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ISSN : 2508-755X(Print)
ISSN : 2288-0178(Online)
Journal of Embryo Transfer Vol.33 No.3 pp.185-194

Effects of ice-binding protein from Leucosporidium on the cryopreservation of boar sperm*

Sang Hyoun Park1, Keon Bong Oh1, Sun-A Ock1, Sung June Byun1, Hwi-Cheul Lee1, Suresh Kumar1, Sung Gu Lee2, Jae-Seok Woo1
1Animal Biotechnology Division, National Institute of Animal Science, RDA, Republic of Korea
2Unit of Polar Genomics, Korea Polar Research Institute, KIOST, Incheon 21990, Korea
Correspondence: Jae-Seok Woo Phone: +82-63-238-7251, Fax: +82-63-238-7297 E-mail:
04/09/2018 20/09/2018 27/09/2018


The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted (1.5×108/ml) in lactose egg yolk (LEY) and cooled at 5°C for 3 h. The cooled semen was then diluted (1×108/ml) in LeIBP containing LEY with 9% glycerol and maintained at 5°C for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen (LN2) vapors for 20 minutes and then plunged into LN2. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.


    Rural Development Administration


    Boar sperm is one of the most important factors in breeding, which is essential for reproduction. However, it is difficult to preserve the semen for long term retention of survival rate and fertilization ability because of decrease in the liquid phase during preservation (Silva and Gadella, 2006). Development of successful methods of cryopreservation of boar spermatozoa for long term could contribute big advances for artificial insemination in farm condition.

    Boar sperm membrane contains a high proportion of unsaturated fatty acids in the phospholipids, which is low in resistance to cold shock due to lipid peroxidation (Darin-Bennet et al., 1976). In addition, the cold shock of spermatozoa is associated with oxidative stress due to the generation of reactive oxygen species (ROS) (Chatterjee and Gagnon, 2001). DNA damage and loss of sperm function have been frequently observed by the oxidative stresses from temperature changes (Malo et al., 2012). Antifreeze proteins (AFPs) functions to inhibit growth and recrystallization of intracellular ice crystals at a sub-zero temperature (Ben, 2001). One of AFPs isolated from Leucosporidium a polar organism, ice-binding protein (LeiBP) has been considered as a reagent to protect damages by frozen (Barrett, 2001), which was known to be useful for frozen foods, cell preservation, and genetic resource conservation (Harding et al., 2003; Fuller, 2004; Venketesh and Dayananda, 2008). Previously, Kim et al (2014) reported that the thermal hysteresis (TH) activity of LeIBP in mass production was similar to the temperature hysteretic activity obtained from the polar organisms.

    To find efficient process for cryopreservation of sperm, we examined influence of LeIBP, supplemented in freezing extender as an intracellular ice crystals inhibitor for boar spermatozoa. In this study, we focused effect of LeIBP on post-thawed sperm in terms of motility, viability, acrosome integrity, and biochemical parameters. Consequently, we discussed optimal concentration of LeIBP for cryopreservation of boar semen.


    1. Chemicals

    All the chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) except stated otherwise. High-purity water (6114VF, Sartorius AG, Goettingen, Germany) was used to prepare solutions.

    2. Preparation of Leucosporidium ice-binding protein (LeIBP)

    LeIBP was kindly provided from Korea Polar Research Institute (Incheon, Korea). LeIBP stocks (0.1, 0.5, 1, 5 and 10 mg/ml) were prepared in triple distilled water and filtered through a 0.22 μm filter (Sartorius, Goettingen, Germany). LeIBP stocks were stored at -20 °C until use.

    3. Collection of boar semen

    Eight boars (Duroc) were used for this experiment. Semen was collected using the gloved-hand technique and filtered through four layers of sterile gauze to remove gel particles. The sperm-rich fractions of ejaculates with greater than 75% motile and 80% morphologically normal spermatozoa were used in this study (Pursel and Johnson, 1975). The collected semen (from 2 or 3 boars) were pooled to reduce the effect of individual differences. The pooled semen was extended ([1 : 1(v : v)]) in Androhep plus (minitube). The diluted spermatozoa were evaluated for volume, sperm concentration, sperm morphology and percentage of motile spermatozoa. Immediately after evaluation, the diluted spermatozoa were stored at 24°C for 2 h.

