Effect of Different Extenders and Dilution Ratios on the Spermatological and Fertilization Parameters of Cyprinus carpio During Cryopreservation

Abstract

Production of quality seeds is a critical need, and cryopreservation serves as an important biotechnological tool in achieving that goal. In the present study, the effects of different extenders and dilution ratios on the spermatological and fertilization parameters of cryopreserved milt were evaluated in common carp brooders. Milt was collected from Cyprinus carpio brooders and diluted and cryopreserved at three dilution ratios such as 1:40, 1:80, and 1:120, using Freshwater Fish Saline (FWFS) and Modified Fish Ringer as extenders and dimethyl sulfoxide as cryoprotectant (90:10). Motility duration, fertilization, and hatching rates were studied after different treatments. Rapid freezing of straws was done for 20 minutes at a freezing rate of −78°C/min. It can be concluded that C. carpio milt can be cryopreserved with FWFS at a dilution ratio of 1:40 to obtain the highest post-thaw motility duration (60.33 ± 1.52 seconds), fertilization rate (87.6% ± 1.52%), and hatching rate (57.1% ± 4.16%). The difference in the values between extenders, dilution ratios, and cryopreserved and fresh milt were statistically significant (p < 0.05) when analyzed using ANOVA.

Introduction

Cryopreservation of gametes is considered an important component in the strategy to save endangered species, by facilitating the storage of their gametes in the gene bank.¹ It is a tool for long-term storage of spermatozoa.²

Salt concentration present in the milt keeps it highly viscous in a liquid state. This high viscosity prevents temperature exchange and causes cryoinjuries. Hence, the milt cannot be frozen directly, and it has to de diluted before freezing. The dilution must be made with a solution that will not harm the spermatozoa during the freezing process. Also, the solution must have salt concentrations similar to that of the seminal plasma of milt. Such a solution is called an “extender.”

An extender is a solution of salts, including organic compounds, which helps maintain the viability of cells during refrigeration.³ Extenders are of two types, viz., ionic extenders or seminal plasma mimicking media⁴ and sugar-based extenders.⁵ While ionic extenders use salts, sugar-based extenders are composed of only sugars such as glucose, sucrose, and so on.

Semen dilution is a process that has been carried out as a means to increase the number of eggs that can be fertilized with a small volume of semen.⁶ In fish spermatozoa cryopreservation, dilution of the sperm fluid is one of the important steps that has been reported to improve fertilization rates when compared with results obtained with undiluted milt.^7,8 Various observations and reports have been noted in the aspects of dilution ratio in Cyprinus carpio spermatozoa cryopreservation.^9–13 The effects of different dilution ratios such as 1:10, 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, and 1:500 on sperm motility of zebra fish were evaluated, and higher motility duration was reported when the milt samples were diluted at 1:10, 1:20, and 1:50, and it was concluded that motility decreased with increased dilution.¹⁴ However, a standardized dilution ratio is not specified for the C. carpio spermatozoa to help in the effective artificial fertilization after cryopreservation.

The present study aimed to study the effects of different extenders and dilution ratios on the spermatological and fertilization parameters of cryopreserved spermatozoa of common carp brooders.

Materials and Methods

C. carpio were selected based on the identification features¹⁵ and matured males and females that released milt and egg, respectively, under slight pressure of the abdomen were selected.¹⁶ The selected males and females with an average body weight of 137 ± 80 g were stocked separately in the net cages erected in the pond for maintenance and collection¹⁷ at a density of 1 kg/m³ of water. The size of the cage was 5 × 3 × 3 feet. Milt was collected in a sterile, prelabeled 1.5 mL cryovial by gentle stripping.^4,12

The spermatological parameters were analyzed under a NIKON E360 phase contrast microscope at 200 × . Suitable motility scores were also assigned.¹⁸ Quality of the spermatozoa in the milt was evaluated through the observation of their motility by placing 1 μL of diluted milt sample and 10 μL of tap water onto a glass slide and observing under the microscope.¹⁷

