Effect of cryopreservation on sperm motility parameters and fertilizing ability of brown trout semen

    Effect of cryopreservation on sperm motility parameters and fertilizing ability of brown trout semen J. Nynca, G.J. Dietrich, S. Dobosz, J. Grudniewska, A. Ciereszko PII: DOI: Reference:

S0044-8486(14)00268-3 doi: 10.1016/j.aquaculture.2014.05.037 AQUA 631197

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

30 January 2014 13 May 2014 26 May 2014

Please cite this article as: Nynca, J., Dietrich, G.J., Dobosz, S., Grudniewska, J., Ciereszko, A., Effect of cryopreservation on sperm motility parameters and fertilizing ability of brown trout semen, Aquaculture (2014), doi: 10.1016/j.aquaculture.2014.05.037

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Effect of cryopreservation on sperm motility parameters and fertilizing ability of brown

T

trout semen

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food

SC R

1

IP

J. Nynca 1*, G.J. Dietrich1, S. Dobosz2, J. Grudniewska2, A. Ciereszko1

Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland 2

Inland Fisheries Institute, Department of Salmonid Research, Rutki, Żukowo, Poland

NU

*Corresponding author: [email protected]

MA

tel: (+48 89) 539 31 34; fax (+48 89) 524 01 24

Abstract

TE

D

The aim of this study was to test the usefulness of 0.18M glucose in 9% methanol as an extender, the ratio 1:5 (semen : extender) and an equilibration time of 15 min for the brown

CE P

trout semen cryopreservation (n=9). The final concentration was 0.15M and 7.5% for glucose and methanol, respectively. Sperm motility parameters, fertilization rate at the eyed and

AC

hatching stage were assessed for post-thaw semen. The applied cryopreservation procedure resulted in remarkably high (73.8%) post-thaw sperm motility. The fertilization rate of cryopreserved brown trout semen was over 90% for sperm-to-egg ratios of both 300 000 and 600 000:1. To our knowledge, this study is the first to report the remarkably high post-thaw fertilization ability of brown trout semen at a sperm-to-egg ratio as low as 300 000:1. The described procedure for brown trout cryopreservation is highly reliable and efficient and therefore can be recommended for hatchery practice after scaling up this technique.

Keywords: Brown trout, sperm, cryopreservation, fertilizing ability 1. Introduction

1

ACCEPTED MANUSCRIPT The brown trout (Salmo trutta m. fario L.) represents a major source of freshwater fish resources in Europe because of its commercial value for aquaculture and extreme importance

T

for angling. The brown trout is naturally subdivided into a large number of reproductively

IP

isolated and genetically distinct populations (Fergusson, 1989). The preservation of the brown

SC R

trout’s genetic variation is not only vital for the recovery of the original genetically unique populations but also allows selective breeding and prevents loss of fitness due to an inbreeding depression (Bartley et al., 1992).

NU

The creation of a cryopreserved sperm bank is an effective strategy for protecting the

MA

biodiversity of the local brown trout population and provides the opportunity to preserve the sperm samples of the most valuable males, which can be used in reproductive technologies in hatchery conditions (Martínez-Páramo et al., 2009). The cryopreservation procedures of

TE

D

brown trout semen were evaluated using different extenders and their concentrations, dilution ratios of sperm and/or volume of straws (Piironen, 1993; Lahnsteiner et al., 1997; Sarvi et al.,

CE P

2006, Martínez-Páramo et al., 2009, Dziewulska and Domagała, 2013). The fertilizing ability of cryopreserved brown trout with the described methods varied from about 45% (Dziewulska

AC

and Domagała, 2013) up to 95% (Lahnsteiner et al., 1997), but the sperm-to-egg ratio used for fertilization was very high, over 2106 spermatozoa/egg (Lahnsteiner et al., 1997; Sarvi et al., 2006). This is about 10 times more than the recommended numbers for fresh semen (Billard 1992). The high sperm-to-egg ratio used for the fertilization with cryopreserved semen limits the routine application of the method in hatchery practice. An elaborated procedure for securing low sperm-to-egg ratio would improve the effectiveness of brown trout cryopreservation and could lead to the implementation of this procedure in hatchery conditions. Recently, an effective cryopreservation procedure with the use of glucose-methanol extender was devised for rainbow trout semen (Ciereszko et al., 2014). However, it is unknown if the described method can be used for cryopreservation of

2

ACCEPTED MANUSCRIPT brown trout sperm. The aim of this study was to test the effect of cryopreservation on sperm motility parameters and fertilizing ability of brown trout semen. In the first experiment, the

T

sperm motility parameters of cryopreserved brown trout semen were evaluated. In the second

IP

experiment, the post-thaw fertilization ability of brown trout semen was examined at 600 000,

SC R

300 000, 100 000 sperm-to-egg ratios.

