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Effect of cholesterol-loaded-cyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation
Animal Reproduction Science 159 (2015) 124–130
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effect of cholesterol-loaded-cyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation Yong-Seung Lee a,1 , Seunghyung Lee a,b,∗,1 , Sang-Hee Lee a , Boo-Keun Yang a , Choon-Keun Park a a b
College of Animal Life Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea Institute of Animal Resources, Kangwon National University, Chuncheon 200-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 10 April 2015 Received in revised form 2 June 2015 Accepted 3 June 2015 Available online 9 June 2015 Keywords: Cholesterol-loaded-cyclodextrin Spermatozoa Viability Cryopreservation
a b s t r a c t This study was undertaken to examine the effect of cholesterol-loaded-cyclodextrin (CLC) on boar sperm viability and spermatozoa cryosurvival during boar semen cryopreservation, and methyl-ˇ-cyclodextrin (MBCD) was treated for comparing with CLC. Boar semen treated with CLC and MBCD before freezing process to monitor the effect on survival and capacitation status by flow cytometry with appropriate fluorescent probes. Sperm viability was higher in 1.5 mg CLC-treated sperm (76.9 ± 1.01%, P < 0.05) than un-treated and MBCD-treated sperm before cryopreservation (58.7 ± 1.31% and 60.3 ± 0.31%, respectively). For CTC patterns, F-pattern was higher in CLC treated sperm than MBCD-treated sperm, for B-pattern was higher in CLC-treated sperm than fresh sperm (P < 0.05). For AR pattern (an acrosome-reacted sperm) was lower in CLC-treated sperm than MBCD-treated sperm (P < 0.05). Moreover, we examined in vitro development of porcine oocytes after in vitro fertilization using CLC-treated frozen–thawed semen, in which CLC treatment prior to freezing and thawing increased the development of oocytes to blastocyst stage in vitro. In conclusion, CLC could protect the viability of spermatozoa from cryodamage prior to cryopreservation in boar semen. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Semen cryopreservation extends the availability of sperm for research, artificial insemination (AI), and in vitro fertilization (IVF) irrespective of time or location. This technique provides the accessibility to sperm, despite of time and location, and permitted the successful development of AI and IVF techniques (Bailey et al., 2008). However,
∗ Corresponding author at: College of Animal Life Science & Institute of Animal Resource, Kangwon National University, Chuncheon 200-701, Republic of Korea. Tel.: +82 33 250 8689. E-mail address: [email protected] (S. Lee). 1 Both the authors contributed equally to this study. http://dx.doi.org/10.1016/j.anireprosci.2015.06.006 0378-4320/© 2015 Elsevier B.V. All rights reserved.
frozen–thawed sperm do not possess the same fertilizing potential as fresh sperm, since the cooling process induces lipid and protein rearrangements within the cell membranes when they are cooled from 22 to 1 ◦ C (Parks and Graham, 1992). This variation was induced by the membrane changing from the fluid to the gel-state at low temperature. Thermotropic phase transitions are considered to be one of the mains reasons for reduced viability and fertility of sperm during the cryopreservation. Especially, boar semen is extremely vulnerable to ‘cold shock’ by cooling during the cryopreservation compared to other species. Sperm sensitivity to cold shock damage is determined primarily by phospholipid constitution and the cholesterol to phospholipid ratio in membrane (Holt, 2000). The cholesterol to phospholipid ratio of the sperm membrane is
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widely considered to be a major determinant in membrane fluidity and stability during cryopreservation. Boar sperm differ from those species that have a high cholesterol to phospholipid ratio in the membrane, such as human and rabbit sperm do not experience this membrane damage when cooled (Darin-Bennett and White, 1977). Membrane damage is considered to be one of the major reasons for cell death (Watson, 1981). Membrane destabilization occurs when the membrane undergoes the phase transition, from the fluid phase to the gel phase, as temperature is decreased. During this phase transition, phospholipids are lost from the plasma membrane leading to increased membrane permeability, membrane disruption, and cell death (Darin-Bennett et al., 1973; Watson, 1981). If the temperature at which the membrane lipid phase transition occurs could be lowered, or if the membrane phase transition could be eliminated completely, membranes would remain fluid at low temperatures and the damage that occur to membranes may be reduced. Previous studies indicated that the cholesterol/phospholipid ratio of the plasma membrane is a major determinant in plasma membrane fluidity and stability during the cryopreservation (Darin-Bennett and White, 1977; Watson, 1981). Cholesterol, a major structural constituent of the membrane, plays an important role as a regulator of membrane function (Yeagle, 1985). Furthermore, cholesterol in the cell membrane has been shown to be an important determinant of membrane fluidity (Hartel et al., 1998) and permeability (McGrath, 1988). Also, cholesterol reduces the phase transition temperature of membrane, and maintains it in a fluid state at reduced temperature thereby reducing the membrane damage that occurs at low temperatures (Amann and Pickett, 1987). Cholesterol can be incorporated readily into or extracted from the membrane of cells using cyclodextrins (Purdy and Graham, 2004; Zeng and Terada, 2001). Cyclodextrins are cyclic heptasaccharides consisting of ˇ (1–4) glucopyranose units, are water soluble but have a hydrophobic center (Purdy and Graham, 2004), and can transport cholesterol in or out of membranes down a concentration gradient (Klein et al., 1995). When cholesterol is loaded into bull sperm membrane prior to cryopreservation with cholesterol-loaded cyclodextrin (CLC), a higher percent sperm motility and membrane were recovered after thawing compare to un-loaded sperm (Purdy and Graham, 2004). However, this procedure has not yet been optimized for boar sperm, nor is it known how the cholesterol regulates the cryosurvival of boar sperm and the function of physiology of the membrane. Additionally, we also used methyl-ˇ-cyclodextrin (MBCD) for this study, since MBCD induces the capacitation and efflux of human sperm (Chiu et al., 2005). Thus, understanding of the regulation of the cholesterol and the protection of sperm membrane and viability are important to the overall understanding cryosurvival during sperm cryopreservation. In this study, we evaluated the effect of cholesterol modification on the cryosurvival of boar sperm during a conventional semen freezing procedure. Further, using cyclodextrin, we investigated in vitro development of porcine oocytes after in vitro fertilization using CLC-treated frozen–thawed semen.
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2. Materials and methods 2.1. Preparation of CLC We prepared cholesterol-loaded-cyclodextrins (CLC) such as method of Purdy and Graham (2004). Briefly, 1 g methyl-ˇ-cyclodextrin (Sigma, St. Louis, MO, USA) was dissolved in 2 mL methanol. 200 mg cholesterol (Sigma) was dissolved in 1 mL chloroform in a separate glass tube. Then a 0.45 mL CLC solution was added to the cyclodextrin solution, and mixed and stirred. The solvents removed using a stream of N2 gas. Finally, the resulting crystals were stored in a glass container at 22 ◦ C, after dry. The working solution was prepared by adding 100 mg of CLC to 1 mL m-Modena B (Lee et al., 2005) at 37 ◦ C. 2.2. Semen collection and treatment with CLC Semen was collected from six miniature pigs (PWG Company, Seoul, Korea) by gloved-hand technique and filtered through cotton gauze to remove the gel particles. All procedures that involved the use of animals were approved by the Kangwon National University Institutional Animal Care and Use Committee (KIACUC-09-0139). The ejaculated sperm were transported to the laboratory at 25 ◦ C within 1 h. The ejaculated sperm were diluted with same volume of extender (m-Modena B). After maintenance at room temperature for 10 min, semen was diluted to 1.2 × 108 sperm in 1 mL of m-Modena B, and incubated with different levels of CLC (0, 0.75, 1.5, 3.0, and 6.0 mg/mL) and 1.5 mg/mL MBCD (negative control) for 15 min at 25 ◦ C. The control group (0 mg/mL) was considered to be a positive control. The incubated semen samples were later centrifuged (400 × g, 10 min) to remove CLC and MBCD from sperm before freezing. 2.3. Semen freezing and thawing Semen treated with CLC and MBCD were processed using the straw freezing procedure described with minor modifications (Kim et al., 2006). The incubated semen was re-suspended with first lactose-egg yolk (LEY) extender (80 mL of 11% lactose and 20 mL egg yolk) to provide 5 × 108 sperm/mL and later centrifuged (400 × g, 10 min, 22 ◦ C) to remove CLC and MBCD from sperm, and were cooled to 5 ◦ C for 3 h. After cooling the semen were diluted 2:1 (v:v) with second LEY extender (LEY extender with 1.5% Orvus Es Paste and 9% glycerol). The sperm were packaged into 0.5 mL straws cooled to −120 ◦ C for 10 min before being plunged into liquid nitrogen for storage using static nitrogen vapor. Frozen-sperm was thawed in a water bath at 50 ◦ C for 10 s. 2.4. Cholesterol incorporation in sperm membrane Semen was extended to 1.2 × 108 sperm/mL in m-Modena B and incubated with 0.75 and 1.5 mg CLC/1.2 × 108 sperm for 15 min at 25 ◦ C. In this experiment, the cyclodextrin has been pre-loaded with cholesterol in which the cholesterol was labeled with the fluorescent molecule 22-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
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Fig. 1. Analysis of sperm viability by flow cytometry. (A) It is the created region of sperm for forward (FSC, relative cell size) and side (SSC, cell internal complexity) scatters. (B) It shows the fluorescence intensity of PI (dead cell stain, a), SYBR-14 + PI (double stained cell stain, b), and SYBR-14 (live cell stain, c) in region of sperm (A).