The milt was diluted with Freshwater Fish Saline (FWFS; NaCl: 7.5 g/L; KCl: 0.2 g/L; NaHCO₃: 0.2 g/L; CaCl₂: 0.2 g/L) and Modified Fish Ringer (MFR; NaCl: 6.5 g/L; KCl: 3.0 g/L; NaHCO₃: 0.2 g/L; CaCl₂: 0.3 g/L). Dimethyl sulfoxide was used as a cryoprotectant and the ratio of extender and cryoprotectant was 9:1. The collected milt was diluted at three dilution ratios such as 1:40, 1:80, and 1:120. No equilibration time was given.^19,20 The milt was loaded into 0.5 mL IMV French Straws and sealed with a polymer powder (IMV, France). Rapid freezing of straws was done for 20 minutes using a floating raft of size 13 × 13 cm by allowing it to float in liquid nitrogen held in a Styrofoam box.²¹ To allow the straws to achieve a uniform freezing effect, the distance between the straws and LN₂ was maintained at 3 cm²² with a freezing rate of −78°C/min.²³ They were then transferred to BA11 model cryocans containing LN_2. Observations were made once every 15 days for 60 days. During sampling, straws were thawed at 30°C for 30 seconds in a serological water bath.²² The thawed milt was observed for the motility duration at room temperature. After 60 days of cryopreservation, the milt was tested for its efficacy in artificial fertilization.

The dry method of in vitro fertilization was practiced.^24,25 Fresh milt and eggs were collected and fertilized with the fresh and cryopreserved milt, which was diluted with FWFS and MFR at dilution ratios of 1:40, 1:80, and 1:120. Eggs from a single female were collected and divided into 6 batches and each batch contained ∼500 eggs. Batches of eggs were inseminated with fresh milt samples. The sperm–egg ratio was ∼250,000 sperm per egg.²⁵ Straws loaded with milt diluted at 1:40 dilution were used in lesser numbers, whereas straws loaded with milt diluted at 1:120 dilution were used in higher numbers to achieve the sperm–egg ratio of 250,000 sperm per egg. Fertilization took place in dry plastic dishes.²⁶ The fertilization rate and hatching rate were calculated.^27,28

All the observations were processed and tabulated. The data were statistically analyzed by SPSS 17.0. To determine whether there were any differences among the means, one-way analysis (ANOVA) and the Duncan multiple range test were applied to the results, and p values <0.05 were regarded as significant.

Results

Spermatological and fertilization parameters with different extenders

The highest mean postthaw motility duration and motility score was obtained with FWFS when compared to MFR as extender. When milt was diluted with FWFS, it exhibited the highest mean post-thaw motility duration of 60.33 ± 1.52 seconds on 60th day (Table 1). The highest mean post-thaw motility duration observed at the end of 60 days in samples diluted with MFR was 41.66 ± 3.05 seconds (Table 2). The values were statistically significant (p < 0.05).

Table 1.

Motility Duration (Seconds) of Milt Collected from Cyprinus carpio and Cryopreserved with Freshwater Fish Saline at Three Dilution Ratios

Dilution ratios	Days of cryopreservation
Dilution ratios	1	15	30	45	60
1:40	69.66 ± 4.72^a	64.66 ± 3.05^ab	63.66 ± 4.5^ab	62.66 ± 2.51^b	60.33 ± 1.52^b
1:80	66.66 ± 3.78^a	57 ± 3^b	61.33 ± 6.65^ab	60.66 ± 1.15^ab	56.33 ± 3.51^b
1:120	53 ± 5^a	49.33 ± 2.51^b	47 ± 1.73^b	46 ± 1^b	46.33 ± 2.08^b

Values in a column with different superscript letters are significantly different from each other at P < 0.05.

Table 2.

Motility Duration (Seconds) of Milt Collected from Cyprinus carpio and Cryopreserved with Modified Fish Ringer at Three Dilution ratios

Dilution ratios	Days of cryopreservation
Dilution ratios	1	15	30	45	60
1:40	48 ± 3^a	53 ± 2^b	42.66 ± 2.08^c	41.66 ± 2.08^c	41.66 ± 3.05^c
1:80	55.33 ± 5.13^b	47 ± 2.64^a	43.33 ± 2.08^ac	43 ± 2^ac	41 ± 1^c
1:120	45.66 ± 2.08^b	44.33 ± 2.08^b	42.33 ± 3.51^ab	37 ± 3.60^ac	36 ± 4.58^c

Values in a column with different superscript letters are significantly different from each other at P < 0.05.