2. Materials and methods

NU

2.1. Collection of semen

MA

The experiments were carried out in November (2013) on sexually matured autumn spawning brown trout (3+ year old) maintained in the Rutki Salmonid Research Laboratory at the Institute of Inland Fisheries in Olsztyn, Poland. The average weight of fish was 920  184 g.

TE

D

The fish were stocked in concrete ponds (56 m3) supplied with water from the river Radunia. The temperature of water was 6-8C. Oxygen saturation levels were maintained at 85-95%.

CE P

Prior to milt collection, fish (n=15) were anesthetized using Propiscin (1 ppm IFI, Żabieniec, Poland). Milt from brown trout males was obtained by abdominal massage, with special care

AC

to avoid blood, urine or feces contamination.

2.2. Cryopreservation

The cryopreservation followed the procedure as previously described (Ciereszko et al., 2014). with the use of 0.18 M glucose and 9% methanol as extender. Final concentration was 0.15M and 7.5% for glucose and methanol, respectively. Semen was diluted with the extender at a ratio of 1:5 (semen: extender) before being drawn into 0.25-ml plastic straws (IMV Technologies, L'Agile, France). The straws with diluted semen were placed on a 3-cm high frame made of Styrofoam and equilibrated for 15 min on ice. Afterwards they floated in liquid

3

ACCEPTED MANUSCRIPT nitrogen for five minutes and then were submerged in it. Straws were then thawed by

T

immersion in a water bath at 40 ºC for 5 s.

IP

2.3. Sperm motility parameters of fresh and cryopreserved semen and fertilizing ability of

SC R

cryopreserved semen

Semen of nine individual males were cryopreserved as described above. The motility parameters of the fresh sperm; fresh-diluted in extender equilibrated for 15 min; and frozen-

NU

thawed sperm were examined with Computer Assisted Sperm Analysis (CASA) using the

MA

Hobson Sperm Cell Tracker (Hobson Vision Ltd, Baslow, UK). Fertilization trials for cryopreserved semen were conducted as presented below (point 2.4). Analyses were run in

TE

D

duplicates for all of the experiments in the present study.

2.4. Fertilization ability of cryopreserved semen

CE P

Fertilization was performed on November 21, 2013. The eggs pooled from two females were divided into batches of 206  15 eggs (21 g) and fertilized with thawed semen with

AC

spermatozoa to egg ratios 600 000; 300 000 and 100 000:1. To test the quality of the eggs, excess fresh semen (50 μl) combined from three males was used at the beginning and at the end of the fertilization trial. The fertilization success was established by calculating the percentage of embryos at the eyed stage (December 24, 2013) and hatching larvae (February 17, 2014). We calculated the percentage of hatching larvae using the initial number of eggs.

2.5. Semen analysis The motility parameters of sperm were examined with Computer Assisted Sperm Analysis using the Hobson Sperm Cell Tracker as described by Dietrich et al. (2005). Sperm was activated at a dilution ratio of 1:300 with 1 mM CaCl2, 20 mM Tris, 30 mM glycine, 125 mM

4

ACCEPTED MANUSCRIPT NaCl, pH 9.0 (Billard 1992) supplemented with 0.5% bovine albumin. The sperm motility parameters: percentage of motile sperm (MOT), straight line velocity (VSL), curvilinear

T

velocity (VCL), average path velocity (VAP), linearity (LIN) and amplitude of lateral head

IP

displacement (ALH) were measured over a 12-second period, between 5 and 17 seconds post

SC R

activation time. Video recordings (two replicates per sample) were made using a microscope Olympus BX40 (Olympus Optical, Tokyo, Japan) with a 10 negative phase objective and a Sony CCD black and white camera (SPT-M108CE), 50 frames per second were used

NU

(Boryshpolets et al., 2013). Sperm concentration were measured in duplicate using

MA

NucleoCounter SP-100 (Chemometec, Denmark) as described by Nynca and Ciereszko (2009). Briefly, semen was diluted 100 times with a sperm immobilizing solution (100 mM

D

NaCl, 40 mM KCl, 3 mM CaCl2, 1.5 mM, MgCl2 and 50 mM Tris, pH 8.5; Morisawa and

TE

Morisawa, 1988) then 51 times with Reagent S100. Osmolality of seminal plasma and extender osmolality was measured using a Minitüb Abfüll-u Labortechnik GmbH &Co.KG

CE P

apparatus (Tiefenbach, Germany).

