Fish Culture – From Research to Production Namur (Belgium), 23 – 24 January, 2008 Sper m quality and cr yopr eser vation in Pe r c a fluviatilis O Linhart 1 , SMH Alavi 1 , M Psenicka 1 , M Rodina 1 , C Rougeot 2 1 University of South Bohemia in Ceske Budejovice Research Institute of Fish Culture and Hydrobiology 389 25 Vodnany, Czech Republic linhart@vurh.jcu.cz www.vurh.jcu.cz/linhart/linhart.htm 2 University of Liege, Aquaculture research and Education Center, B-4500 Tihange, Belgium

University of South Bohemia Research Institute of Fish Culture and Hydrobiology Vodnany - Czech Republic

www.vurh.jcu.cz

Fish Sperm is Immotile in Seminal Plasma

Cascade of events at motility triggering of fresh water species timing Transfer of sperm in fresh water Zero time Decerase of external osmolality Triggering of osmoregulation Decrease of internal osmolality Rice of internal ionic concentration Less than Switch on of dynenin activity one second ATP store decreases Internal ionic concentration too low for dynein activity One to several minutes Motility stops

General Characteristics of Fish Sperm Motile cells(%) Velocity Beat frequency ATP Sperm parameters Time after activation of sperm motility

Aims of this study Determining biological aspects of the spermatozoa Sperm volume and density Seminla plasma ionic contents Seminal plasma osmolality Provide us with gamete management in hatchery Effects of ions and osmolality on sperm motility For understanding of mechanism of initiation of sperm motility Cryopreservation of sperm of normal and neomales For applied aquaculture porposes and gene resource banking

Methods After Sperm and Eggs Collections Ionic composition Sperm Density Sperm Motility Hatching test and osmolality Experimental Counting Dark-filed Centrifugation of Semen Haemocytometer chamber incubation trays 3000 rpm for 3 min microscope 10000 rpm for 10 min •testicular sperm of neomales (TSN) cryopreservation •stripped sperm of normal males (SSNM) - sperm quality

Method for Analysing Sperm Motility •TSN/SSNM were diluted in tube 1:200 with 300 mM glucose solution or 200 mM NaCl •1 µl prediluted sperm with 49 µl of 30 mM NaCl (or others) solution with 0.1 % of BSA •final dilution on activated sperm was 1:10000 •10 s after mixing a video recording was made for 1-min •Velocity and motility were assessed at 10, 15, 20, 25, 30, 35 and 40 s post activation

Determination of the freezing conditions Determination of the freezing conditions and hatching hatching test test and Collection of sperm, short-term storage as minimum as possible at 0 o C, then diluted with 300 mM glucose 1+6 and no equilibrated added 10 % of methanol, Control of transferred to 0.5 ml spermatozoa 0.5 ml straws , no equilibration motility 90 % equilibration straws •Styrofoam box with liquid nitrogen • Styrofoam box with liquid nitrogen •3 3- -cm cm- -high floating frame high floating frame • •Freezing 10 minutes Freezing 10 minutes • •Plunged into liquid nitrogen Plunged into liquid nitrogen • Finally, transfer to liquid nitrogen Hatching rate – 1 g (451) eggs+ fresh and thawed Thawing Thawing - - TSN/SSNM 120000 or 240000 per egg + 1 ml water w ater bath bath hatchery water + 1 min shaking; incubation 15 o C; at 40 o o C for C for at 40 hatching rate calculated from total eggs 8 s and and used used 8 s fertilization fertilization

Sperm Morphology and Fine Structure (Lahnstainer et al., 1995) Head 1.87 µm 1 mitochondria Lenght around 20 µm (Ginsburg 1968) Head 1.7 µm Lenght 54 µm

Comparison sperm of perch and barb Perch Barbus barbus

Sperm Volume and Density, Parameters of Seminal Plasma and Sperm Motility Seminal plasma Alavi et al (2007) Lahnsteiner et al (1995) 130.97 ± 2.19 124.01 ± 21.68 Sodium (mM) 106.75 ± 2.37 106.75 ± 2.37 Chloride (mM) 10.70 ± 0.61 10.22 ± 1.11 Potassium (mM) 2.41 ± 0.09 0.72 ± 0.26 Calcium (mM) 298.07 ± 5.09 283.90 ± 37.19 Osmolality (mOsmol Kg -1 ) Percentage of sperm motility (%) 91.90 ± 1.27a 85.00 ± 2.7 15 sec post sperm activation 35.49 ± 2.17b 30 sec post sperm activation 7.04 ± 0.71c 45 sec post sperm activation Sperm velocity µm/s 115.54 ± 1.25a 122.4 ± 21.9 15 sec post sperm activation 12.96 ± 0.20b 30 sec post sperm activation 6.68 ± 0.20c 45 sec post sperm activation Sperm volume (ml) - 2.75±0.51 Sperm density (x10 9 spz ml -1 ) - 29.19±3.15

