Elsevier Editorial System(tm) for Cryobiology Manuscript Draft Manuscript Number CRYO-D-12-00078R1 Title Hypotonic treatment prior to freezing improves spermatozoa survival to cryoinjuries in common carp (Cyprinus carpio L) Article Type Brief Communication Keywords Carp sperm hypotonic treatment cryoresistance Corresponding Author Dr Boris Dzyuba Corresponding Authors Institution IPCampC NAS First Author Boris Dzyuba Order of Authors Boris Dzyuba Jacky Cosson Gunes Yamaner Marek Rodina David Gela Volodymir Bondarenko Anna Shaliutina Otomar Linhart Abstract Our results revealed new findings that post-thaw spermatozoa motility percent curvilinear velocity (VCL) and fertilizing ability parameters of carp sperm could be improved by application of a short-time sperm treatment with moderately hypotonic media prior to freezing Before cryopreservation carp sperm samples were treated with NaCl solutions of different osmolality ranging from 100 to 300 mOsmkg-1 then 10 seconds after final osmolality was adjusted to 300mOsmkg-1 Thereafter the resultant sperm suspension was diluted 11 with cryoprotective medium and frozen using conventional freezing techniques Control samples were submitted to the same protocol except that the predilution step was omittedPost-thaw motility percent in samples pretreated by 200 mOsmkg1 NaCl was significantly higher (44plusmn10 ) than in control (21plusmn15 ) or samples pretreated by 100 mOsmkg-1 (25plusmn15 ) or 300 mOsmkg-1 (25plusmn12 ) NaCl solutions Significantly higher values of mean VCL were observed in samples pretreated by media of 100 150 and 200 mOsmkg-1 (119plusmn 24 118 plusmn22 and 115plusmn 32microms-1 respectively) in comparison with control (92plusmn27 microms-1) Fertilization rate of eggs fertilized by frozen-thawed sperm samples treated with 200 mOsmkg-1 solution with following osmolality increase by 2M NaCl (25plusmn18 ) were significantly higher than in eggs fertilized with sperm samples treated by 300 mOsmkg-1 NaCl solutions (12plusmn7 ) or control (9plusmn6 )

COVERING LETTER

Dear Dr David Rawson

Thank you very much for revising our manuscript

We appreciate the suggestions from the referees and hope our answers to their questions

will improve the manuscript

Please find our answers bellow

Yours sincerely

Boris Dzyuba

THE ANSWERS TO REFEREES COMMENTS

Reviewer 1 The paper is generally well written

The only clarification I would like to have is what is the osmolality of the activation

medium (line 94)

Boris Dzyuba (BD)

Added to the text

ldquo(147 mOsmkg-1

)rdquo Line (L) 114-115

Reviewer 2 Hypertonic treatment prior to freezing improves spermatozoa survival to

cryoinjuries in common carp (Cyprinus carpio)

The manuscript reports that cryoresistance of carp sperm can be improved by short term

exposure to hypertonic media with an osmolality of 200 mosmlkg an osmolality which

is circa 70 mosmolkg lower than the osmolality of the seminal plasma

These results are of general interest As also stated by the authors they are preliminary

however they might be used as a base for further investigations

The fertilization rates are very low and for samples pre-treated with 200 mosmolkg NaCl

also highly variable It is uncertain if they are representative One should consider that

not only too high sperm to egg ratios are non-discriminate but also too low and they

might mask the effect of experiments

(BD)

Regarding statistics please see my answer below on ldquoSpecific commentsrdquo

Added to the text of discussion

Response to Reviewers

ldquoSince in our study quite low spermeggs ratio was used it should be

considered that applied in our study too low spermegg ratio might

mask the effect of experimentsrdquo L 177-178

For carp sperm cryopreservation there exist several efficient and optimized protocols As

seen from the results and as stated by the authors themselves not the optimal protocol

was used The intention is difficult to understand as several of the authors are experts in

sperm cryopreservation This failure reduces the value of the study

(BD)

Added to text of Introduction

rdquoWe hypothesized that results of carp sperm cryopreservation could be

improved by sperm hypotonic treatment prior to freezing and focused

our study exactly to this point rather than the selection of optimal

cryopreservation method required for available for us males from our

own facilitiesrdquo L71-74

Specific comments

The state of art (the optimal cryopreservation methods postthaw motility and fertility

rates) should be shortly summarized in introduction

(BD)

Added to text of Introduction

ldquoFor this reason many cryobiological studies were conducted in carp

sperm These studies resulted in the designing of some cryoprotective

media cryoprotectants and freezingthawing protocols allowing the

achievement of quite a high sperm post-thaw motility parameters and

fertilization rate [58] However the selection (or even modification) of

optimal method for certain breeders is sometimes required because of

huge diversity of sperm cryoresistance arising probably from

conditions of malesrsquo maintaining before stripping [6]rdquo L 57-62

Generally the sample number is low for this kind of experiment considering that in carp

neither semen quantities nor male fish are limiting

(BD)

We consider that low sample number (n=5) could be used in pilot study

because non-parametric statistical analysis applied for small ldquonrdquo

allows a correct estimation of the significance of observed differences

This is mentioned in the text L 142-145

Semen samples with 270 mosmolkg were diluted 1 4 with solutions of different

osmolalities (200 250 and 300 mosmolkg) What was the final osmolality For

dilutions with 200 250 and 300 mosmolkg the osmolality differences to undiluted semen

should be relatively low (I estimate in the range of 30 mosomlkg and even lower) and

close to the range of osmolality variations of fresh semen This should be discussed

(BD)

The values of osmolality for resultant sperm suspension is added to text

ldquoAfter this dilution the resultant cell suspension osmolalities were

within the ranges of 132-137 174-177 214-217 254-257 294-298

mOsmkg-1

respectively (min-max values within a range correspond to

the sperm sample with lowest and highest osmolalities)rdquo L 87-90

The seminal plasma osmolality was 270 mosmolkg Why was semen rediluted to 300

mosmolkg The discrepancy is high when considering the slight osmolality changes due

to predilution Should also be explained better

(BD)

Added to text ldquoThis osmolality was selected as a minimum one required

for immediate sperm motility stop for a sample pre-diluted with any

media used in the experimentsrdquo L 91-93

What was the time span between re-dilution and cryopreservation

(BD)

Added to the text

rdquoTime span between sperm samples treatment and freezing onset was

about 15 minrdquo L 97-98

What were the final dilution ratios of semen Seminal plasma may have a protective

effect and to high dilution ratios may decrease sperm viability

(BD)

Added to text

ldquoFinal dilution ratio was 1 9 This dilution rate was quite low to avoid

the loss of possible protective effect of seminal plasma [8]rdquo L 100-101

Here the reference [8] (F Lahnsteiner B Berger A Horvath B

Urbanyi T Weismann Theriogenology 54 (2000) 1477ndash1498) was

changed to the reference ldquoF Lahnsteiner B Berger T Weismann

Theriogenology 60 (2003) 829-841rdquo This citation is required as the most

informative for our ldquoBrief Communicationrdquo paper (restrictions in the

rules for authors 10 publications maximum)