AC

2.6. Statistical analysis

All the results are presented as mean ± SD. All analyses were performed at a significance level of 0.05 using GraphPad Prism software (GraphPad Software Inc, San Diego, CA, USA). For statistical procedures data percentages were transformed by arcsin square root transformation. Data were subjected to repeated measures one-way ANOVA followed by Tukey’s post hoc test.

3. Results 3.1. Sperm motility parameters of fresh and cryopreserved semen and fertilizing ability of cryopreserved semen

5

ACCEPTED MANUSCRIPT The percentage of sperm motility of cryopreserved semen was high, above 74%, which was significantly lower in comparison to fresh semen (90%; Fig. 1). The equilibration period of

T

fresh semen diluted with extender for 15 min did not influence the sperm motility parameters

IP

of fresh semen. VSL and VAP declined simultaneously in cryopreserved semen by 28% and

SC R

by 14% respectively, but VCL, LIN and ALH values were not affected by cryopreservation. The average sperm concentration and osmolality of fresh undiluted semen were 16.60  2.87  109 and 271  26 mOsmol/kg, respectively. The extender osmolality was 2808 mOsmol/kg.

NU

An increased percentage of eyed embryos for the cryopreserved samples was observed

MA

with an increase in the sperm-to-egg ratio used for fertilization (Fig. 2A). The highest percentage of eyed embryos (more than 90%) was found at the spermatozoa to egg ratios

D

300 000 and 600 000:1. At the 100 000:1 ratio, the fertilization ability of cryopreserved

TE

semen was significantly lower compared to 300 000 and 600 000:1 ratios, but still very high (about 87%; Fig. 2A). Very little mortality (1.2-2.1%) was observed at the hatching stage.

CE P

Similarly, the highest percentage of hatched larvae were observed for 300 000 and 600 000:1 sperm-to egg ratios. (Fig. 2B). The fertilization rate of eggs fertilized with an excess of fresh

AC

semen at the beginning and at the end of fertilization trial were 99.6% and 98.6%, respectively, for the hatching stage 99.1% and 98.6%, respectively.

4. Discussion In this study we demonstrated the usefulness of glucose-methanol extender for brown trout semen cryopreservation. Using 0.18M glucose in 9% methanol as an extender, the ratio 1:5 (semen : extender) and an equilibration time of 15 min resulted in remarkably high (73.8%) post-thaw sperm motility. The fertilization success of cryopreserved brown trout semen was over 90% for both 300 000 and 600 000:1 sperm-to-egg ratio.

6

ACCEPTED MANUSCRIPT According to our knowledge, this study is the first to report a very high post-thaw fertilization ability of brown trout semen at a sperm-to-egg ratio as low as 300 000:1.

T

Furthermore, our results demonstrate that a sperm-to-egg ratio as low as 100 000:1 also

IP

produced a very high percentage of eyed embryos. It must be emphasized that for salmonid

SC R

fish a ratio 300 000:1 is recommended for fertilization with fresh semen (Billard, 1992). Therefore, the fertilizing ability of cryopreserved semen is suggested to be close to that of fresh semen. In the previous studies regarding fertilization with thawed brown trout semen,

NU

the lowest sperm-to-egg ratios (1.2-2.4106 spermatozoa per egg) assured a high post-thaw

MA

fertilization rate (95.7%) was applied by Lahnsteiner et al. (1997). In recent studies, the sperm-to-egg ratio used in the fertilization trials with cryopreserved brown trout sperm was as

D

high as 4106 (Dziewulska and Domagała, 2013) up to 6.2106 (Sarvi et al., 2006) and

TE

resulted in 45% and 66.6% fertilization rates, respectively. Summing up, our procedure decreased the sperm to egg ratio by a 10 factor compared to the results previously published.

CE P

Our results strongly suggest that the described procedure for brown trout cryopreservation is highly reliable and efficient and therefore can be recommended for hatchery practice after

AC

scaling up this technique.

In this study, we have demonstrated that the use of a extender containing 0.18M glucose in 9% methanol resulted in remarkably high 73.8% post-thaw sperm motility. Recently, Dziewulska and Domagała (2013) and Horvath et al. (2013) using a glucosemethanol extender for the brown trout sperm cryopreservation obtained 12.5% and 55.0% post-thaw sperm motility, respectively. It is difficult at present to determine which specific factor is responsible for the discrepancy between results of the present study and the data of the above authors, due to significant differences in cryopreservation technologies regarding extender composition and equilibration period.

