Effect of different avian egg yolk types on fertilizationability of cryopreserved common carp (Cyprinus carpio)spermatozoa

Yusuf Bozkurt • Ilker Yavas • Cengiz Yıldız

Received: 29 January 2013 / Accepted: 10 November 2013 / Published online: 17 November 2013� Springer Science+Business Media Dordrecht 2013

Abstract In spite of the fact that egg yolk from different avian species has successfully

been used as an additive for the cryopreservation of sperm in mammalian species, its

efficacy for cryopreserving fish sperm has not previously been tested comparatively.

Therefore, the present study was carried out to determine the effect of egg yolks from

different avian species, namely domestic chicken (Gallus gallus domesticus), turkey

(Meleagris gallopavo) and quail (Coturnix coturnix), on post-thaw motility and fertiliza-

tion ability of cryopreserved common carp spermatozoa. Egg yolks from chicken, turkey

and quail were analysed for moisture, total fat, protein, cholesterol and phospholipid

profile. Total fat and cholesterol contents of the turkey egg yolk were higher than chicken

and quail egg yolks (p \ 0.05). Semen was frozen according to conventional slow freezing

procedure. The extender contained 350 mM glucose, 30 mM Tris and 5 % glycerol sup-

plemented with different ratios of avian egg yolk (10, 15 and 20 %). Semen was equili-

brated at 4 �C for 15 min and placed into 0.25-ml straws and frozen in liquid nitrogen

vapour (for 10 min at -120 �C) and finally stored in liquid nitrogen (-196 �C) tank. The

frozen spermatozoa were thawed in a water bath at 35 �C for 30 s. Fertilization was

conducted using a ratio of 1 9 105 spermatozoa/egg. Cryopreservation experiments

resulted in higher post-thaw motility and fertilization rates. Mean post-thaw motility of

cryopreserved spermatozoa was between 45 and 80 %, and fertilization rates, expressed as

the percentage of eyed embryos, ranged from 70 to 95 %. In conclusion, the present study

showed that turkey and quail egg yolks are suitable alternatives to the chicken egg yolk for

the cryopreservation of common carp spermatozoa.

Y. Bozkurt (&)Department of Aquaculture, Faculty of Marine Science and Technology, University of Mustafa Kemal,Iskenderun, Hatay, Turkeye-mail: yfbozkurt@hotmail.com

I. Yavas � C. YıldızDepartment of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Universityof Mustafa Kemal, Antakya, Hatay, Turkey

123

Aquacult Int (2014) 22:131–139DOI 10.1007/s10499-013-9728-4

Keywords Cyprinus carpio � Cryopreservation � Egg yolk � Sperm quality �Fertilization

Introduction

Long-term storage of sperm in liquid nitrogen is a valuable technique for genetic resources

preservation. Research on fish sperm cryopreservation has achieved great advances since

the first successful sperm cryopreservation in herring (Blaxter 1953). It provides many

benefits such as ease of global germplasm shipping and supply (Tiersch et al. 2004),

selective breeding and hybridization with desirable characteristics and also conservation of

genetic diversity (Ohta et al. 2001). Furthermore, cryopreservation is considered as one

component in an effective strategy to save endangered species by facilitating the storage of

their gametes in gene banks (Akcay et al. 2004).

Even though many successes have been achieved in fish semen cryopreservation, the

technique remains as a method that is difficult to be standardized and use in all types of

fishes. This is due to the fact that cryopreservation of sperm from different fish species

required different conditions, where the protocol needs to be established individually.

Cryopreservation techniques involve addition of cryoprotectants, freezing and thawing of

sperm samples, all of which may result in some damage to the spermatozoa and may

decrease egg fertilization rate (Kopeika et al. 2003). Therefore, before cryopreservation of

sperm, a thorough evaluation of different extender solutions, cryoprotectants, straw sizes

and thawing rates is essential to develop optimum cryopreservation protocols for various

species (Yavas and Bozkurt 2011).

The species-specific cryopreservation procedure needs a suitable extender, as undiluted

semen is not suitable for long-term preservation. Similarly, addition of optimum amount of

cryoprotectant reduces cell damages associated with dehydration, cellular injuries and ice

crystal formation (Leung and Jamieson 1991). Although cryoprotectants help to prevent

cryoinjuries during freezing and thawing, they can become toxic to the cells when the

exposure time and concentration are more (Tekin et al. 2007). In addition, type of cryo-

protectants is also very specific to many species.