Similarly, the highest percentage of fertilized eggs and the highest hatching rate were obtained with FWFS compared with MFR. When FWFS was used as the diluent, the higher percentage of fertilized eggs that was obtained with cryopreserved milt was 87.6% ± 1.52% at 1:40 dilution, and with fresh milt, it was 91.3% ± 1.52% (Table 3). The highest fertilization rate obtained using MFR with cryopreserved milt was 75.6% ± 2.08% at 1:40 dilution, whereas it was 87.6% ± 2.51% in fresh milt diluted with the same diluents (Table 3). There was a significant difference between the fertilization rates of the two extenders (p < 0.05).

Table 3.

Fertilization Rate (%) of Eggs Fertilized with Fresh and Cryopreserved Milt

Dilution ratio	Fresh milt		Cryopreserved milt
Dilution ratio	FWFS	MFR	FWFS	MFR
1:40	91.3 ± 1.52^a	87.6 ± 2.51^a	87.6 ± 1.52^b	75.6 ± 2.08^c
1:80	89.3 ± 2.51^c	83.6 ± 2.51^a	82.3 ± 2.08^c	74.3 ± 1.52^a
1:120	86.6 ± 2.51^d	84.6 ± 3.05^d	76.3 ± 2.51^b	71.6 ± 1.52^c

Values in a column with different superscript letters are significantly different from each other at P < 0.05.

FWFS, Freshwater Fish Saline; MFR, Modified Fish Ringer.

The hatching rates obtained with both the extenders using cryopreserved milt and fresh milt are presented in Table 4. The higher percentage of hatching rate observed, when milt cryopreserved with FWFS was used for fertilization, was 57.3% ± 4.16%, while with MFR, it was 41.6% ± 1.52%. The hatching rate between two extenders was statistically significant (p < 0.05).

Table 4.

Hatching Rate (%) of Eggs Fertilized with Fresh and Cryopreserved Milt

Dilution ratio	Fresh milt		Cryopreserved milt
Dilution ratio	FWFS	MFR	FWFS	MFR
1:40	76.6 ± 3.05^a	71.6 ± 3.51^ab	57.3 ± 4.1^c	39.3 ± 4.16^d
1:80	73.6 ± 2.5^e	69.3 ± 1.52^e	55.3 ± 2.5^c	37.6 ± 3.51^d
1:120	68.3 ± 2.51^e	63.3 ± 2.51^e	50.6 ± 2.08^e	35.6 ± 1.5^cd

Values in a column with different superscript letters are significantly different from each other at P < 0.05.

Spermatological and fertilization parameters with different dilution ratios

The highest mean post-thaw motility duration, motility score, percentage of fertilized eggs, and hatching rate was obtained with the 1:40 dilution. The mean motility duration of milt diluted with FWFS at a ratio of 1:40, 1:80, and 1:120 at the end of the experiment was 60.33 ± 1.52, 56.33 ± 3.51, and 46.33 ± 2.08 seconds, respectively. When diluted with MFR at a ratio of 1:40, 1:80, and 1:120, the mean motility duration observed was 41.66 ± 3.05, 41 ± 1, and 36 ± 4.58 seconds (Tables 1 and 2). There was a significant difference between the three dilution ratios and the values were statistically significant (p < 0.05).

The fertilization and hatching rates obtained with cryopreserved milt and fresh milt at different dilution ratios are given in Tables 3 and 4, respectively. The fertilization rate was always higher for fresh milt when compared to cryopreserved milt. When FWFS was used, there was a significant difference between the fertilization rate of three dilution ratios (p < 0.05). However, there was no statistical significance in the fertilization rate between dilution ratios when MFR was used. This was the case in both cryopreserved and fresh milt.

Discussion

Spermatological and fertilization parameters with different extenders

The differences in the highest mean post-thaw motility duration between FWFS and MFR were in accordance with the findings of other reserachers^29,30 who mentioned that motility duration is known to vary with each extender and the value decreases as the storage period increases.

Decline in the motility duration and score from the initial value is generally an accepted phenomenon for all the fish spermatozoa under cryopreservation.^31–34 Previous studies³⁵ have observed a decrease in motility duration following cryopreservation. These studies also reported that penetrating cryoprotectants could affect the percentage of motile sperm.