aminl-23,24-bisnor-5-cholen-3B-ol (NBD cholesterol, Invitrogen, Korea). The incubated semen samples were centrifuged (400 × g, 10 min) to remove CLC and MBCD from sperm, then resuspended sperm with phosphate buffer saline. The amount of NBD-labeled cholesterol was measured using flow cytometry. NBD was excited using an argon laser tuned to 488 nm. A total of 10,000 cells were analyzed per sample. The fluorescent image of cholesterol incorporation in the membrane of sperm was monitored by fluorescence microscope. After suspension of semen, 5 L drops of sample were placed onto pre-heated glass slides (37 ◦ C) and examined using a microscope (OLYMPUS LX70 Tokyo, Japan) with an argon laser tuned to 488 nm, a 505 nm dichroic mirror and a 520 nm long-pass filter, to determine sperm membrane that contained NBD fluorescence. 2.5. Assessment of sperm viability and acrosome state Frozen–thawed sperm (1.2 × 108 sperm/mL) were stained with 5 L SYBR-14 (20 M solution in DMSO), and 5 L propidium iodide (2.4 mM solution in water; Molecular Probes, Eugene, OR) for flow cytometric viability analysis. The samples were incubated for 10 min at 37 ◦ C before being analyzed (10,000 cells per sample) using a flow cytometer equipped with an argon laser tuned to 488 nm (Fig. 1). The acrosomal state and capacitation of sperm were assessed using a cyclotetracyclin (CTC) staining method (Abeydeera et al., 1997). Each cell was observed under ultraviolet (UV) illumination (emission at 420 nm). The three fluorescent staining patterns identified were: F pattern (an uncapacitated sperm), with uniform fluorescence over the whole sperm head; B pattern (a capacitated sperm), with a fluorescence-free band in the postacrosome region; and AR pattern (an acrosome-reacted
sperm), with almost no fluorescence over the sperm head. 2.6. In vitro fertilization (IVF) Oocytes were selected and cultured, and used for IVF as described (Lee and Park, 2015). Briefly, we collected ovaries from prepubertal gilts at a slaughter house (Pocheonfarm, Pochoen, Korea) and moved to our laboratory within 2 h. Oocytes were cultured in modified TCM-199 with EGF, hCG, pFF, LH, and FSH in a humidified, 5% CO2 atmosphere at 38.5 ◦ C for 44 h. Oocytes were separated from the enclosing cumulus cells in maturation medium with 0.1% hyaluronidase after 44 h for IVF. After fertilization, extra spermatozoa were removed from the oocytes by repetitive pipetting action and oocytes were washed three times in culture medium (porcine zygote medium: PZM-3 with 0.3% BSA) and incubated at 38.5 ◦ C for 144 h under 5% CO2 in humidified air. The oocyte cleavage and blastocyst rates were counted using microscope. 2.7. Statistical analysis The results were analyzed using Statistical Analysis System (SAS) software version 8.01. Treatment groups were compared for differences using of Duncan’s modified multiple range tests. Data were presented as mean ± SEM, and a 5% probability was considered significant. 3. Results 3.1. Cholesterol incorporation in sperm membrane Flow cytometric analysis of sperm treated with 0.75 mg and 1.5 mg of NBD-labeled CLC indicated that the fluorescence of NBD was increased by cholesterol level (Fig. 2).
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Table 1 In vitro development of porcine oocytes fertilized with addition of CLC frozen–thawed semen at 144 h after in vitro fertilization. Treatments
No. of oocytes in IVF
Cleavage (%)
Control CLC
266 249
209 (79.5 ± 2.6) 199 (80.2 ± 4.3)
No. of embryo development to (%) 2 cell ∼ morula
Blastocytes
172 (80.5 ± 5.7) 144 (75.3 ± 3.1)
37 (17.0 ± 1.8)b 55 (28.3 ± 3.8)a
Values in the same column with different superscripts letters (a, b) differ significantly (P < 0.05). Data are presented as the mean ± SEM (n = 3).