The temperature for diluents motility activation and handling is missing

(BD)

COVERING LETTER

Dear Dr David Rawson

Thank you very much for revising our manuscript

We appreciate the suggestions from the referees and hope our answers to their questions

will improve the manuscript

Please find our answers bellow

Yours sincerely

Boris Dzyuba

THE ANSWERS TO REFEREES COMMENTS

Reviewer 1 The paper is generally well written

The only clarification I would like to have is what is the osmolality of the activation

medium (line 94)

Boris Dzyuba (BD)

Added to the text

ldquo(147 mOsmkg-1

)rdquo Line (L) 114-115

Reviewer 2 Hypertonic treatment prior to freezing improves spermatozoa survival to

cryoinjuries in common carp (Cyprinus carpio)

The manuscript reports that cryoresistance of carp sperm can be improved by short term

exposure to hypertonic media with an osmolality of 200 mosmlkg an osmolality which

is circa 70 mosmolkg lower than the osmolality of the seminal plasma

These results are of general interest As also stated by the authors they are preliminary

however they might be used as a base for further investigations

The fertilization rates are very low and for samples pre-treated with 200 mosmolkg NaCl

also highly variable It is uncertain if they are representative One should consider that

not only too high sperm to egg ratios are non-discriminate but also too low and they

might mask the effect of experiments

(BD)

Regarding statistics please see my answer below on ldquoSpecific commentsrdquo

Added to the text of discussion

Response to Reviewers

ldquoSince in our study quite low spermeggs ratio was used it should be

considered that applied in our study too low spermegg ratio might

mask the effect of experimentsrdquo L 177-178

For carp sperm cryopreservation there exist several efficient and optimized protocols As

seen from the results and as stated by the authors themselves not the optimal protocol

was used The intention is difficult to understand as several of the authors are experts in

sperm cryopreservation This failure reduces the value of the study

(BD)

Added to text of Introduction

rdquoWe hypothesized that results of carp sperm cryopreservation could be

improved by sperm hypotonic treatment prior to freezing and focused

our study exactly to this point rather than the selection of optimal

cryopreservation method required for available for us males from our

own facilitiesrdquo L71-74

Specific comments

The state of art (the optimal cryopreservation methods postthaw motility and fertility

rates) should be shortly summarized in introduction

(BD)

Added to text of Introduction

ldquoFor this reason many cryobiological studies were conducted in carp

sperm These studies resulted in the designing of some cryoprotective

media cryoprotectants and freezingthawing protocols allowing the

achievement of quite a high sperm post-thaw motility parameters and

fertilization rate [58] However the selection (or even modification) of

optimal method for certain breeders is sometimes required because of

huge diversity of sperm cryoresistance arising probably from

conditions of malesrsquo maintaining before stripping [6]rdquo L 57-62

Generally the sample number is low for this kind of experiment considering that in carp

neither semen quantities nor male fish are limiting

(BD)

We consider that low sample number (n=5) could be used in pilot study

because non-parametric statistical analysis applied for small ldquonrdquo

allows a correct estimation of the significance of observed differences

This is mentioned in the text L 142-145

Semen samples with 270 mosmolkg were diluted 1 4 with solutions of different

osmolalities (200 250 and 300 mosmolkg) What was the final osmolality For

dilutions with 200 250 and 300 mosmolkg the osmolality differences to undiluted semen

should be relatively low (I estimate in the range of 30 mosomlkg and even lower) and

close to the range of osmolality variations of fresh semen This should be discussed

(BD)

The values of osmolality for resultant sperm suspension is added to text

ldquoAfter this dilution the resultant cell suspension osmolalities were

within the ranges of 132-137 174-177 214-217 254-257 294-298

mOsmkg-1

respectively (min-max values within a range correspond to

the sperm sample with lowest and highest osmolalities)rdquo L 87-90

The seminal plasma osmolality was 270 mosmolkg Why was semen rediluted to 300

mosmolkg The discrepancy is high when considering the slight osmolality changes due

to predilution Should also be explained better

(BD)

Added to text ldquoThis osmolality was selected as a minimum one required

for immediate sperm motility stop for a sample pre-diluted with any

media used in the experimentsrdquo L 91-93

What was the time span between re-dilution and cryopreservation

(BD)

Added to the text

rdquoTime span between sperm samples treatment and freezing onset was

about 15 minrdquo L 97-98

What were the final dilution ratios of semen Seminal plasma may have a protective

effect and to high dilution ratios may decrease sperm viability

(BD)

Added to text

ldquoFinal dilution ratio was 1 9 This dilution rate was quite low to avoid

the loss of possible protective effect of seminal plasma [8]rdquo L 100-101

Here the reference [8] (F Lahnsteiner B Berger A Horvath B

Urbanyi T Weismann Theriogenology 54 (2000) 1477ndash1498) was

changed to the reference ldquoF Lahnsteiner B Berger T Weismann

Theriogenology 60 (2003) 829-841rdquo This citation is required as the most

informative for our ldquoBrief Communicationrdquo paper (restrictions in the

rules for authors 10 publications maximum)

The temperature for diluents motility activation and handling is missing

(BD)

ldquoSince in our study quite low spermeggs ratio was used it should be

considered that applied in our study too low spermegg ratio might

mask the effect of experimentsrdquo L 177-178

For carp sperm cryopreservation there exist several efficient and optimized protocols As

seen from the results and as stated by the authors themselves not the optimal protocol

was used The intention is difficult to understand as several of the authors are experts in

sperm cryopreservation This failure reduces the value of the study

(BD)

Added to text of Introduction

rdquoWe hypothesized that results of carp sperm cryopreservation could be

improved by sperm hypotonic treatment prior to freezing and focused

our study exactly to this point rather than the selection of optimal

cryopreservation method required for available for us males from our

own facilitiesrdquo L71-74

Specific comments

The state of art (the optimal cryopreservation methods postthaw motility and fertility

rates) should be shortly summarized in introduction

(BD)

Added to text of Introduction

ldquoFor this reason many cryobiological studies were conducted in carp

sperm These studies resulted in the designing of some cryoprotective

media cryoprotectants and freezingthawing protocols allowing the

achievement of quite a high sperm post-thaw motility parameters and

fertilization rate [58] However the selection (or even modification) of

optimal method for certain breeders is sometimes required because of

huge diversity of sperm cryoresistance arising probably from

conditions of malesrsquo maintaining before stripping [6]rdquo L 57-62

Generally the sample number is low for this kind of experiment considering that in carp

neither semen quantities nor male fish are limiting

(BD)