Even though diluents are necessary to enhance the lifespan of milt during cryopreservation,^36–38 a decrease in fertilization rate has been widely reported when diluted and cryopreserved sperm are used.^4,39

When diluted cryopreserved common carp milt was used, a fertilization rate of 31% was achieved, while in the case of undiluted fresh milt, it was 83%.⁴ Similarly, a 66% fertilization rate from cryopreserved milt and a 74% fertilization rate from undiluted milt were obtained.³⁹ In the present study also, similar results were noticed between cryopreserved and fresh sperm.

Fertilization and hatching rates can be decreased after cryopreservation when compared with fresh sperm.⁴⁰ Studies⁴¹ have found significant differences in the fertilization of fresh (68 + − 11%) and thawed spermatozoa (56 + − 10%) of common carp with a hatching rate of 50 + − 18% and 52 + − 9%, respectively. A reason for low hatching rate from cryopreserved spermatozoa in this study might be due to the cryoinjury that could happen during freezing and thawing of the spermatozoa.⁴²

In the present study, two extenders, namely FWFS⁴³ and MFR,⁹ were used. Both were ionic extenders which used only salts.⁴ There was a statistical difference in the motility duration between these two extenders. FWFS was used for tilapia sperm,⁴³ whereas MFR was used for rainbow trout and chum salmon sperm,⁹ only under refrigerated conditions. The changes in the results in the current study with respect to motility duration, fertilization, and hatching rate might be due to species difference because MFR does not work well with C. carpio milt when compared with FWFS.

The components of both extenders included NaCl, KCl, NaHCO₃, and CaCl₂. Potassium is known to induce motility of common carp.⁴⁴ There is not much difference in the composition of salts in the extenders used for the study. However, the KCl level is higher in the MFR solution (3 g/L) and lower in FWFS (0.2 g/L). This might have been a reason for the reduction in motility and fertilization ability of spermatozoa cryopreserved with MFR.

Studies have found⁴⁵ that the ratio of Na:K ions in the extender was an important factor for the successful cryopreservation of rainbow trout sperm.⁴⁶ It has been reported that the duration of sperm motility in summer sand whiting (Sillago ciliate) significantly increased with a Na:K ratio >2:1.⁴⁷

In the present study, FWFS used 7.5 g/L of NaCl and 0.2 g/L of KCl, where Na:K ratio is very meager and sodium can easily inhibit the action of potassium.⁴⁴ Studies have found⁴⁴ that sodium, calcium, and magnesium reduced the inhibiting action of potassium. However, MFR contained a Na:K ratio of ∼2:1. In this case, the level of sodium and calcium present in the solution might not be enough to inhibit the action of potassium. Hence, MFR could not maintain the motility of spermatozoa compared with FWFS because the presence of a high amount of potassium would have depleted the energy of spermatozoa during storage, due to its inducing action.

Spermatological and fertilization parameters with different dilution ratios

Dilution of milt is essential for fish sperm to survive after cryopreservation.⁴⁸ Dilution is used to increase the number of eggs that can be fertilized with a small volume of semen during artificial fertilization.⁶ In the present study, the highest post-thaw motility duration was observed only in samples diluted at 1:40 dilution ratio. This is because at higher cell concentrations, the post-thaw survival of milt significantly decreases, which is attributed to cell compression because of limited intracellular space.^14,49

When dilutions of 1:25, 1:50, and 1:100 were evaluated on Perca fluviatilis, the best result was obtained at the 1:50 dilution.⁵⁰ Similarly, in the present study, the best result was obtained at the 1:40 dilution.

The fertilization rate in the present study decreased with increasing dilution ratios (Table 3). This fact was confirmed by researchers who found that the motility and fertility of deep frozen spermatozoa of C. carpio were reported to be significantly improved when the dilution ratio was reduced from 1:100 to 1:2.⁵ The milt to dilution ratio had a strong effect on postthaw motility and fertility.^9,10

Differences in the fertilization rate between cryopreserved and fresh milt were noticed in the present study. Since cryopreservation techniques involve the addition of cryoprotectants, freezing, and thawing of sperm samples, the fertilization rate is decreased with respect to the damage of spermatozoa.⁵¹ A higher thawing temperature is necessary to recover the membrane stability or metabolism of spermatozoa.³⁵

Researchers²¹ have studied a sperm–egg ratio of 500,000 sperm per egg⁵² for rainbow trout and stated that the number of spermatozoa used in the study was too high, which resulted in camouflaging of the fertilizing capacity of spermatozoa. Since the sperm–egg ratio used in the present study was 250,000 sperm per egg,²⁵ good results were obtained with the fertilization rate, and it can be suggested that 250,000 sperm per egg will be optimum for fertilization studies in common carp.