An important component of both cooling and freezing mediums for cryopreservation of

sperm from several species is avian egg yolk. The discovery that egg yolk has a beneficial

effect on fertility in semen extenders led to its widespread use with bull semen (Phillips

1939). Egg yolk diluents used to cryopreserve spermatozoa protect them against cold shock

during cooling, freezing and thawing (Watson 1981; Aboagla and Terada 2004).

Currently, egg yolk is a common component of most semen cryopreservation extenders

for most domestic animals. Traditionally, chicken egg yolk has been used as additive for

the freeze preservation of spermatozoa because of its wide availability (Bathgate et al.

2006). On the other hand, egg yolk from other bird species has successfully been used as an

additive for the cryopreservation of sperm in some mammalian species. For instance,

freezing of stallion semen with duck (Anas domesticus) yolk invests thawed sperm cells

with significantly greater motility and post-thaw longevity (Clulow et al. 2004). The post-

thaw motility of stallion semen frozen in diluents with chukar (Alectoris chukar) egg yolk

appears to be even greater than when chicken egg yolk is used (Humes and Webb 2006).

Trimeche et al. (1997) showed an improvement in the motility of thawed Poitou jackass (a

threatened domestic jackass breed) spermatozoa when quail (Coturnix coturnix) egg yolk

was used as an additive rather than chicken egg yolk.

132 Aquacult Int (2014) 22:131–139

123

The chemical composition of the egg yolks from different avian species varies partic-

ularly in terms of the cholesterol, fatty acid and phospholipid contents, and this influences

the protection they afford during cooling, freezing and thawing. The phospholipid, cho-

lesterol and the low-density lipoprotein contents of avian egg yolk specifically have been

identified as the protective components (Watson 1976). On the other hand, the exact

mechanism of egg yolks on common carp sperm during the freeze/thaw process is

unknown.

To the best of our knowledge, there is limited information regarding protective role of

egg yolk types on cryopreservation of fish sperm. For this reason, the main aim of the

present study was to determine the effect of chicken, turkey and quail egg yolks on post-

thaw motility and fertilization ability of the cryopreserved common carp sperm.

Materials and methods

Broodstock management

The common carp broodstock was collected from wintering ponds by seining and trans-

ported into the hatchery 48 h prior to gamete collection. In the hatchery, male and female

broodfish were held separately in shadowed tanks (V = 1,000 L) supplied with continu-

ously (2.5 L min-1) well-aerated water at 23 �C.

Gamete collection

Male and female common carp broodstock was anaesthetized with MS 222 at a ratio of

0.1 g/L before stripping. Semen was collected from ten mature males by manual

abdominal stripping 12 h after a single injection of 2 mg/kg of carp pituitary extract (CPE)

at 23 �C water temperature. Eggs were collected from five mature females by hand

stripping 10–12 h after a double injection of 3.5 mg/kg of CPE. The first injection of 10 %

(0.35 mg/kg) CPE was given 10 h before the second (3.15 mg/kg).

For sperm collection, the urogenital papillae of mature male fish were carefully dried,

and sperm was hand-stripped directly into test tubes. Following this, the tubes containing

sperm were placed in a styrofoambox containing crushed ice (4 �C). Contamination of

sperm with water, urine or faeces was carefully avoided. Sperm was transported to the

laboratory within 15 min. For the collection of eggs, females were wiped dry and stripped

by gentle abdominal massage, and the eggs from each female were collected in a dry metal

bowl. Eggs were checked visually, and only those lots of homogenous shape, colour and

size were used in fertilization experiments.

Sperm quality

Motility was determined subjectively using light microscope (Olympus, Japan) with a

4009 magnification. Samples were activated by mixing 1 ll of sperm with 20 ll activation

solution (0.3 % NaCl) on a glass slide. The percentage of motility was defined as the

percentage of spermatozoa moving in a forward motion every 20 % motile increment (i.e.

0, 20, 40, 60, 80 and 100 %) (Vuthiphandchai and Zohar 1999). Motility measurements

were performed within 15 s after activation. Sperm cells that vibrated in place were not

considered to be motile. Sperm motility was analysed with three replicates of samples by

Aquacult Int (2014) 22:131–139 133

123

the same observer. For cryopreservation experiments, samples below 80 % motile sper-

matozoa were discarded. Duration of sperm motility was determined using a sensitive

chronometer (sensitivity 1/100 s) by recording the time following the addition of activation

solution to the sperm samples.