    4. Sperm freezing-thawing protocol

    The freezing extenders used in the experiments were composed of freezing extender 1 (FE 1) and freezing extender 2 (FE 2). FE 1 was composed of lactose egg yolk (LEY) extender (80 ml of lactose solution, 20 ml of egg yolk and 0.1% antibiotic-antimycotic in 100 ml sterile non-pyrogenic water). FE 2 consisted of LEY extender supplemented with 9% (v : v) glycerol and 1.5% (v : v) Equex STM. Semen was processed according to the freezing procedure (Guthrie and Welch, 2006). Briefly, Semen was diluted in Androhep plus and centrifuged at 850 ×g for 15 min at room temperature. After removing supernatant, the sperm pellet was re-suspended with FE 1 to a concentration of 1.5 x 108/ml. The sperm suspensions were cooled gradually from 24°C to 5°C for 3 h. The sperm was maintained at 5°C for 30 min after a second dilution step to 1 x 108/ml with FE 2 at 5°C. The cooled sperm was loaded into 0.5 ml straws and sealed.

    A styrofoam box (29.5 × 18.7 × 24 cm3) was filled with LN2 to a depth of 5 cm and a rack with two bars was set to 7 cm from the surface of the LN2. The straws were then aligned horizontally for 20 min on the rack in the LN2 vapor and then plunged into LN2 for storage. Straws from each group were thawed by immersion in a circulating water bath at 37°C for 25 sec before use in experiments.

    5. Assessment of sperm motility

    The percentage rate of total motile spermatozoa was evaluated and recorded using computer-assisted sperm analysis (CASA) system (SCA Production v 5.3.: MICROPTIC S.L., Barcelona, Spain) with a phase contrast microscope Eclipse E200 (Nikon, Tokyo, Japan) at 200 magnification when five fields of view per straw were evaluated at least. Chambers (20 um; Leja, Nieuw Vennep, The Netherlands) were loaded with semen and maintained at 37 °C. Semen samples were evaluated immediately after thawing (Jeong et al., 2009).

    6. FITC-PSA/PI staining for Sperm viability and acrosome integrity

    The fluorescein isothiocyanate-pisumsativum agglutinin (FITC-PSA; green fluorescence)/PI dual-staining method was used to assess the integrity of the acrosome plasma membrane as described previously (Subas et al., 2014; Gurler et al., 2016).

    7. Sperm intracellular ROS level

    2′, 7′-dichlorodihydrofluorescein diacetate ([H2DCFDA (DCF); Molecular Probes Inc.]) was used to detect H2O2 (Park and Yu, 2005). The working solution of 20 mM DCF was prepared in dimethyl sulfoxide. Aliquots of 500 μL of semen sample (1 × 105 sperm/mL) were mixed with DCF to a final concentration of 200 μM. For simultaneous differentiation of living from dead sperm, propidium iodide (PI; final concentration, 2 μM) was added to DCF sperm. Samples stained with DCF and PI were incubated at 25°C for 60 min.

    8. BODIPY-C11 581/591 staining for sperm lipid peroxidation (LPO)

    The fluorescent fatty acid analog BODIPY-C11 581/591 was used to detect LPO in the plasma membrane of spermatozoa (Ball and Vo, 2002), the intact probe fluoresces red when intercalated into the membrane and its fluorescence shifts towards green (520 mM) emission after the attack with oxidative radicals. To quantify the LPO of the plasma membrane, intact sperm 12.5 ul of BODIPY-C11 581/591 (2mM), and 7.5 ul of PI (2.4mM) were added to 980 ul of diluted sperm suspension with 1x Dulbecco’s phosphate buffered saline. Samples stained with BODIPY-C11 581/591 and PI were incubated at 37°C for 30 min.

