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

Sper m quality and cr yopr eser vation in Pe r

c a fluviatilis

O Linhart 1, SMH Alavi 1, M Psenicka1, M Rodina1, C Rougeot2

1University 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

2University of Liege, Aquaculture research and Education

Center, B-4500 Tihange, Belgium

Percid Fish Culture – From Research to Production

Namur (Belgium), 23 – 24 January, 2008

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

Transfer of sperm in fresh water Decerase of external osmolality Triggering of osmoregulation Decrease of internal osmolality Rice of internal ionic concentration Switch on of dynenin activity ATP store decreases Internal ionic concentration too low for dynein activity Motility stops timing

Zero time

Less than

• ne second

One to several minutes

General Characteristics of Fish Sperm

Time after activation of sperm motility Sperm parameters

Motile cells(%) Velocity Beat frequency ATP

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

Centrifugation of Semen 3000 rpm for 3 min 10000 rpm for 10 min

Ionic composition and osmolality

Sperm Density

Sperm Motility

Counting

Haemocytometer chamber

Dark-filed microscope

Hatching test

Experimental incubation trays

• testicular sperm of neomales (TSN)
• stripped sperm of normal males (SSNM) - sperm quality

cryopreservation

Method for Analysing Sperm Motility

• 1 µl prediluted sperm with 49

µl of 30 mM NaCl (or others) solution with 0.1 % of BSA

• TSN/SSNM were diluted in

tube 1:200 with 300 mM glucose solution or 200 mM NaCl

• 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

added 10 % of methanol, transferred to 0.5 ml 0.5 ml straws straws, no equilibration equilibration

• 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

Determination of the freezing conditions Determination of the freezing conditions and and hatching hatching test test

Control of spermatozoa motility 90 %

Thawing Thawing -

• w

water ater bath bath at 40 at 40 o

• C for

C for 8 s 8 s and and used used fertilization fertilization Collection of sperm, short-term storage as minimum as possible at 0 oC, then diluted with 300 mM glucose 1+6 and no equilibrated Hatching rate – 1 g (451) eggs+ fresh and thawed TSN/SSNM 120000 or 240000 per egg + 1 ml hatchery water + 1 min shaking; incubation 15 oC; hatching rate calculated from total eggs

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

Barbus barbus Perch

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

Sperm Volume and Density, Parameters of Seminal Plasma and Sperm Motility Sperm volume (ml) - 2.75±0.51 Sperm density (x109 spz ml-1) - 29.19±3.15

Dilution Ratio Effect on Sperm Motility

a a a b b a a a a

10 20 30 40 50 60 70 80 90 100 15 30 45 Time post sperm activation (sec)

IS25 IS50 IS100

Immobilizing solution containing NaCl 200 mM, NaHCO3 2.38 mM (osmolality 380 mOsmol Kg-1) in a ratio 1:50

Osmolality Effect on Sperm Motility and Velocity

bB b a A A

20 40 60 80 100

Control 100 200 Osmolality (mOsmol kg-1)

40 80 120 160 200 Sperm velocity (_m s-1)

Sperm motility %

Potassium Effect on Sperm Motility

a a a a A BC CD E

20 40 60 80 100 Control 5.0 20.0 50.0 Potassium concentration (mM) 40 80 120 160 200

Sperm velocity (_m s-1)

Sperm motility %

Calcium Effects on Sperm Motility

a aC a a B A B 10 20 30 40 50 60 70 80 90 100

Control 0.5 2.5 5.0

Calcium concentartion (mM) 40 80 120 160 200 Sperm velocity (_m s-1)

Sperm motility %

Inhibitory factor of Sperm Motility in Seminal Plasma

In seminal plasma: In seminal plasma:

Osmolality: 298.07 Osmolality: 298.07 mOsmol mOsmol Kg Kg-

• 1

1

K K+

+ concentrations: 10.70

concentrations: 10.70 mM mM

The major inhibitory factor of sperm motility is osmolality of the seminal plasma

Spermatozoa motility was prevented in medium with Osmolality > 300mOsmol kg-1

Mechanism of Initiation of Sperm Motility

Stripped sperm and TSN

Stripped sperm and TSN

TSN SSNM

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

Marek Rodina David Gela Jana Peknicova

Martin Psenicka

S.M.Hadi Alavi Martin Hulak Jacky Cosson Jana Nebesařová

Vojta Kašpar

Boryshpolets Sergey 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 (x109 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 (%) 15 sec 91.90±1.27 a 30 sec 35.49±2.17 b 45 sec 7.04±0.71 c Sperm velocity (μm s-1) 15 sec 115.54±1.25 a 30 sec 12.96±0.20 b 45 sec 6.68±0.20 c

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