We consider that low sample number (n=5) could be used in pilot study

because non-parametric statistical analysis applied for small ldquonrdquo

allows a correct estimation of the significance of observed differences

This is mentioned in the text L 142-145

Semen samples with 270 mosmolkg were diluted 1 4 with solutions of different

osmolalities (200 250 and 300 mosmolkg) What was the final osmolality For

dilutions with 200 250 and 300 mosmolkg the osmolality differences to undiluted semen

should be relatively low (I estimate in the range of 30 mosomlkg and even lower) and

close to the range of osmolality variations of fresh semen This should be discussed

(BD)

The values of osmolality for resultant sperm suspension is added to text

ldquoAfter this dilution the resultant cell suspension osmolalities were

within the ranges of 132-137 174-177 214-217 254-257 294-298

mOsmkg-1

respectively (min-max values within a range correspond to

the sperm sample with lowest and highest osmolalities)rdquo L 87-90

The seminal plasma osmolality was 270 mosmolkg Why was semen rediluted to 300

mosmolkg The discrepancy is high when considering the slight osmolality changes due

to predilution Should also be explained better

(BD)

Added to text ldquoThis osmolality was selected as a minimum one required

for immediate sperm motility stop for a sample pre-diluted with any

media used in the experimentsrdquo L 91-93

What was the time span between re-dilution and cryopreservation

(BD)

Added to the text

rdquoTime span between sperm samples treatment and freezing onset was

about 15 minrdquo L 97-98

What were the final dilution ratios of semen Seminal plasma may have a protective

effect and to high dilution ratios may decrease sperm viability

(BD)

Added to text

ldquoFinal dilution ratio was 1 9 This dilution rate was quite low to avoid

the loss of possible protective effect of seminal plasma [8]rdquo L 100-101

Here the reference [8] (F Lahnsteiner B Berger A Horvath B

Urbanyi T Weismann Theriogenology 54 (2000) 1477ndash1498) was

changed to the reference ldquoF Lahnsteiner B Berger T Weismann

Theriogenology 60 (2003) 829-841rdquo This citation is required as the most

informative for our ldquoBrief Communicationrdquo paper (restrictions in the

rules for authors 10 publications maximum)

The temperature for diluents motility activation and handling is missing

(BD)

should be relatively low (I estimate in the range of 30 mosomlkg and even lower) and

close to the range of osmolality variations of fresh semen This should be discussed

(BD)

The values of osmolality for resultant sperm suspension is added to text

ldquoAfter this dilution the resultant cell suspension osmolalities were

within the ranges of 132-137 174-177 214-217 254-257 294-298

mOsmkg-1

respectively (min-max values within a range correspond to

the sperm sample with lowest and highest osmolalities)rdquo L 87-90

The seminal plasma osmolality was 270 mosmolkg Why was semen rediluted to 300

mosmolkg The discrepancy is high when considering the slight osmolality changes due

to predilution Should also be explained better

(BD)

Added to text ldquoThis osmolality was selected as a minimum one required

for immediate sperm motility stop for a sample pre-diluted with any

media used in the experimentsrdquo L 91-93

What was the time span between re-dilution and cryopreservation

(BD)

Added to the text

rdquoTime span between sperm samples treatment and freezing onset was

about 15 minrdquo L 97-98

What were the final dilution ratios of semen Seminal plasma may have a protective

effect and to high dilution ratios may decrease sperm viability

(BD)

Added to text

ldquoFinal dilution ratio was 1 9 This dilution rate was quite low to avoid

the loss of possible protective effect of seminal plasma [8]rdquo L 100-101

Here the reference [8] (F Lahnsteiner B Berger A Horvath B

Urbanyi T Weismann Theriogenology 54 (2000) 1477ndash1498) was

changed to the reference ldquoF Lahnsteiner B Berger T Weismann

Theriogenology 60 (2003) 829-841rdquo This citation is required as the most

informative for our ldquoBrief Communicationrdquo paper (restrictions in the

rules for authors 10 publications maximum)

The temperature for diluents motility activation and handling is missing

(BD)

Added to text ldquoAll the procedures of pre-dilution prior to freezing were

carried out at 4ordmC and motility was analyzed at room temperature (18-

20ordmC) ldquo L 105-106

It is described that KCl and NaCl are used for dilution However the procedure is not

described clearly and the results are shown only partly (eg Fig 1)

(BD)

Motility parameters were studied in sperm samples treated with 100

150 200 250 300 mOsmkg-1

media while fertilization tests were

performed for the samples treated with 100 200 300 mOsmkg-1

media

Added to text

rdquoFor fertilization tests the sperm samples were treated prior to

cryopreservation with 100 200 and 300 mOsmkg-1 (as described

previously) following osmolality increase by 2 M KCl or 2 M NaCl The

use of KCl or NaCl was applied for elucidation the question rdquoWhether

use of Na+ or K

+ ions for sperm treatment prior to cryopreservation

could influence the fertilization rate in the present experimentsrdquo L

106-111

The motility analysis procedure needs clear description and definition of analyzed

parameters

(BD)

Added to text

ldquoVideo records were analyzed to estimate spermatozoa curvilinear

velocity (VCL) percent of motile cells (motility) by microimage

analyzer (Olympus Micro Image 401 for Windows) Microimage

analyzer allows to measure VCL and motility by overlapping five

successive video frames Due to this the overlapping tracks of

spermatozoa heads became visible allowing the calculation of VCL

(defined as total point-to-point distance traveled by the sperm over the

time period between the first and fifth frames) and motility percentage

[9]rdquo L118-124

Hypotonic treatment prior to freezing improves spermatozoa survival to cryoinjuries in common 1

carp (Cyprinus carpio L) 2

3

Borys Dzyuba1 Jacky Cosson

1 Gunes Yamaner

2 Olga Bondarenko

1 Marek Rodina

1 David 4

Gela1 Volodymir Bondarenko

1 Anna Shaliutina

1 and Otomar Linhart

1 5

6

7

1ndash Faculty of Fisheries and Protection of Waters University of South Bohemia in Ceske 8

Budejovice South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses 9

Vodnany Czech Republic 10

2 ndash Istanbul University Faculty of Fisheries BeyazitEminonu 34452 Istanbul Turkey 11

12

13

14

Corresponding author Tel +42 0773476453 fax (420) 387 774 634 15

e-mail bdzyubafrovjcucz (B Dzyuba) 16

17

University of South Bohemia in Českeacute Budějovice Faculty of Fisheries and Protection of Waters 18

Research Institute of Fish Culture and Hydrobiology Zaacutetišiacute 728II 389 25 Vodňany Czech 19