There was significant difference between the hatching rates of cryopreserved and fresh milt (p < 0.05). In both the cases, milt diluted at a 1:40 ratio alone gave higher hatching rate (Table 4). The hatching rate obtained with milt cryopreserved with FWFS at different dilution ratios alone was statistically significant (p < 0.05).

The fertilization rate of cryopreserved spermatozoa was similar with the fresh sperm although differences were observed in the hatching rate.¹³ A reason for the low hatching rate from cryopreserved spermatozoa might be due to the cryoinjury that could happen during freezing and thawing of the spermatozoa.⁴² At the higher dilution rate of 1:120, the postthaw motility, fertilization, and hatching rates were much less when compared with the 1:40 dilution. A possible explanation for this could be that, as found in rainbow trout, dilution is known to reduce sperm motility by removing the protective components of seminal plasma.^53,54

Conclusion

From the present study, it can be inferred that milt cryopreserved with FWFS at a dilution ratio of 1:40 alone exhibited the highest motility duration, fertilization, and hatching rates. Hence, FWFS at the 1:40 ratio can be used for further cryopreservation and artificial fertilization programs of common carp spermatozoa to obtain quality fish seeds.

Footnotes

Acknowledgments

University Grants Commission, New Delhi, is greatly acknowledged for providing funding support through Maulana Azad National Fellowship to carry out the research work.

Author Disclosure Statement

No conflicting financial interests exist.

References

Gausen

. The Norwegian gene bank programme for Atlantic salmon (Salmo salar). In: Cloud JG, Thorgaard GH (eds). Genetic Conservation of Salmonid Fishes. New York: Plenum; 1993: 181–187.

Cloud

, Miller

, Levanduski

. Cryopreservation of sperm as a means to store salmonid germplasm and to transfer genes from wild fish to hatchery populations. Prog Fish Cult, 1990; 52:51–53.

Graybill

, Horton

. Limited fertilisation of steelhead trout eggs with cryopreserved sperm. J Fish Res Brd Canada, 1969; 26:1400–1404.

Kurokura

, Hirano

, Tomita

, Iwahashi

. Cryopreservation of carp sperm. Aquaculture, 1984; 37:267–273.

Cognie

, Billard

, Chao

. Freezing of the milt of the common carp, Cyprinus carpio. J Appl Ichthyol, 1989; 5:165–176.

Billard

. Artificial insemination in salmonids. In: Iwamotoa RN, Sewer S (eds). Salmonid Reprodution. Seattle, WA: Sea Grants Publication; 1985: 116–128.

Poon

, Johnson

. The effect of delayed fertilization on transported salmon eggs. Prog Fish Cult, 1970; 32:81–84.

Rieniets

, Millard

. Use of saline solutions to improve fertilization of northern pike eggs. Prog Fish Cult, 1987; 48:117–119.

Rana

, McAndrew

. The viability of cryopreserved tilapia spermatozoa. Aquaculture, 1989; 76:335–345.

10.

Gwo

, Strawn

, Longnecker

, Arnold

. Cryopreservation of Atlantic croaker spermatozoa. Aquaculture, 1991; 94:355–375.

11.

Reenaselvi. Cryopreservation of carp spermatozoa. PhD Thesis, TANUVAS; 1994: 166.

12.

Lubzens

, Daube

, Pekarskay

, et al. Carp (Cyprinus carpio L.) spermatozoa cryobanks—Strategies in research and application. Aquaculture 1997;155:13–30.

13.

Lahnsteiner

, Berger

, Weismann

. Effects of media, fertilization technique, extender straw volume and sperm to egg ratio on hatchability of cyprinid embryos, using cryopreserved semen. Theriogenology, 2003; 60:829–841.

14.

Jing

, Huang

, Bai

, Tanguay

, Dong

. Optimization of activation, collection, dilution and storage methods for zebrafish sperm. Aquaculture, 2009; 290: 65–171.

15.

Thomas

, Rath

SCh.

, Mohapatra

. Breeding and Seed Production of Fin Fish and Shell Fish. Delhi: Daya Publishing House; 2003: 402.