    9. Apoptosis rate [Phosphatidylserine (PS) translocation]

    An Annexin V-FITC apoptosis detection kit I (BD Pharmingen, San Diego, CA, USA) was used according to the manufacturer's instructions. The sperm suspension was centrifuged at 300 x g for 5 min and the supernatant was removed. The sperm pellet was resuspended in 1X Annexin V binding buffer ([10 mM HEPES/NaOH (pH 7.4], 140 mM NaCl and 2.5 mM CaCl2]) at room temperature to a concentration of 1 x 106 sperm/mL. Aliquots (100 μL of 1 x 105 cells) of the sperm suspension were transferred to 5 mL culture tubes. Annexin V-FITC (5 μL) and 5 μL of propidium iodide (PI) or nothing were added to the samples. The tubes were gently mixed and incubated at room temperature for 15 min in the dark. After incubation, an additional 1X binding buffer (400 μL) was added to each tube and flow cytometric analysis was conducted within 1 h. The PS translocation rate was only measured for the live sperm population. The different labeling patterns in the Annexin V (AN)/PI analysis were classified as follows: viable (AN−/PI−); viable but PS translocated (AN+/PI−); nonviable and PS translocated (AN+/PI+) and nonviable and late necrotic sperm (AN−/PI+). We defined the ratio between AN+/PI− sperm and total living (PI−) sperm as the PS translocation index.

    10. Flow cytometry

    All fluorescence signals of labeled spermatozoa were analyzed with a (FACS Calibur) flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 15 mW air-cooled 488 nm argon-ion laser. This technique was used in the assays for determining viability, acrosome integrity, PS externalization events, lipid peroxidation, and ROS levels. Data were collected from 1 x 104 sperm, and then analyzed using Cell Quest Pro software (Becton Dickinson, San Jose, CA, USA).

    11. Statistical analyses

    Percentage data were subjected to arcsine transformation before analysis. All data are presented as mean ± SE and were analyzed using ANOVA followed by Duncan’s multiple range test. Statistical Analysis System Ver. 8x software (SAS, Cary, NC, USA) was used and P < 0.05 was considered statistically significant. Two-tailed tests were used and statistical significance was set at P < 0.05.


    1. Effect of LeIBP supplementation in LEY extender on sperm motility of boar spermatozoa after cooling and post-thaw

    Sperm parameters the according to LeIBP concentrations after cooling and post-thaw are shown in Fig 1. The motility of spermatozoa after cooling ([control (50.6 ± 4.4%), 0.001 (52.6 ± 4.9%), 0.005 (54.3 ± 9.2%), 0.01 (65.4 ± 6.0%), 0.05 (58.4 ± 5.9%) and 0.1 (45.2 ± 3.8%)]) and post-thaw ([control (24.3 ± 1.6%), 0.001 (27.6 ± 3.3%), 0.005 (24.9 ± 1.2%), 0.01 (22.8 ± 0.6%), 0.05 (21.8 ± 1.6%) and 0.1 (23.9 ± 2.3%)]) was not significantly different among the groups.

    2. Effects of LeIBP supplementation on viability and acrosome integrity

    As shown in Fig 2, the percentage of viable sperm in the 0.05 (49.0 ± 1.6%) and 0.1 mg/ml (48.3 ± 04%) LeIBP groups were significantly higher than the other groups ([control (39.6 ± 0.6%), 0.001(35.6 ± 0.3%), 0.005 (41.0 ± 0.2%) and 0.01 (41.3 ± 0.7%) mg/ml LeIBP group (P < 0.05)]). However, 0.001(35.6 ± 0.3%) mg/ml LeIBP group was significantly lower among all the groups (P < 0.05).

    Acrosomal status of sperm detected by FITC/PSA stain using the FACS system is summarized in Fig 2. Acrosome integrity rate in 0.005 (89.1 ± 0.6%), 0.01(87.7 ± 0.1%), 0.05 (87.2 ± 0.1%) and 0.1 (90.3 ± 0.8%) mg/ml LeIBP groups were significantly higher than the control (83.7 ± 0.2 %) group (P < 0.05). However, 0.001 (85.3 ± 0.6%) mg/ml LeIBP group was not significantly different with control group.