Republic 20

21

22

23

24

25

26

27

28

ManuscriptClick here to view linked References

Hypotonic treatment prior to freezing improves spermatozoa survival to cryoinjuries in common 1

carp (Cyprinus carpio L) 2

3

Borys Dzyuba1 Jacky Cosson

1 Gunes Yamaner

2 Olga Bondarenko

1 Marek Rodina

1 David 4

Gela1 Volodymir Bondarenko

1 Anna Shaliutina

1 and Otomar Linhart

1 5

6

7

1ndash Faculty of Fisheries and Protection of Waters University of South Bohemia in Ceske 8

Budejovice South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses 9

Vodnany Czech Republic 10

2 ndash Istanbul University Faculty of Fisheries BeyazitEminonu 34452 Istanbul Turkey 11

12

13

14

Corresponding author Tel +42 0773476453 fax (420) 387 774 634 15

e-mail bdzyubafrovjcucz (B Dzyuba) 16

17

University of South Bohemia in Českeacute Budějovice Faculty of Fisheries and Protection of Waters 18

Research Institute of Fish Culture and Hydrobiology Zaacutetišiacute 728II 389 25 Vodňany Czech 19

Republic 20

21

22

23

24

25

26

27

28

ManuscriptClick here to view linked References

Abstract 29

Our results revealed new findings that post-thaw spermatozoa motility percent curvilinear velocity 30

(VCL) and fertilizing ability parameters of carp sperm could be improved by application of a short-31

time sperm treatment with moderately hypotonic media prior to freezing Before cryopreservation 32

carp sperm samples were treated with NaCl solutions of different osmolality ranging from 100 to 33

300 mOsmkg-1

then 10 seconds after final osmolality was adjusted to 300mOsmkg-1

Thereafter 34

the resultant sperm suspension was diluted 11 with cryoprotective medium and frozen using 35

conventional freezing techniques Control samples were submitted to the same protocol except that 36

the predilution step was omittedPost-thaw motility percent in samples pretreated by 200 37

mOsmkg1 NaCl was significantly higher (44plusmn10 ) than in control (21plusmn15 ) or samples 38

pretreated by 100 mOsmkg-1

(25plusmn15 ) or 300 mOsmkg-1

(25plusmn12 ) NaCl solutions 39

Significantly higher values of mean VCL were observed in samples pretreated by media of 100 150 40

and 200 mOsmkg-1

(119plusmn 24 118 plusmn22 and 115plusmn 32microms-1

respectively) in comparison with 41

control (92plusmn27 microms-1

) Fertilization rate of eggs fertilized by frozen-thawed sperm samples 42

treated with 200 mOsmkg-1

solution with following osmolality increase by 2M NaCl (25plusmn18 ) 43

were significantly higher than in eggs fertilized with sperm samples treated by 300 mOsmkg-1 44

NaCl solutions (12plusmn7 ) or control (9plusmn6 ) 45

46

Keywords 47

Carp sperm hypotonic treatment cryoresistance 48

49

50

51

52

53

54

55

Carp sperm cryopreservation is considered as potentially powerful tool in carp aquaculture 56

and conservation programs [6] For this reason many cryobiological studies were conducted in carp 57

sperm These studies resulted in the designing of some cryoprotective media cryoprotectants and 58

freezingthawing protocols allowing the achievement of quite a high sperm post-thaw motility 59

parameters and fertilization rate [58] However the selection (or even modification) of optimal 60

method for certain breeders is sometimes required because of huge diversity of sperm 61

cryoresistance arising probably from conditions of malesrsquo maintaining before stripping [6] Carp 62

(Cyprinus carpio) which possess hypotonic mode of sperm motility activation provides several 63

advantages for cryobiological studies applied to freshwater fish with external fertilization Previous 64

researches have shown that carp spermatozoa quickly (within one second) respond on environment 65

hypotonicity by activation of their motility which is associated with rearrangement in intracellular 66

ion concentration changes in membrane structure [7] and spermatozoa swelling [3] Additionally 67

these changes could be reversed by osmolality increase leading to immediate motility cessation 68

Therefore carp sperm spermatozoa could be considered as an appropriate model for studies on the 69

relationships between cell membrane parameters changes occurring before freezing and cell 70

cryoresistance We hypothesized that results of carp sperm cryopreservation could be improved by 71

sperm hypotonic treatment prior to freezing and focused our study exactly to this point rather than 72

the selection of optimal cryopreservation method required for available for us males from our own 73

facilities In present study taking into account specific features of carp sperm we have 74

investigated the possibility to manipulate sperm cryoresistance by sperm pre-incubation in solutions 75

of different osmolalities prior to freezing As carp sperm treatment by hypotonic solutions leads to 76

quick cell hydration associated with drastic decrease of ATP content we treated sperm samples for 77

only very brief period and then reversed the surrounding osmolality to isotonic level 78

Sperm from males of common carp Cyprinus carpio was stripped by abdominal massage 79

and collected into plastic syringes 24 h after injection with carp pituitary extract (1 mgkg-1

of 80

body weight) Sperm samples from 5 males possessing 95plusmn3 motility percent and 276plusmn6 81

mOsmkg-1

seminal fluid osmolality were selected for experiments Prior to cryopreservation 82

sperm samples were treated with 5 different solutions (pre-dilution medium) of osmolality 100 150 83

200 250 and 300 mOsmkg-1

containing 10mM Tris-HCl buffer pH 80 and NaCl and applied at a 84

dilution rate 14 Prior to cryopreservation sperm samples were treated with 5 different solutions 85

(pre-dilution medium) of osmolality 100 150 200 250 and 300 mOsmkg-1

containing 10mM 86

Tris-HCl buffer pH 80 and NaCl and applied at a dilution rate 14 After this dilution the resultant 87

cell suspension osmolalities were within the ranges of 132-137 174-177 214-217 254-257 294-88

298 mOsmkg-1

respectively (min-max values within a range correspond to the sperm sample with 89

lowest and highest osmolalities) Ten seconds after dilution a 2 M KCl or 2 M NaCl solution was 90

added to adjust final osmolality to 300mOsmkg-1

This osmolality was selected as a minimum one 91

required for immediate sperm motility stop for a sample pre-diluted with any media used in the 92

experiments The resultant sperm suspension was diluted 11 with cryoprotective medium 93

Cryoprotective medium consisted of 59 mM NaCl 63 mM KCl 068 mM CaCl2 21 mM MgCl2 94

27mM NaHCO3 34 mM sucrose 69 mM manitol 118 mM Tris pH 82 16 ethylene glycol [5] 95

Ten min after dilution sperm samples were frozen in 05 ml straws 3 cm above liquid nitrogen level 96

for 20 min then followed by plunging of straws into liquid nitrogen Time span between sperm 97

samples treatment and freezing onset was about 15 min Sperm samples which were not pre-diluted 98

with any NaCl solutions before freezing were considered as a control group and frozen after sperm 99

dilution with cryprotective medium (dilution rate 11) Final dilution ratio was 1 9 This dilution 100

rate was quite low to avoid the loss of possible protective effect of seminal plasma [8] Frozen 101

sperm samples were thawed at 40degC for 6 s and immediately subjected to estimation of motility 102

parameters and ability for fertilization Post-thaw spermatozoa motility percent and average 103

velocity were estimated for sperm samples treated with 100 150 200 250 and 300 mOsmkg-1