16.

Zaniboni

, Weingartner

. Induced breeding in migratory fishes. Rev Bras Reprod Anim, 2007; 31:367–373.

17.

Secer

, Tekin

, Bozkurt

, Bukan

, Akcay

. Correlation between biochemical and spermatological parameters in rainbow trout (Oncorhynchus mykiss) semen. Isr J Aquacult, 2004; 56:274–280.

18.

Betsy

, Stephen

JSK

. New classification of motility score in fishes to determine the quality of spermatozoa. Int J Fish Aquat Stud, 2014; 1:20–13.

19.

Jamieson

. Fish Evolution and Systematic: Evidence from Spermatozoa. New York: Cambridge University Press; 1991: 319.

20.

Babiak

, Glogowski

, Goryczko

, et al. Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa. Theriogenelogy, 2001; 56:177–192.

21.

Judycka

, Ciereszko

, Dobosz

, Zalewski

, Dietrich

. Effect of dilution in sperm maturation media and time of storage on sperm motility and fertilizing capacity of cryopreserved semen of sex-reversed female rainbow trout. Gen Comp Endocrinol, 2017; 245:89–93.

22.

Bozkurt

, Akcay

, Tekin

, Secer

. Effect of freezing techniques, extenders and cryoprotectants on the fertilization rate of frozen rainbow trout (Oncorhynchus mykiss) sperm. Isr J Aquacult Bamid, 2005; 57:125–130.

23.

Kurokura

, Hirano

, Tomita

. Cryopreservation of rainbow trout sperm. Bull Jpn Soc Sci Fish, 1980; 46:1493–1495.

24.

Sultana

, Nahiduzzaman

, Hassan

, Khanam

MUH

, Hossain

MAR

. Fertility of cryopreserved common carp (Cyprinus carpio) spermatozoa. Univ J Zool Rajshahi Univ, 2010; 28:51–55.

25.

Aliniya

, Khara

, Noveiri

, Dadras

. Influence of age of common Carp (Cyprinus carpio) brood stock on reproductive traits and fertilization. Turk J Fish Aquat Sci, 2013; 13:19–25.

26.

Tekin

, Secer

, Akcay

, Bozkurt

, Kayam

. The effect of age on spermatological properties in rainbow trout (Oncorhynchus mykiss W. 1792). Turk J Vet Anim Sci, 2003; 27:37–44.

27.

Bromage

, Cumaranatunga

. Egg production in the rainbow trout. In: Muir JF, Roberts RJ (eds). Recent Advances in Aquaculture. London: Croom Helm; 1988: 63–138.

28.

Hanjavanit

, Kitancharoen

, Rakmanee

. Experimental infection of aquatic fungi on eggs of African catfish (Clarias gariepinus Burch). KKU Sci, 2008; 36:36–43.

29.

Keeran

, Woods

. An evaluation of extenders for the short-term storage of striped bass milt. N Am J Aquacult, 2002; 64:248–256.

30.

Şahin

, Kurtoğlu

, Balta

. Effect of different extenders and storage periods on motility and fertilization rate of rainbow trout (Oncorhynchus Mykiss) semen. Univ J Agric Res, 2013; 1:65–69.

31.

Hammerstedt

, Graham

, Nolan

. Cryopreservation of mammalian sperm: What we ask them to survive. Andrology, 1990; 11:73–88.

32.

Ohta

, Shimma

, Hirose

. Relationship between fertility and motility of cryopreserved spermatozoa of the amago salmon Oncorhynchus masou ishikawae. Fish Sci, 1995; 61:886–887.

33.

Chao

, Liao

. Cryopreservation on finfish and shellfish gametes and embryos. Aquaculture, 2001; 197:161–189.

34.

Viveiros

TMA

, Taffarel

, Leal

. Osmolality and composition of the extender during the cold storage of Prochilodus lineatus (Characiformes: Prochilodontidae) sperm. Neotrop Ichthyol, 2014; 12:643–648.

35.

Bozkurt

, Yavas

, Karaca

. Cryopreservation of

Brown trout

(Salmo trutta macrostigma) and Ornamental Koi carp (Cyprinus carpio) sperm. In: Katkov I (ed). Current Frontiers in Cryopreservation. InTech;, 2012:293–304. DOI: 10.5772/32284.