    3. Effect of LeIBP supplementation on ROS level

    As shown in Fig 3, ROS levels in the LeIBP groups were significantly lower than the control group (P < 0.05). Live cells labeled with H2DCFDA fell in the lower left and the right quadrant.

    4. Effect of LeIBP supplementation on lipid peroxidation

    As shown in Fig 4, the undetected population of spermatozoa from BODIPY stain for lipid peroxidation in the 0.001 (40.4 ± 1.7%) mg/ml group was significantly higher than 0.005 (30.0 ± 1.5%), 0.01(33.3 ± 1.6%), 0.05 (34.2 ± 1.4%) and 0.1 (33.7 ± 0.5%) mg/ml group (P < 0.05). However, the LeIBP supplementation had a linear increase compared with those in the control (23.± 0.3%) group (P < 0.05).

    5. Effect of LeIBP supplementation on phosphatidylserine (PS) translocation

    Apoptosis density detected by FACS system analysis, resulted in as presented in Fig 5. Apoptosis rate significantly increased in 0.005 mg/ml LeIBP group (0.48 ± 0.04%). However, 0.05, 0.001, 0.01 and 0.1 mg/ml LeIBP group was a significant linear increase (0.43 ± 0.02 %), (0.33 ± 0.0%), (0.33 ± 0.01%) and (0.38 ± 0.05%) (P < 0.05). No significant responses were observed for control (0.23 ± 0.02%) group (P < 0.05).


    It is well known that the organisms living in polar regions possess specific proteins to inhibit ice crystal growth which results in reduction of freezing point of water, and to prevent at low temperature (Cheng, 1998; Tomczak et al., 2002; Venketesh and Dayananda, 2008). In our knowledge, in this study we first attempted analyses of LeIBP effect on cryopreservation of boar sperm.

    1. Effect of LeIBP on motility

    The effects of LeIBP supplementation on sperm motility have been reported in different mammalian species. Panyne et al. (1994) reported that sperm motility decreased during the cold stage, while it improved after thawing in the ram (Panyne et al,, 1994). On the other hand, Younis et al, (1998), literature has reported that improvement in post-thaw motility was reported when the freezing extender was supplemented with AFP III in chimpanzee sperm. In the present study, the LeIBP supplementation did not result in an enhancement of motility rate of spermatozoa after cooling. Moreover, there were no significant differences in sperm motility when comparing the cryopreserved spermatozoa.

    Variable results have been reported on the capacity of AFPs to protect sperm during the different freeze-thaw procedure in mammals with both positive and negative results (Panyne et al., 1994; Prathalingam et al., 2006). It seems that there are differences in the effects of AFPs according to types and animals species. Therefore, we suggest that the LeIBP used in this experiment does not seem to affect the motility of frozen-thawed boar sperm.

    2. Effect of LeIBP on viability and acrosome integrity

    In the current study, the viability of boar spermatozoa was improved (live sperm with intact acrosome) when 0.05 and 0.1 mg/ml LeIBP were supplemented in freezing extender (Fig 2). Our results were in agreement Younis et al. (1998) who observed that the viability of frozen bull spermatozoa was improved with the exposure of AFP III. On the contrary, Qadeer et al. (2014) reported that the viability of frozen a buffalo bull spermatozoa was not improved when AFP was supplemented in the extender.

    The acrosomal intactness of sperm is crucial in carrying hydrolytic enzymes required for oocyte penetration and thus aids in fertilization (McLeskey et al., 1998). It has been reported that AFP has a positive effect in protecting the plasma membrane of bovine oocytes and pig oocytes. AFP also stabilizes the buffalo spermatozoa (Rubinsky et al., 1991; Arav et al., 1993; Qadeer et al., 2014). The varying effects of AFPs on acrosome integrity in different species might be attributed to different cooling methods, different membrane composition and phase transition temperature of the lipid membrane of different species. The improvement in post-thaw plasma membrane integrity of boar spermatozoa with supplementation of LeIBP may be attributed to the interaction of LeIBP with unsaturated fatty acid, stabilizing the plasma membrane organization during cryopreservation processes. Therefore, we conclude that LeIBP has a function to stabilize the acrosome of sperm in cryopreservation processes.