104

osmolality adjusted by adequate addition of 2 M KCl All the procedures of pre-dilution prior to 105

freezing were carried out at 4ordmC and motility was analyzed at room temperature (18-20ordmC) For 106

fertilization tests the sperm samples were treated prior to cryopreservation with 100 200 and 300 107

Carp sperm cryopreservation is considered as potentially powerful tool in carp aquaculture 56

and conservation programs [6] For this reason many cryobiological studies were conducted in carp 57

sperm These studies resulted in the designing of some cryoprotective media cryoprotectants and 58

freezingthawing protocols allowing the achievement of quite a high sperm post-thaw motility 59

parameters and fertilization rate [58] However the selection (or even modification) of optimal 60

method for certain breeders is sometimes required because of huge diversity of sperm 61

cryoresistance arising probably from conditions of malesrsquo maintaining before stripping [6] Carp 62

(Cyprinus carpio) which possess hypotonic mode of sperm motility activation provides several 63

advantages for cryobiological studies applied to freshwater fish with external fertilization Previous 64

researches have shown that carp spermatozoa quickly (within one second) respond on environment 65

hypotonicity by activation of their motility which is associated with rearrangement in intracellular 66

ion concentration changes in membrane structure [7] and spermatozoa swelling [3] Additionally 67

these changes could be reversed by osmolality increase leading to immediate motility cessation 68

Therefore carp sperm spermatozoa could be considered as an appropriate model for studies on the 69

relationships between cell membrane parameters changes occurring before freezing and cell 70

cryoresistance We hypothesized that results of carp sperm cryopreservation could be improved by 71

sperm hypotonic treatment prior to freezing and focused our study exactly to this point rather than 72

the selection of optimal cryopreservation method required for available for us males from our own 73

facilities In present study taking into account specific features of carp sperm we have 74

investigated the possibility to manipulate sperm cryoresistance by sperm pre-incubation in solutions 75

of different osmolalities prior to freezing As carp sperm treatment by hypotonic solutions leads to 76

quick cell hydration associated with drastic decrease of ATP content we treated sperm samples for 77

only very brief period and then reversed the surrounding osmolality to isotonic level 78

Sperm from males of common carp Cyprinus carpio was stripped by abdominal massage 79

and collected into plastic syringes 24 h after injection with carp pituitary extract (1 mgkg-1

of 80

body weight) Sperm samples from 5 males possessing 95plusmn3 motility percent and 276plusmn6 81

mOsmkg-1

seminal fluid osmolality were selected for experiments Prior to cryopreservation 82

sperm samples were treated with 5 different solutions (pre-dilution medium) of osmolality 100 150 83

200 250 and 300 mOsmkg-1

containing 10mM Tris-HCl buffer pH 80 and NaCl and applied at a 84

dilution rate 14 Prior to cryopreservation sperm samples were treated with 5 different solutions 85

(pre-dilution medium) of osmolality 100 150 200 250 and 300 mOsmkg-1

containing 10mM 86

Tris-HCl buffer pH 80 and NaCl and applied at a dilution rate 14 After this dilution the resultant 87

cell suspension osmolalities were within the ranges of 132-137 174-177 214-217 254-257 294-88

298 mOsmkg-1

respectively (min-max values within a range correspond to the sperm sample with 89

lowest and highest osmolalities) Ten seconds after dilution a 2 M KCl or 2 M NaCl solution was 90

added to adjust final osmolality to 300mOsmkg-1

This osmolality was selected as a minimum one 91

required for immediate sperm motility stop for a sample pre-diluted with any media used in the 92

experiments The resultant sperm suspension was diluted 11 with cryoprotective medium 93

Cryoprotective medium consisted of 59 mM NaCl 63 mM KCl 068 mM CaCl2 21 mM MgCl2 94

27mM NaHCO3 34 mM sucrose 69 mM manitol 118 mM Tris pH 82 16 ethylene glycol [5] 95

Ten min after dilution sperm samples were frozen in 05 ml straws 3 cm above liquid nitrogen level 96

for 20 min then followed by plunging of straws into liquid nitrogen Time span between sperm 97

samples treatment and freezing onset was about 15 min Sperm samples which were not pre-diluted 98

with any NaCl solutions before freezing were considered as a control group and frozen after sperm 99

dilution with cryprotective medium (dilution rate 11) Final dilution ratio was 1 9 This dilution 100

rate was quite low to avoid the loss of possible protective effect of seminal plasma [8] Frozen 101

sperm samples were thawed at 40degC for 6 s and immediately subjected to estimation of motility 102

parameters and ability for fertilization Post-thaw spermatozoa motility percent and average 103

velocity were estimated for sperm samples treated with 100 150 200 250 and 300 mOsmkg-1

104

osmolality adjusted by adequate addition of 2 M KCl All the procedures of pre-dilution prior to 105

freezing were carried out at 4ordmC and motility was analyzed at room temperature (18-20ordmC) For 106

fertilization tests the sperm samples were treated prior to cryopreservation with 100 200 and 300 107

mOsmkg-1

seminal fluid osmolality were selected for experiments Prior to cryopreservation 82

sperm samples were treated with 5 different solutions (pre-dilution medium) of osmolality 100 150 83

200 250 and 300 mOsmkg-1

containing 10mM Tris-HCl buffer pH 80 and NaCl and applied at a 84

dilution rate 14 Prior to cryopreservation sperm samples were treated with 5 different solutions 85

(pre-dilution medium) of osmolality 100 150 200 250 and 300 mOsmkg-1

containing 10mM 86

Tris-HCl buffer pH 80 and NaCl and applied at a dilution rate 14 After this dilution the resultant 87

cell suspension osmolalities were within the ranges of 132-137 174-177 214-217 254-257 294-88

298 mOsmkg-1

respectively (min-max values within a range correspond to the sperm sample with 89

lowest and highest osmolalities) Ten seconds after dilution a 2 M KCl or 2 M NaCl solution was 90

added to adjust final osmolality to 300mOsmkg-1

This osmolality was selected as a minimum one 91

required for immediate sperm motility stop for a sample pre-diluted with any media used in the 92

experiments The resultant sperm suspension was diluted 11 with cryoprotective medium 93

Cryoprotective medium consisted of 59 mM NaCl 63 mM KCl 068 mM CaCl2 21 mM MgCl2 94

27mM NaHCO3 34 mM sucrose 69 mM manitol 118 mM Tris pH 82 16 ethylene glycol [5] 95