7

ACCEPTED MANUSCRIPT According to Morisawa et al. (1983) the presence of potassium in the extender is required to maintain salmonid sperm in a quiescent state. In our study glucose-methanol

T

extender did not activate sperm motility. Probably the osmolality of the extender is

IP

responsible for the maintaining of the spermatozoa in the quiescent stage, but further studies

SC R

are needed to elucidate which specific component of the extender is responsible for this phenomenon.

Our study indicated limited differences between sperm motility characteristics of

NU

fresh-diluted and cryopreserved brown trout semen. A significant decrease in the values of

MA

sperm motility, VSL, VCL, VAP of fresh-diluted semen were noticed in brown trout postthaw sperm, but at the same time LIN and ALH were not affected by the freezing-thawing procedures. This is in agreement with data obtained previously for the rainbow trout

TE

D

cryopreserved semen with the exception of VCL and ALH, the former did not change and the latter was lower after cryopreservation (Ciereszko et al., 2014). These differences can reflect

CE P

species-specific variations between properties of brown and rainbow trout spermatozoa. Furthermore, it should be underlined that curvilinear velocity of brown trout fresh semen did

AC

not differ significantly from cryopreserved semen. Since VCL is considered to be a sperm quality indicator (Lahnsteiner et al., 1998), it might partially determine the high reproductive success confirming the effectiveness of the glucose-methanol cryopreservation procedure. The high quality of post-thaw brown trout semen cryopreserved according to the procedure established recently for rainbow trout sperm in our laboratory (Ciereszko et al., 2014) suggests that the glucose-methanol extender can be universal for the freezing of salmonid fish sperm. This is in agreement with data obtained by Lahnsteiner et al. (1997) who demonstrated that semen of salmonid fish species can effectively be cryopreserved using identical extenders. Perhaps, the salmonid fish spermatozoa respond in a similar pattern to the conditions of the cryopreservation procedure. Further studies should be aimed to the unravel

8

ACCEPTED MANUSCRIPT the mechanism of changes in salmonid fish sperm during cryopreservation. Such studies should include morphological and biochemical analysis, including flow cytometry,

T

microscopy and ATP measurements.

IP

In conclusion, brown trout semen can efficiently be cryopreserved with the application

SC R

of the procedure elaborated recently in our laboratory for rainbow trout (Ciereszko et al., 2014). To our knowledge this study is the first to report remarkably high post-thaw fertilization ability of brown trout semen at the sperm-to-egg ratio as low as 300 000:1.

NU

Further studies should be focused on scaling up this efficient cryopreservation technique for

MA

application in hatchery conditions.

Aknowledgements

TE

D

This work was supported by Iuventus grant IP2011 0390 71 from Polish Ministry of Higher Education, funds of the National Science Centre granted on research project nr

CE P

2011/01/D/NZ9/03738, and funds appropriated to the Institute of Animal Reproduction and

AC

Food Research, Polish Academy of Sciences.

References

Bartley, D., Bagley, M., Gall, G., Bentley, B., 1992. Use of linkage disequilibrium to estimate effective size of hatchery and natural fish populations. Conserv. Biol. 6, 365-375. Billard, R., 1992. Reproduction in rainbow trout: sex differentiation, dynamics of gametogenesis, biology and preservation of gametes. Aquaculture 100, 263-298. Boryshpolets, S., Kowalski, R.K., Dietrich, G.J., Dzyuba, B., Ciereszko, A., 2013. Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters. Theriogenology 80, 758-765.

9

ACCEPTED MANUSCRIPT Ciereszko, A., Dietrich, G.J., Nynca, J., Dobosz, S., Zalewski, T., 2014. Cryopreservation of rainbow trout semen using a glucose-methanol extender. Aquaculture 420-421, 275-

T

281.

IP

Dietrich, G.J, Kowalski, R., Wojtczak, M., Dobosz, S., Goryczko, K., Ciereszko, A., 2005.

SC R

Motility parameters of rainbow trout (Oncorhynchus mykiss) spermatozoa in relation to sequential collection of milt, time of post-mortem storage and anesthesia. Fish Physiol. Biochem. 31, 1-9.

NU

Dziewulska, K., Domagała, J., 2013. Spermatozoa concentration influences cryopreservation

MA

success in sea trout (Salmo trutta m. trutta L.). Theriogenology 80, 659-664. Fergusson, A., 1989. Genetic differences among brown trout, Salmo trutta, stocks and their importance for the conservation and management of the species. Freshwater Biol. 21,

TE

D

35-46.

Horváth, Á., Labbé, C., Jesenšek, D., Bernáth, G., Kaczkó, D., Bokor, Z., Urbányi, B., 2013.