Spermatozoa density was determined according to the haemacytometric method. Sperm

was diluted at a ratio of 1:1,000 with Hayem solution (5 g Na2SO4, 1 g NaCl, 0.5 g HgCl2and 200 ml bicine), and density was determined using a 100-lm-deep Thoma haemocy-

tometer (TH-100, Hecht-Assistent, Sondheim, Germany) at 4009 magnification with

Olympus BX50 phase contrast microscope (Olympus, Japan) and expressed as sperma-

tozoa 9109 ml-1 (three replicates). Counting chambers were always kept in a moist

atmosphere for at least 10 min before cell counting. Sperm pH was measured using

indicator papers (Merck 5.5–9) within 30 min of sampling.

Egg yolk analysis

Three yolks of each species were separated from the albumin. Filter paper was used to

remove all traces of albumin from the yolk, and the yolks were broken and combined. The

protocols for the determination of total moisture, total fat and protein were performed as

described by Bair and Marion (1978). For the aim of the analysis of cholesterol, a final 1-g

subsample of yolk was taken and used for the cholesterol assay. The yolk was diluted in

10 ml warmed PBS. A 1-ml aliquot was assayed for the cholesterol content using a

commercially available enzymatic, colorimetric assay at the Mustafa Kemal University,

Central Research Laboratory, Hatay. Phospholipid analysis was adapted according to the

protocol established by Bathgate et al. (2006).

Moisture content

A 2-g subsample of egg yolk was taken, weighed and dried in an oven at 95 �C for 24 h.

The resulting desiccate was weighed to determine the moisture content of the yolk. The

amount of moisture per gram of yolk was calculated.

Total fat content

Another 2-g subsample was taken for total fat content determination. The yolk was

transferred to a 50-ml tube, and the fat was extracted by agitating the yolk in 1:1:0.9

chloroform/methanol/water solvent for 10 min. The solvent and yolk mix were then

centrifuged at 5 �C and 2,000g for 15 min. The lower chloroform layer was aspirated into

an aluminium weigh boat, of known weight. The chloroform was evaporated off over 24 h.

The total fat was determined gravimetrically and the proportion of fat per gram of yolk

calculated.

Protein content

The protein content of the yolk was estimated by subtracting the average total lipid and

moisture weights from the total yolk weight. This was done on the assumption that the ash

and carbohydrate contents of the yolks were \1.5 % of the yolk combined.

134 Aquacult Int (2014) 22:131–139

123

Sperm dilution and cryopreservation procedure

Collected semen from males showing [80 motility was pooled into equal aliquots

according to the required semen volume and sperm density needed to eliminate the effects

of individual variability of the donors. The extender containing 350 mM glucose, 30 mM

Tris, 5 % glycerol and 100 ml distilled water was used as the basic extender. The pooled

semen was diluted at a ratio of 1:3 (semen/extender) with the glucose-based extender

containing 10 %, 15 and 20 % egg yolk from each of the three avian species, i.e. the

domestic chicken (Gallus gallus domesticus), turkey (Meleagris gallopavo) and quail

(C. coturnix), thus resulting in nine different extenders.

The diluted samples were drawn into 0.25-ml plastic straws (IMV, France) and were

sealed with polyvinyl alcohol (PVA). Having been diluted, the samples were equilibrated

for 15 min at 4 �C. After equilibration, the straws were placed on a styrofoam rack that

floated on the surface of liquid nitrogen in a styrofoam box. The straws were frozen in

liquid nitrogen vapour 3 cm above the surface of liquid nitrogen (-140 �C) for 10 min.

Following freezing, the straws were plunged into the liquid nitrogen (-196 �C) and stored

for several days. For thawing, the straws were removed from liquid nitrogen and immersed

in 35 �C water for 30 s. Thawed sperm was activated using 0.3 % NaCl and observed

under microscope for the determination of spermatozoa motility and motility durations.

Fertilization experiments

For fertilization, pooled eggs from mature females were used to determine fertilization

rates. Egg samples (about 1,000 eggs) were inseminated in dry Petri dishes with fresh

sperm or frozen sperm immediately after thawing at a spermatozoa/egg ratio of 1 9 105:1.