36.

Stoss

. Fish gamete preservation and spermatozoa physiology. In: Hoar WS, Randall DJ, Donaldson EM (eds). Fish Physiology (IX b). New York: Academic Press; 1983: 305–350.

37.

Ohta

, Izawa

. Diluent for cool storage of the Japanese eel (Anguilla japonica) spermatozoa. Aquaculture, 1996; 142:107–118.

38.

Mengumphan

, Whangchai

, Amornlerdpison

. Effects of extender type, sperm volume, cryoprotectant concentration, cryopreservation and time duration on motility, survival and fertilisation rates of Mekong giant catfish sperm. Maejo Int J Sci Technol, 2010; 4:417–427.

39.

Mongkonpunya

, Pupipat

, Pholprasith

, et al. Sperm cryopreservation of Mekong giant catfish Pangasinodon gigas (Chevy). In: Proceedings of a Network Meeting on Aquaculture. Washington, DC: National Academy Press; 1992: 56–60.

40.

Honeyfield

, Krise

. Measurement of milt quality and factors affecting viability of fish spermatozoa. In: Tiersch TR, Mazik PM (eds). Cryopreservation in Aquatic Species. Baton Rouge, LA: World Aquaculture Society; 2000: 49–58.

41.

Linhart

, Mims

, Gomelsky

, et al. Spermiation of paddlefish (Polyodon spathula) stimulated with injection of LHRH analogue and carp pituitary extract. Aquat Liv Res, 2000; 13:1–6.

42.

Muchlisin

. Current status of extenders and cryoprotectants on fish spermatozoa cryopreservation. Biodiversitas, 2004; 6:66–69.

43.

Chao

, Chan

, Liu

, Liao

. The properties of Tilapia sperm and its cryopreservation. J Fish Biol, 1987; 30:107–119.

44.

Billard

, Cosson

. Some problems related to the assessment of sperm motility in freshwater fish. J Exp Zool, 1992; 261:122–131.

45.

Kurokura

. Studies of Preservation of Salmon and Trout sperm. PhD Dissertation, Tokyo University, 1979: 164.

46.

Hara

, Canto

. JT, Almendras JME. A comparative study of various extenders for milkfish, Chanos chanos (Forsskal), sperm preservation. Aquaculture, 1982; 28:339–346.

47.

Goodall

, Blackshaw

, Capra

. Factors affecting the activation and duration of motility of the spermatozoa of the summer whiting (Sillago ciliata). Aquaculture, 1989; 77:243–250.

48.

McPartlin

, Littell

, Mark

, Nelson

, Travis

, Bedford-Guaus

. A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis. Theriogenology, 2008; 69:639–650.

49.

Sarder

MRI

, Rahman

AKMA

, Samad

, Nazrul

KMS

, Bhuiyan

MKJ

. Cryopreservation of sperm of Labeo rohita (Hamilton, 1822) and its use in fertilization and hatching of eggs. Prog Agric, 2011; 22:123–137.

50.

Alavi

SMH

, Rodina

, Policar

, Kozak

, Psenicka

, Linhart

. Semen of Perca fluviatilis L.: Sperm volume and density, seminal plasma indices and efefcets of dilution ratio, ions and osmolality on sperm quality. Theriogenology 2007;68:276–283.

51.

Kopeika

, Kopeika

, Zhang

, Rawson

. Studies on the toxicity of dimethyl sulfoxide, ethylene glycol, methanol and glycerol to loach (Misgurnus fossilis) sperm and the effect on subsequent embryo development. Cryo Letters, 2003; 24:365–374.

52.

Dietrich

, Nynca

, Dobosz

, Zalewski

, Ciereszko

. Application of glucose-methanol extender to cryopreservation of semen of sex-reversed females rainbow trout results in high post-thaw sperm motility and fertilizing ability. Aquaculture, 2014; 434:27–32.

53.

Lahnsteiner

. Characterization of seminal plasma proteins stabilizing the sperm viability in rainbow trout (Oncorhynchus mykiss). Anim Reprod Sci, 2007; 97:151–164.

54.

Lahnsteiner

, Mansour

, Berger

. Seminal plasma proteins prolong the viability of rainbow trout (Oncorhynchus mykiss) spermatozoa. Theriogenology, 2004; 62:801–808.