Dilution Ratio Effect on Sperm Motility a 100 a a IS25 90 IS50 80 IS100 70 60 b 50 40 a 30 a b 20 10 a a 0 15 30 45 Time post sperm activation (sec) Immobilizing solution containing NaCl 200 mM, NaHCO 3 2.38 mM (osmolality 380 mOsmol Kg -1 ) in a ratio 1:50

Osmolality Effect on Sperm Motility and Velocity b 100 bB 200 Sperm motility % Sperm velocity (_m s -1 ) 80 160 A A 60 120 a 40 80 20 40 0 0 Control 100 200 Osmolality (mOsmol kg -1 )

Potassium Effect on Sperm Motility a a 100 200 a a Sperm motility % E 80 160 Sperm velocity (_m s -1 ) CD BC 60 A 120 40 80 20 40 0 0 Control 5.0 20.0 50.0 Potassium concentration (mM)

Calcium Effects on Sperm Motility aC 200 100 a a a 90 B Sperm motility % 160 80 B Sperm velocity (_m s -1 ) 70 A 120 60 50 80 40 30 40 20 10 0 0 Control 0.5 2.5 5.0 Calcium concentartion (mM)

Inhibitory factor of Sperm Motility in Seminal Plasma In seminal plasma: In seminal plasma: -1 1 Kg - Osmolality: 298.07 mOsmol mOsmol Kg Osmolality: 298.07 + concentrations: 10.70 K + concentrations: 10.70 mM mM K Spermatozoa motility was prevented in medium with Osmolality > 300mOsmol kg -1 The major inhibitory factor of sperm motility is osmolality of the seminal plasma

Mechanism of Initiation of Sperm Motility

Stripped sperm and TSN

SSNM Stripped sperm and TSN TSN

Sperm motility fresh SSNM a TSN

Sperm velocity fresh SSNM a TSN

Hatching rate with thawed sperm of SSNM a TSN

Acknowledgments to my collegues David Gela S.M.Hadi Alavi Boryshpolets Sergey Marek Rodina Martin Psenicka Jana Jana Peknicova Nebesa ř ová Martin Hulak Jacky Cosson Vojta Kašpar Li Ping

Thanks for your attention!

Future Studies on Perch Sperm Biology

Perca fluviatilis Sperm quality

Sperm Volume and Density, Parameters of Seminal Plasma and Sperm Motility Parameter Mean±S.E.M Sperm volume (ml) 2.75±0.51 Sperm density (x10 9 spz ml -1 ) 29.19±3.15 Sodium (mM) 130.97±2.19 Chloride (mM) 106.75±2.37 Potassium (mM) 10.70±0.61 Calcium (mM) 2.41±0.09 Osmolality (mOsmol kg -1 ) 298.07±5.09 Percentage of motility (%) 91.90±1.27 a 15 sec 35.49±2.17 b 30 sec 7.04±0.71 c 45 sec Sperm velocity ( μ m s -1 ) 115.54±1.25 a 15 sec 12.96±0.20 b 30 sec 6.68±0.20 c 45 sec

Published Articles Alavi, S.M.H., Rodina, M., Policar, T., Kozak, P., Psenicka, M., Linhart, O. 2007. Semen of Perca fluviatilis L: Sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm motility. Theriogenology, 68: 276-283. Alavi, S.M.H., Rodina, M., Policar, T., Cosson, J., Kozak, P., Psenicak, M., Linhart, O., 2007. Physiology and behavior of stripped and testicular sperm in Perca fluviatilis L. 1758. Cybium. In press. Rodina, M., Policar, T., Linhart, O., Redought, C., 2007. Cryopreservation of sperm of normal and nemales Perca fluviatilis Journal of Applied Ichthyology, In press

Members

The 1st International Workshop on the Biology of Fish Sperm Vodnany, Czech Republic, 29 - 31 August 2007 Proceedings in the Journal of Applied Ichthyology The Second will be Held in Spain (University of Valencia), 2009

Broodfish and Collection of Sperm