Eggs were inseminated by the dry fertilization technique using a solution of 3 g urea and

4 g NaCl in 1 L distilled water. The sperm and eggs were slightly stirred for 30 min,

washed with hatchery water (23 �C, 9 mg/L O2) and gently transferred to labelled Zuger

glass incubators with running water (23 �C) where they were kept until hatching

(3–4 days). Living and dead eggs were counted in each incubator during incubation, and

dead eggs were removed. When the fertilized eggs developed to embryos at the gastrula

stage, the fertilization rate (number of gastrula stage embryos/number of total eggs) was

calculated.

Statistical analysis

Results are presented as mean ± SE. Data for the percentage of sperm motility and fer-

tilization were transformed by angular transformation prior to statistical analysis by SPSS

10.0 software. Differences between parameters were analysed by repeated analysis of

variance (ANOVA). Significant means were subjected to a multiple comparison test

(Duncan) for post hoc comparison at a level of a = 0.05. All analyses were carried out

using SPSS 10 for Windows statistical software package.

Results

The moisture, total fat, total protein and cholesterol contents of chicken, turkey and quail

egg yolks are summarized in Table 1. The total moisture and protein contents were similar

(p [ 0.05), but the lipid and cholesterol fractions revealed differences in yolk composition.

Aquacult Int (2014) 22:131–139 135

123

Turkey egg yolk contained more cholesterol than the other two types, and quail egg yolk

contained more total fat than chicken egg yolk (p \ 0.05). The phospholipid contents of

the different avian egg yolks are summarized in Table 2.

The one-way analysis of variance indicated significant differences between egg yolks

regarding sperm motility, motility period and fertilization. According to the results, sperm

frozen in the presence of 15 % chicken egg yolk showed the highest motility rate

(p \ 0.05). The highest spermatozoa motility period was determined with 10 % chicken,

quail egg and turkey egg yolks compared to in all studied extender groups (p \ 0.05;

Table 3). Fertilization rates were determined higher than 70.0 % in all sperm extenders.

According to the fertilization results, fresh sperm had the highest fertilization rate com-

pared to extenders containing 10, 15 and 20 % different avian egg yolks (p \ 0.05;

Table 4). Likewise, cryopreserved sperm with extender containing 15 % chicken egg yolk

provided highest result (94 %) in terms of fertilization rate when compared with the other

tested groups (p \ 0.05).

Discussion

As far as we know, the present paper describes the first attempt testing the effect of

different avian egg yolk types on cryopreservation of common carp sperm. There is limited

information on the effect of avian egg yolks, other than chicken egg yolk, on freezability of

the common carp sperm. Egg yolk is one of the most commonly used components of

cryoprotectants utilized during freezing and thawing processes. The beneficial effect of egg

yolks in the cryopreservation of sperm can be attributed to a resistance factor which helps

to protect sperm against cold shock and storage factors.

Table 1 Component of egg yolks from different avian species

Egg origin Yolk contents (/g yolk)

Moisture (%) Total fat (%) Protein (mg/g yolk) Cholesterol (mg)

Chicken 1.396 27.674a 0.224 8.264a

Turkey 1.359 32.752b 0.185 11.672b

Quail 1.495 31.048b 0.177 8.264a

Different superscripts indicate significant difference within columns (p \ 0.05)

Table 2 Proportion of phospholipid types in the yolks of different avian species

Phospholipid type (% detected phospholipid) Egg yolk sources

Chicken Turkey Quail

Phosphatidic acid 13.0 ± 0.3a 13.6 ± 0.2a 14.4 ± 0.1a

Phosphatidylethanolamine (PE) 15.5 ± 1.6a 11.8 ± 0.6a 12.5 ± 0.5a

Phosphatidylinositol 13.4 ± 0.7a 21.4 ± 0.8b 12.2 ± 0.0a

Phosphatidylserine 22.7 ± 0.3b 20.8 ± 0.6b 25.0 ± 0.2b

Phosphatidylcholine (PC) 15.6 ± 0.1a 14.5 ± 0.2a 19.6 ± 0.5b

Lysophosphatidylcholine 19.9 ± 0.9b 15.2 ± 0.4a 15.6 ± 0.5a

Different superscripts indicate significant difference within columns (p \ 0.05)