Ten min after dilution sperm samples were frozen in 05 ml straws 3 cm above liquid nitrogen level 96

for 20 min then followed by plunging of straws into liquid nitrogen Time span between sperm 97

samples treatment and freezing onset was about 15 min Sperm samples which were not pre-diluted 98

with any NaCl solutions before freezing were considered as a control group and frozen after sperm 99

dilution with cryprotective medium (dilution rate 11) Final dilution ratio was 1 9 This dilution 100

rate was quite low to avoid the loss of possible protective effect of seminal plasma [8] Frozen 101

sperm samples were thawed at 40degC for 6 s and immediately subjected to estimation of motility 102

parameters and ability for fertilization Post-thaw spermatozoa motility percent and average 103

velocity were estimated for sperm samples treated with 100 150 200 250 and 300 mOsmkg-1

104

osmolality adjusted by adequate addition of 2 M KCl All the procedures of pre-dilution prior to 105

freezing were carried out at 4ordmC and motility was analyzed at room temperature (18-20ordmC) For 106

fertilization tests the sperm samples were treated prior to cryopreservation with 100 200 and 300 107

mOsmkg-1

(as described previously) following osmolality increase by 2 M KCl or 2 M NaCl The 108

use of KCl or NaCl was applied for elucidation the question rdquoWhether use of Na+ or K

+ ions for 109

sperm treatment prior to cryopreservation could influence the fertilization rate in the present 110

experimentsrdquo 111

Osmolalities of media used in experiments before and after KCl addition were evaluated 112

using a Vapor Pressure Osmometer 5520 (Wescor USA) and expressed in mOsmkg-1

113

Saline solution composed of 45 mM NaCl 5 mM KCl 30 mM Tris-HCl pH 82 (147 114

mOsmkg-1

) was used as hypotonic activating media (AM) Sperm was added to the AM using the 115

tip of a dissecting needle thereafter the sperm suspensions were thoroughly dispersed for 2 s 116

Motility was recorded for 1ndash2 min post-activation using video microscopy techniques combined 117

with stroboscopic illumination using ExposureScopereg Video records were analyzed to estimate 118

spermatozoa curvilinear velocity (VCL) percent of motile cells (motility) by microimage analyzer 119

(Olympus Micro Image 401 for Windows) Microimage analyzer allows to measure VCL and 120

motility by overlapping five successive video frames Due to this the overlapping tracks of 121

spermatozoa heads became visible allowing the calculation of VCL (defined as total point-to-point 122

distance traveled by the sperm over the time period between the first and fifth frames) and motility 123

percentage [9] To assess fertility rate of eggs fertilized with cryopreserved sperm samples the 124

pooled ova from three females were used Egg samples (2 g) were inseminated in a dish with either 125

fresh or frozen-thawed sperm sample immediately after thawing Eggs were fertilized at a 126

spermova ratio of 1051 This spermova ratio was chosen in order to avoid excess of sperm which 127

would lead to maximal fertilization rate in all experimental trials and in turn would impede 128

detection the differences between experimental samples Spermatozoa concentration of each sperm 129

sample was estimated using a Burker cell hemocytometer (Meopta Czech Republic) at 200times 130

magnification on Olympus BX 50 phase contrast microscope (Olympus Japan) Dishes were placed 131

on an orbital agitator (200 rpm 10 mm deflection) Gametes were activated with 10 ml of hatchery 132

water and after 1 min samples of ova were transferred to three Petri dishes Approximately 100ndash133

200 eggs were placed into each dish and incubated with aerated dechlorinated and UV-sterilized 134

tap water Living and dead eggs were counted in each Petri dish during incubation and dead eggs 135

were removed Living embryos were counted at the eyed stage after 2 days incubation at 20ndash21oC 136

and fertilization rate was expressed as the percent of living embryos from the initial number of eggs 137

incubated 138

The values of parameters were checked for distribution characteristics and homogeneity of 139

dispersions using the ShapirondashWilks and Levenersquos tests respectively For normally distributed 140

sperm velocity values before and after cryopreservation parametric ANOVA was applied and 141

Fisher LSD test was used as criteria for differences between subgroups Nonparametric statistics 142

using the KruskalndashWallis test followed by the MannndashWhitney U-test were conducted for 143

comparison of motility percent motility duration and fertilization rate values because of small 144

amount of values were obtained in the study (n=5) Statistical significance was considered at P lt 145

005 Mean values and standard error of the mean (SEM) are presented on figures and meanplusmn 146

standard deviation values are mentioned in the text All analyses and plotting were conducted using 147

Statistica V91 computer program (Statsoft Inc USA) 148

In our study the highest post-thaw motility percent spermatozoa velocity and fertilization 149

rate were obtained after sperm treatment with hypotonic solutions applied prior to freezing in 150

comparison with non-treated sperm or sperm treated with isotonic solution Post-thaw motility 151

percent in samples pretreated by 200 and 150 mOsmkg-1

NaCl was significantly higher (44plusmn10 152

and 41plusmn10 respectively) than that of control (21plusmn15 ) or samples treated by 100 mOsmkg-1 153

(25plusmn15 ) or 300 mOsmkg-1

(25plusmn12 ) solutions (Fig 1A) Significantly higher values of mean 154

VCL were observed in samples treated by media of 100 150 and 200 mOsmkg-1

NaCl (119plusmn 24 155

118 plusmn22 and 115plusmn 32micromc-1

respectively) in comparison with control (92plusmn27 microms-1

) (Fig 1B) 156

Fertilization rate of eggs submitted to fresh sperm was 87plusmn6 Fertilization rate of eggs 157

submitted to frozen-thawed sperm samples treated with 200 mOsmkg-1

solution (21plusmn15) with 158

following osmolality increase by either 2 M KCl (17plusmn6 ) or 2 M NaCl (25plusmn18 ) were 159

significantly higher than in eggs fertilized by sperm samples pretreated by 300 mOsmkg-1

NaCl 160

solutions (12plusmn7 ) or control (9plusmn6 ) sperm samples Fertilization rate of eggs fertilized with 161

sperm samples pretreated by 100 mOsmkg-1

NaCl was not significantly different from that for eggs 162

fertilized by sperm samples pretreated with 200 mOsmkg-1

NaCl 300 mOsmkg-1

NaCl and non-163

treated (control) samples (Fig 1B) Altogether these new results revealed that post-thaw 164

spermatozoa motility percent VCL and fertilizing ability parameters of carp sperm could be 165

improved by short-time sperm pretreatment with hypotonic media For this purpose pretreatment 166

by a medium with moderate osmolality should be applied Moreover observed phenomenon is not 167

depending on chemical nature of ion by which osmolality is reversed to hypotonic level before 168

cryopreservation 169

Considering the results already obtained in field of carp sperm cryopreservation results on 170

post-thaw sperm characteristics obtained in the present study could be compared with whose 171

already published Spermatozoa post-thaw motility percent velocity and fertilizing ability are 172

comparable with results obtained by Boryshpolets et al [1] Results of motility percentage and 173

fertilization tests obtained in our present study are lower than reported previously [4] This 174

diversity of results could arise from differences in sperm quality procedures of cryopreservation 175

sperm dilution rates etc [6] and they indicate that non-optimal cryopreservation procedures were 176

applied Since in our study quite low spermeggs ratio was used it should be considered that 177

applied in our study too low spermegg ratio might mask the effect of experiments However to 178

our knowledge we report for the first time the possibility of improvement of post-thaw sperm 179

motility parameters by hypotonic treatment of spermatozoa prior to freezing even when applying 180

non-optimal procedure of freezing We found no information in literature regarding the influence of 181

hypotonic treatment before freezing on fish sperm cryoresistance Available information about 182

negative effect of hypotonic treatment before freezing on mammalian cells cryoresistance [10] 183

could be considered as indication of large taxonomical differences in membrane response to 184

hypotonicity probably because in contrast to mammals evolution of physiological activity of fresh 185