CE P

Post-thaw storage of sperm from various salmonid species. 4th International Workshop on the Biology of Fish Gametes. 17-20 Sept. 2013, Albufeira, Portugal,

AC

126-127.

Lahnsteiner, F., Patzner, R.A., Weismann, T., 1997. Methanol as cryoprotectant and the suitability of 1.2 ml and 5 ml straws for cryopreservation of semen from salmonid fishes. Aquac. Res. 28, 471-479. Lahnsteiner, F., Berger, B., Weismann, T., Patzner, RA., 1998. Determination of semen quality of the rainbow trout, Oncorhynchus mykiss, by sperm motility, seminal plasma parameters, and spermatozoal metabolism. Aquaculture 163, 163–181. Martínez-Páramo, S., Pérez-Cerezales, S., Gómez-Romano, F., Blanco, G., Sánchez, J.A., Herráez, M.P., 2009. Cryobanking as tool for conservation of biodiversity: effect of

10

ACCEPTED MANUSCRIPT brown trout sperm cryopreservation on the male genetic potential. Theriogenology 71, 594-604.

T

Morisawa, M., Suzuki, K., Morisawa, S., 1983. Effects of potassium and osmolality on

IP

spermatozoan motility of salmonid fishes .J. Exp. Biol. 107, 105-113.

SC R

Morisawa, S., Morisawa, M., 1988. Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon. J. Exp. Biol. 136, 13-22. Nynca, J., Ciereszko, A., 2009. Measurement of concentration and viability of brook trout

NU

(Salvelinus fontinalis) spermatozoa using computer-aided fluorescent microscopy.

MA

Aquaculture 292, 256-258.

Piironen, J., 1993. Cryopreservation of sperm from brown trout (Salmo trutta m. lacustris L.) and Arctic charr (Salvelinus alpinus L.). Aquaculture 116, 275-285.

TE

D

Sarvi, K., Niksirat, H., Mojazi Amiri, B., Mirtorabi, S.M., Rafiee, G.R., Bakhtiyari, M., 2006. Cryopreservation of semen from the endangered Caspian brown trout (Salmo trutta

AC

Figure captions

CE P

caspius). Aquaculture 256, 264-269.

Fig. 1. Sperm motility of fresh, fresh–diluted and cryopreserved semen of brown trout (n=9). Results are expressed as mean  SD. Different superscripts indicate significant differences (P<0.05) between fresh and cryopreserved semen.

Fig. 2. The sperm fertilization ability of cryopreserved semen A) the percentage of eyed embryos; B) the percentage of hatched larvae in relation to different sperm-to-egg ratio (n=9). Results are expressed as mean  SD. Different superscripts indicate significant differences (P<0.05) between used sperm-to-egg ratio.

11

a

b

a a

50

a

D

40 30 10 0

a

120

b

90 60 30

Fresh-diluted Cryopreserved

Fresh

Fresh-diluted Cryopreserved

25 20

a

a

a

15 10 5 0 Fresh

Fresh-dilutedCryopreserved

AC

CE P

Fresh

TE

LIN (%)

Fresh-dilutedCryopreserved

0

Fresh-diluted Cryopreserved

20

Fig. 1.

b

SC R

ab

80 60

a

150

a

Fresh

70

a

Fresh

VAP ( m s1)

200 180 160 140 120 100 80 60 40 20 0

Fresh-diluted Cryopreserved

NU

VCL (m s-1)

Fresh

100 90 80 70 60 50 40 30 20 10 0

T

VSL ( m s-1)

b

IP

a

a

ALH ( m)

100 90 80 70 60 50 40 30 20 10 0

MA

Motility (%)

ACCEPTED MANUSCRIPT

12

ACCEPTED MANUSCRIPT

100 000:1

300 000:1

600 000:1

100 90 80 70 60 50 40 30 20 10 0

b

a

b

T

b

IP

b

SC R

a

100 000:1

300 000:1

600 000:1

NU

100 90 80 70 60 50 40 30 20 10 0

B Hatching rate (%)

Eyed embryos (%)

A

AC

CE P

TE

D

MA

Fig. 2.

13

ACCEPTED MANUSCRIPT Highlights

-Brown trout semen can be effectively cryopreserved with the use of glucose-methanol

T

extender in a ratio 1:5 (semen : extender)

IP

-Remarkably high post-thaw fertilization ability (more than 90%) of brown trout semen at the sperm-to-egg ratio as low as 300 000:1.

AC

CE P

TE

D

MA

NU

SC R

-Glucose-methanol extender can be universal for cryopreservation of salmonid fish

14