136 Aquacult Int (2014) 22:131–139

123

Analysis of the composition of the different yolks showed that the basic components of

moisture and total protein were present in similar levels. However, analysis of total fat,

cholesterol and phospholipid profiles revealed significant differences between the egg yolk

types. Components of the yolks used in the present study showed that the positive effect of

supplementing extender with turkey yolk may be due to the relatively high cholesterol

content in the yolk from the chicken and quail yolks. Buhr et al. (1994) demonstrated that

the freeze–thawing process resulted in changes to the sperm membrane, including a loss of

fluidity, which may be related to the level of cholesterol in the membrane. This has been

proposed as one reason for compromised ova fertilization rate after the use of frozen–

thawed sperm.

The composition of phospholipid in the yolks did not help explain the post-thaw results.

Although differences were found between the yolks from different sources, none could be

used to explain the differences in sperm quality after freezing. It would be interest to

further study the classes of phospholipids in the yolks, as even the degree of saturation of

the fatty acids that make up the phospholipids would potentially effect the interaction

between the sperm and yolk during freeze–thawing (Graham and Foote 1987).

The significant effects of egg yolk in cryopreservation of sperm can be attributed to a

resistance factor, which helps to protect sperm against cold shock effects and storage

Table 3 Post-thaw motility and motility periods of common carp semen cryopreserved by using differentavian egg yolks (n = 3; mean ± SE)

Egg yolk types Rates (%) Post-thaw motility (%) Post-thaw motility periods (s)

Quail 10 53.2 ± 1.6a,b 25 ± 1.2a

15 45.3 ± 1.7a 15 ± 1.9b

20 55.2 ± 2.1a,b 15 ± 2.4b

Chicken 10 67.4 ± 2.4a,b 27 ± 1.6a

15 75.2 ± 1.6b 24 ± 1.2a

20 55.9 ± 1.4a,b 15 ± 2.3b

Turkey 10 56.1 ± 1.9a,b 26 ± 1.5a

15 59.5 ± 1.2a,b 25 ± 1.4a

20 48.6 ± 2.4a,b 24 ± 1.8a

Different superscripts indicate significant difference within columns (p \ 0.05)

Table 4 Fertilization rates ofcommon carp semen cryopre-served by using different avianegg yolks (n = 3; mean ± SE)

Different superscripts indicatesignificant difference withincolumns (p \ 0.05)

Egg yolk types Rates (%) Fertilization rates (%)

Quail 10 80.0 ± 5.7a,b,c

15 74.0 ± 2.3a

20 90.7 ± 2.3a,b,c

Chicken 10 90.0 ± 2.9a,b,c

15 94.0 ± 1.0b,c

20 91.7 ± 1.7a,b,c

Turkey 10 93.3 ± 1.7b,c

15 83.5 ± 3.4a,b,c

20 76.8 ± 2.5a,b

Control (fresh sperm) – 98.3 ± 4.2c

Aquacult Int (2014) 22:131–139 137

123

factors that help to maintain sperm quality parameters and fertility capacity. Additionally,

the cryoprotective effect of egg yolk may also be specific to the individual formulation of

extenders. For instance, Bozkurt et al. (2012) described the positive effects of chicken egg

yolk on brown trout and koi carp sperm with emphasis on post-thaw motility and fertility.

On the other hand, most of the cryopreservation studies in aquaculture have not tested the

composition of the egg yolks used in the extender formulations. Furthermore, experiments

have been mainly carried out with chicken egg yolks rather than the other avian species

egg yolks.

Finally, the most important finding of the present study was that semen frozen in the

extender containing quail and turkey egg yolk recorded high sperm quality like the semen

frozen in the chicken egg yolk. The present study shows that different avian egg yolks

(chicken, turkey and quail) have similar cryoprotective actions on common carp sperm

freezing. Based on the results of this study, it is recommended that common carp sperm can

be cryopreserved using a glucose-based extender containing turkey or quail egg yolks

instead of chicken. On the other hand, further investigation is required on the effect of

adjusting the composition of the yolk used in freeze–thawing of sperm.

Acknowledgments This research was funded by a Grant from Scientific Research Institute of MustafaKemal University (MKU-08-E-0203). The authors would like to thank the staff of the State HydraulicWorks (SHW) Fish Production Station in Adana (Turkey) for their technical assistance. Also, the valuablecomments and suggestions from anonymous reviewers are deeply thanked.