200 eggs were placed into each dish and incubated with aerated dechlorinated and UV-sterilized 134

tap water Living and dead eggs were counted in each Petri dish during incubation and dead eggs 135

were removed Living embryos were counted at the eyed stage after 2 days incubation at 20ndash21oC 136

and fertilization rate was expressed as the percent of living embryos from the initial number of eggs 137

incubated 138

The values of parameters were checked for distribution characteristics and homogeneity of 139

dispersions using the ShapirondashWilks and Levenersquos tests respectively For normally distributed 140

sperm velocity values before and after cryopreservation parametric ANOVA was applied and 141

Fisher LSD test was used as criteria for differences between subgroups Nonparametric statistics 142

using the KruskalndashWallis test followed by the MannndashWhitney U-test were conducted for 143

comparison of motility percent motility duration and fertilization rate values because of small 144

amount of values were obtained in the study (n=5) Statistical significance was considered at P lt 145

005 Mean values and standard error of the mean (SEM) are presented on figures and meanplusmn 146

standard deviation values are mentioned in the text All analyses and plotting were conducted using 147

Statistica V91 computer program (Statsoft Inc USA) 148

In our study the highest post-thaw motility percent spermatozoa velocity and fertilization 149

rate were obtained after sperm treatment with hypotonic solutions applied prior to freezing in 150

comparison with non-treated sperm or sperm treated with isotonic solution Post-thaw motility 151

percent in samples pretreated by 200 and 150 mOsmkg-1

NaCl was significantly higher (44plusmn10 152

and 41plusmn10 respectively) than that of control (21plusmn15 ) or samples treated by 100 mOsmkg-1 153

(25plusmn15 ) or 300 mOsmkg-1

(25plusmn12 ) solutions (Fig 1A) Significantly higher values of mean 154

VCL were observed in samples treated by media of 100 150 and 200 mOsmkg-1

NaCl (119plusmn 24 155

118 plusmn22 and 115plusmn 32micromc-1

respectively) in comparison with control (92plusmn27 microms-1

) (Fig 1B) 156

Fertilization rate of eggs submitted to fresh sperm was 87plusmn6 Fertilization rate of eggs 157

submitted to frozen-thawed sperm samples treated with 200 mOsmkg-1

solution (21plusmn15) with 158

following osmolality increase by either 2 M KCl (17plusmn6 ) or 2 M NaCl (25plusmn18 ) were 159

significantly higher than in eggs fertilized by sperm samples pretreated by 300 mOsmkg-1

NaCl 160

solutions (12plusmn7 ) or control (9plusmn6 ) sperm samples Fertilization rate of eggs fertilized with 161

sperm samples pretreated by 100 mOsmkg-1

NaCl was not significantly different from that for eggs 162

fertilized by sperm samples pretreated with 200 mOsmkg-1

NaCl 300 mOsmkg-1

NaCl and non-163

treated (control) samples (Fig 1B) Altogether these new results revealed that post-thaw 164

spermatozoa motility percent VCL and fertilizing ability parameters of carp sperm could be 165

improved by short-time sperm pretreatment with hypotonic media For this purpose pretreatment 166

by a medium with moderate osmolality should be applied Moreover observed phenomenon is not 167

depending on chemical nature of ion by which osmolality is reversed to hypotonic level before 168

cryopreservation 169

Considering the results already obtained in field of carp sperm cryopreservation results on 170

post-thaw sperm characteristics obtained in the present study could be compared with whose 171

already published Spermatozoa post-thaw motility percent velocity and fertilizing ability are 172

comparable with results obtained by Boryshpolets et al [1] Results of motility percentage and 173

fertilization tests obtained in our present study are lower than reported previously [4] This 174

diversity of results could arise from differences in sperm quality procedures of cryopreservation 175

sperm dilution rates etc [6] and they indicate that non-optimal cryopreservation procedures were 176

applied Since in our study quite low spermeggs ratio was used it should be considered that 177

applied in our study too low spermegg ratio might mask the effect of experiments However to 178

our knowledge we report for the first time the possibility of improvement of post-thaw sperm 179

motility parameters by hypotonic treatment of spermatozoa prior to freezing even when applying 180

non-optimal procedure of freezing We found no information in literature regarding the influence of 181

hypotonic treatment before freezing on fish sperm cryoresistance Available information about 182

negative effect of hypotonic treatment before freezing on mammalian cells cryoresistance [10] 183

could be considered as indication of large taxonomical differences in membrane response to 184

hypotonicity probably because in contrast to mammals evolution of physiological activity of fresh 185

significantly higher than in eggs fertilized by sperm samples pretreated by 300 mOsmkg-1

NaCl 160

solutions (12plusmn7 ) or control (9plusmn6 ) sperm samples Fertilization rate of eggs fertilized with 161

sperm samples pretreated by 100 mOsmkg-1

NaCl was not significantly different from that for eggs 162

fertilized by sperm samples pretreated with 200 mOsmkg-1

NaCl 300 mOsmkg-1

NaCl and non-163

treated (control) samples (Fig 1B) Altogether these new results revealed that post-thaw 164

spermatozoa motility percent VCL and fertilizing ability parameters of carp sperm could be 165

improved by short-time sperm pretreatment with hypotonic media For this purpose pretreatment 166

by a medium with moderate osmolality should be applied Moreover observed phenomenon is not 167

depending on chemical nature of ion by which osmolality is reversed to hypotonic level before 168

cryopreservation 169

Considering the results already obtained in field of carp sperm cryopreservation results on 170

post-thaw sperm characteristics obtained in the present study could be compared with whose 171

already published Spermatozoa post-thaw motility percent velocity and fertilizing ability are 172

comparable with results obtained by Boryshpolets et al [1] Results of motility percentage and 173

fertilization tests obtained in our present study are lower than reported previously [4] This 174

diversity of results could arise from differences in sperm quality procedures of cryopreservation 175