    3. Effect of LeIBP on ROS level

    To the best of our knowledge, the present study is the first to examine the ROS level in response to LeIBP in boar spermatozoa. As shown in Fig 3, LeIBP supplementation in freezing medium significantly decreased ROS levels in boar sperm. LeIBP has a function of antioxidant due to the ability to inhibit ROS formation (level) by the enzymatic and non-enzymatic system. LeIBP has also the ROS scavenging activity, which helps to explain our results. ROS level in this study was similar to those of antioxidants used to reduce oxidative stress during freezing process (Malo et al.,2011; Shen et al., 2015; Park et al., 2017). Although AFP is known to lower the freezing point, it is presumed that LeIBP also has the function of inhibiting the generation of ROS during the freezing and thawing process.

    4. Effect of LeIBP on lipid peroxidation (LPO)

    If the generation of oxidized substances is not prevented, it may lower the motility of sperm, induce the lipid peroxidation in the sperm membrane and eventually damage sperm DNA. Antioxidants have been used to prevent these conditions (Armstrong et al., 1999; Chatterjee and Gagnon, 2001; Fraser and Strzezek, 2005). Frozen spermatozoa are sensitive to LPO which is formed following the oxidation of membrane lipid by ROS (Bucaket al., 2010). In addition, we observed for LeIBP in the freezing extender increased undetected LPO percentage compared to control group. As shown in Fig 4, LeIBPs treatment significantly increased the percentage of undetected sperm due to LPO stain from FACS, presumably due to their lower ROS level, as described above.

    5. Effect of LeIBP on PS translocation

    Apoptosis is the programmed cell death that affects single cells (Wyllie et al., 1980). It has been demonstrated that PS translocation can be used as a marker for disturbed membrane function of post-thawing human and bovine spermatozoa (Kemal Duru et al., 2001; Anzar et al., 2002). Moustafa (2004) and Kotwicka (2011) reported that ROS leads to PS translocation in human sperm. In agreement with these studies, our result also revealed that 0.005, 0.05 and 0.1 mg/ml LeIBP groups significantly increased the percentage of apoptotic sperm then control. Similarly, Li et al. (2018) reported that Resveratrol (Res) possesses stronger antioxidant activity with toxicity. Although Res is an antioxidant, as the concentration of Res increases, the percentage of apoptotic sperm also increases than the control. However, the percentage of apoptotic sperm increased within 0.5% in total sperm population in our FACS results, when we compare with other results (Pena et al., 2003; Li et al., 2018). We suggested that though LeIBP could reduce the ROS but induced cell death program in freezing and thawing processes. The mechanism of LeIBP activity seems to be different from apoptosis and ROS.

    6. Conclusion

    In conclusion, our results obtained in the present study revealed that treatment with the different concentration of LeIBP supplementation was significantly decreased the levels of ROS, lipid peroxidation, and protected acrosome integrity and viability. However, LeIBP has increased the apoptotic sperm in frozen-thawed boar sperm.



    Effect of LeIBP on motility after cooling and thawing boar spermatozoa.

    LeIBP: Leucosporidium ice binding protein, Values are not significantly different (P > 0.05).


    Effect of LeIBP on viability and acrosome integrity after cooling and thawing boar spermatozoa.

    Values are expressed as mean ± SE a,b,c, d different superscripts differ significantly (P < 0.05).


    Effect of LeIBP on ROS levels after cooling and thawing boar spermatozoa.

    ROS: reactive oxidative species, Values are expressed as mean ± SE. a,b,c different superscripts differ significantly (P < 0.05).


    Effect of LeIBP on LPO rate after cooling and thawing boar spermatozoa.

    LPO: lipid peroxidation, Values are expressed as mean ± SE. a,b,c different superscripts differ significantly (P < 0.05).


    Effect of LeIBP on PS translocation rate after cooling and thawing boar spermatozoa.

    PS: phosphatidylserine, Values are expressed as mean ± SE. a,b,c different superscripts differ significantly (P < 0.05).



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