References

Aboagla EM, Terada T (2004) Effects of egg yolk during the freezing step of cryopreservation on theviability of goat spermatozoa. Theriogenology 62:1160–1172

Akcay E, Bozkurt Y, Secer S, Tekin N (2004) Cryopreservation of mirror carp semen. Turk J Vet Anim Sci28(5):837–843

Bair CW, Marion WW (1978) Yolk cholesterol in eggs from various avian species. Poult Sci 57:1260–1265Bathgate R, Maxwell WMC, Evans G (2006) Studies on the effect of supplementing boar semen cryo-

preservation media with different avian egg yolk types on in vitro post-thaw sperm quality. ReprodDomest Anim 41:68–73

Blaxter JHS (1953) Sperm storage and cross fertilization of spring and autumn spawning herring. Nature172:1189–1190

Bozkurt Y, Yavas I, Karaca F (2012) Cryopreservation of brown trout (Salmo trutta macrostigma) andornamental koi carp (Cyprinus carpio) sperm. Current frontiers in cryopreservation. In: Katkov I (ed)Section IV, pp 293–304, Celltronix and Sanford–Burnham Institute for Medical Research USA, ISBN:978-953-51-0302-8, p 462

Buhr MM, Curtis EF, Somnapan KN (1994) Composition and behaviour of head membrane lipids of freshand cryopreserved boar sperm. Cryobiology 31:224–238

Clulow J, Maxwell WMC, Evans G, Morris LHA (2004) A comparison between duck and chicken egg yolkfor the cryopreservation of stallion spermatozoa. In: Proceed. 15th Int. Congr. Anim. Reprod PortoSeguro, Brazil, 2004, p 506

Graham JK, Foote RH (1987) Effect of several lipids, fatty acyl chain length, and degree of unsaturation onthe motility of bull spermatozoa after cold shock and freezing. Cryobiology 24:42–52

Humes R, Webb G (2006) Use of chicken or chukar egg yolk with two cryoprotectants for preservation ofstallion semen. Anim Reprod Sci 94:62–63

Kopeika J, Kopeika E, Zhang T, Rawson DM, Holt WV (2003) Detrimental effects of cryopreservation ofloach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubatingfertilised eggs in caffeine. Cryobiology 46:43–52

Leung LKP, Jamieson BGM (1991) Live preservation of fish gametes. In: Jamieson BGM (ed) Fish evo-lution and systematics: evidence from spermatozoa. Cambridge University Press, Cambridge,pp 245–295

138 Aquacult Int (2014) 22:131–139

123

Ohta H, Kawamura K, Unuma T, Takegoshi Y (2001) Cryopreservation of the sperm of the Japanesebitterling. J Fish Biol 58:670–681

Phillips PH (1939) Preservation of bull semen. J Biol Chem 130:415Tekin N, Secer S, Akcay E, Bozkurt Y, Kayam S (2007) Effects of glycerol additions on post-thaw fertility

of frozen rainbow trout sperm, with an emphasis on interaction between extender and cryoprotectant.J Appl Ichthyol 23(1):60–63

Tiersch TR, Wayman WR, Skapura DP, Neidig CL, Grier HJ (2004) Transport and cryopreservation ofsperm of the common snook, Centropomus undecimalis (Bloch). Aquac Res 35:278–288

Trimeche A, Anton M, Renard P, Gandemer G, Tainturier D (1997) Quail egg yolk: a novel cryoprotectantfor the freeze preservation of Poitou Jackass sperm. Cryobiology 34:385–393

Vuthiphandchai V, Zohar Y (1999) Age-related sperm quality of captive striped bass Morone saxatilis.J World Aquac Soc 30(1):65–72

Watson PF (1976) The protection of ram and bull spermatozoa by the low density lipoprotein fraction of eggyolk during storage at 5 �C and deep freezing. J Thermal Biol 1:137–141

Watson PF (1981) The roles of lipid and protein in the protection of ram spermatozoa at 5�C by egg-yolklipoprotein. J Reprod Fertil 62:483–492

Yavas I, Bozkurt Y (2011) Effect of different thawing rates on motility and fertilizing capacity of cryo-preserved grass carp (Ctenopharyngodon idella) sperm. Biotechnol Biotechnol Equip 25(1):2254–2257

Aquacult Int (2014) 22:131–139 139

123