sperm dilution rates etc [6] and they indicate that non-optimal cryopreservation procedures were 176

applied Since in our study quite low spermeggs ratio was used it should be considered that 177

applied in our study too low spermegg ratio might mask the effect of experiments However to 178

our knowledge we report for the first time the possibility of improvement of post-thaw sperm 179

motility parameters by hypotonic treatment of spermatozoa prior to freezing even when applying 180

non-optimal procedure of freezing We found no information in literature regarding the influence of 181

hypotonic treatment before freezing on fish sperm cryoresistance Available information about 182

negative effect of hypotonic treatment before freezing on mammalian cells cryoresistance [10] 183

could be considered as indication of large taxonomical differences in membrane response to 184

hypotonicity probably because in contrast to mammals evolution of physiological activity of fresh 185

water fish sperm took place in conditions of hypotonic environment Apart from motility activation 186

little is known about the consequences of sperm transfer from isotonic condition of seminal fluid to 187

hypotonic environment However quick increase in membrane fluidity in response to hypotonicity 188

observed in carp sperm [7] could be favorable for sperm surviving watercryoprotectant fluxes 189

occurring during freeze-thawing and following fertilization in hypotonic condition This speculation 190

is also supported by observation made in rainbow trout in which increased membrane fluidity was 191

correlated with higher post-thaw sperm fertilizing ability (for review see [2]) 192

We suppose that the phenomenon observed in the present study could arise from sperm volume 193

increase which leads to sperm membrane modification improving the ability of spermatozoa to 194

survive freeze-thawing processes We consider our results as preliminary and requiring further 195

studies for application of this methodology to become useful in fish sperm cryopreservation 196

AKNOWLEDGMENTS 197

Special thanks are expressed to the projects IAA 608030801 ME10015 QH82119 LC06073 198

CZ1052100010024 GACR P502110090 GAJU 0462010Z 199

200

201

202

203

204

205

206

207

208

209

210

211

REFERENCES 212

[1] S Boryshpolets B Dzyuba M Rodina P Li M Hulak D Gela O Linhart Cryobiology 59 213

(2009) 291-296 214

[2] A Ciereszko J Glogowski K Dabrowski Biochemical characteristics of seminal plasma and 215

spermatozoa of freshwater fishes in TR Tiersh and CC Green (Eds) Cryopreservation in 216

Aquatic Species 2nd Edition World Aquaculture Society Baton Rouge Louisiana 2011 pp 217

46 -79 218

[3] B Dzuba G Bozhok S Rudenko Aquacult Res 32 (2001) 51-56 219

[4] A Horvath E Miskolczi S Mihalffy K Osz K Szabo B Urbanyi Cryobiology 54 (2007) 220

251ndash257 221

[5] EF Kopeika Instruction on Low Temperature Preservation of Carp Sperm VNIPRKh 222

Moscow 1986 223

[6] E Kopeika J Kopeika Variability of sperm quality after cryopreservation in fish in SMH 224

Alavi J Cosson K Coward (Eds) Fish Spermatology Alfa Science Oxford UK 2008 pp 225

347ndash396 226

[7] Z Krasznai M Morisawa Z T Krasznai S Morisawa K Inaba Z K Bazsane B 227

Rubovszky B Bodnar A Borsos T Maria Cell Motil Cytoskeleton 55 (2003) 232ndash243 228

[8] F Lahnsteiner B Berger T Weismann Theriogenology 60 (2003) 829-841 229

[9] O Linhart SMH Alavi M Rodina D Gela J Cosson J Appl Ichthyol 24 (2008) 386ndash392 230

[10] D E Pegg and M P Diaper Cryobiology 28 (1991) 18-35 231

232

233

234

235

236

237 238

REFERENCES 212

[1] S Boryshpolets B Dzyuba M Rodina P Li M Hulak D Gela O Linhart Cryobiology 59 213

(2009) 291-296 214

[2] A Ciereszko J Glogowski K Dabrowski Biochemical characteristics of seminal plasma and 215

spermatozoa of freshwater fishes in TR Tiersh and CC Green (Eds) Cryopreservation in 216

Aquatic Species 2nd Edition World Aquaculture Society Baton Rouge Louisiana 2011 pp 217

46 -79 218

[3] B Dzuba G Bozhok S Rudenko Aquacult Res 32 (2001) 51-56 219

[4] A Horvath E Miskolczi S Mihalffy K Osz K Szabo B Urbanyi Cryobiology 54 (2007) 220

251ndash257 221

[5] EF Kopeika Instruction on Low Temperature Preservation of Carp Sperm VNIPRKh 222

Moscow 1986 223

[6] E Kopeika J Kopeika Variability of sperm quality after cryopreservation in fish in SMH 224

Alavi J Cosson K Coward (Eds) Fish Spermatology Alfa Science Oxford UK 2008 pp 225

347ndash396 226

[7] Z Krasznai M Morisawa Z T Krasznai S Morisawa K Inaba Z K Bazsane B 227

Rubovszky B Bodnar A Borsos T Maria Cell Motil Cytoskeleton 55 (2003) 232ndash243 228

[8] F Lahnsteiner B Berger T Weismann Theriogenology 60 (2003) 829-841 229

[9] O Linhart SMH Alavi M Rodina D Gela J Cosson J Appl Ichthyol 24 (2008) 386ndash392 230

[10] D E Pegg and M P Diaper Cryobiology 28 (1991) 18-35 231

232

233

234

235

236

237 238

Figures legends 239 240 Figure 1 Post-thaw sperm motility percent (A) and spermatozoa curvilinear velocity (B) in carp 241

sperm samples treated with NaCl solutions of different osmolalities before cryopreservation Values 242

with different letters are significantly different (P lt 005) C ndash corresponds to control sperm 243

samples which were not pre-diluted with any NaCl solutions before freezing 244

245

Figure 2 Fertilization rate of eggs fertilized by frozen-thawed sperm samples treated with NaCl 246

solutions of different osmolalities before cryopreservation Values with different letters are 247

significantly different (P lt 005) ldquoKClrdquo and ldquoNaClrdquo inside the columns indicate that sperm 248

samples osmolality after 10 s treatment by pre-dilution medium was increased to 300 mOsmkg-1

249

by addition of 2 M KCl or NaCl solution respectively Column corresponding to 300 mOsmkg-1

250

refers to sperm treated by 300 mOsmkg-1

condition omitting any further addition C ndash corresponds 251

to control sperm samples which were not pre-diluted with any NaCl solutions prior to freezing 252

253

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bull E-mail address

bull Full postal address

bull Telephone and fax numbers

All necessary files have been uploaded and contain

bull Keywords

bull All figure captions

bull Manuscript has been spell-checked and grammar-checked

bull References are in the correct format for this journal

bull All references mentioned in the Reference list are cited in the text and vice versa

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