CLC compared to untreated or treated with 1.5 mg MBCD (Fig. 4). The higher percent sperm viability (76.9 ± 1.01%, P < 0.05) was revealed (Fig. 5) after thawing, when 1.5 mg CLC was added to sperm before freezing compared to other CLC levels (3.0 and 6.0 mg) and sperm treated with 1.5 mg MBCD (60.3 ± 0.31%) and untreated sperm (58.7 ± 1.31%). Also, 1.5 mg CLC-added sperm was higher percentages of capacitated acrosome-intact spermatozoa (B pattern) and significant (P < 0.05) lower percentage of acrosome-reacted spermatozoa (AR pattern) than untreated sperm (control) and MBCD-treated samples (Fig. 6). In F pattern (uncapacitated acrosome-intact spermatozoa), CLC-treated sample (P < 0.05) was higher than MBCD-treated sample, not than fresh semen. 3.3. Oocyte cleavage and blastocyst formation rates Fig. 2. Single parameter histogram for the log green fluorescence of boar sperm. The sperms treated with 0.5 and 2 mg cholesterol-loadedcyclodextrin (CLC) per 1.2 × 108 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Micrographs of sperm cells treated with 0.75 mg and 1.5 mg of fluorescently labeled cholesterol indicated that cholesterol was incorporated into the membrane compartment (Fig. 3). The sperm cells treated with 0.75 mg NBD labeled CLC showed acrosomal compartment to be more heavily labeled, but cells treated with 1.5 mg NBD labeled CLC showed fluorescence in the acrosomal and mitochondrial compartments. 3.2. Effect of cholesterol on viability and acrosome state of frozen–thawed sperm Flow cytometric dotplots data for the viability assay of frozen–thawed sperm showed higher sperm viability in the live cell zone (blue quadrant) when treated with 1.5 mg
The embryo cleavage and blastocyst formation rates at 144 h after IVF are summarized in Table 1. 226 oocytes used for control and 249 for CLC-treated samples, respectively. The oocyte cleavage rate in the CLC-treated sperm did not significantly differ, but the blastocyst formation rate was significantly higher in CLC-treated samples (28.3 ± 3.8%) than without CLC (17.0 ± 1.8%, P < 0.05). 4. Discussion Boar spermatozoa are more sensitive to events associated with cryopreservation than other species such as sheep, human, and bull (Medeiros et al., 2002; Padilha et al., 2012). Cryopreservation induces many forms of stress on sperm, including destabilizing the membrane, dilution, storage condition, heat stress, osmotic stress, cold shock, and intracellular ice formation. Cell death is observed during the freezing process especially by formation of intracellular ice crystals and membrane damage
Fig. 3. Fluorescent imaging of cholesterol incorporation in the membrane of sperm as observed by fluorescent microscope; non-treated cholesterol-loadedcyclodextrin (CLC); 0.75 mg CLC treated; 1.5 mg CLC treated.
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Fig. 4. Flow cytometric dot plots show fluorescence signals in boar sperms. The sperms stained with PI and SYBR-14 for viability assay, and control was unstained with PI and SYBR-14, and treated with non-treated cholesterol-loaded-cyclodextrin (CLC), 1.5 mg CLC and 1.5 mg methyl-ˇ-cyclodextrin (MBCD) during the freezing process.
(Hammerstedt et al., 1990; Leeuw et al., 1993; Mazur, 1977; Maxwell and Johnson, 1997; Steponkus et al., 1983). Cholesterol reduces the transition temperature of membranes, and maintains them in a fluid state at reduced temperatures thereby reducing the membrane damage that occurs at low temperatures (Amann and Pickett, 1987; Chakrabarty et al., 2007; Glazar et al., 2009). Cholesterol can easily be incorporated into or extracted from the plasma membranes of cells using cyclodextrins. Li et al. (2006) and Purdy (2004) reported that intact sperm membrane was recovered in frozen–thawed sperm with cholesterol.
Fig. 5. Effect of cholesterol-loaded-cyclodextrin (CLC), methyl-ˇcyclodextrin (MBCD) on viability of frozen–thawed boar sperm. Bars with different superscripts (a–c) within the CLC levels category differ significantly (P < 0.05). Asterisks (*) shows significant difference between CLC- and MBCD-treated groups (P < 0.05).
Similar results have also been reported for stallion sperm treated with CLCs (Moore et al., 2005). In this study, higher proportion of viability and acrosome state of boar sperm treated with CLC was observed than sperm untreated or treated with MBCD (Figs. 5 and 6). The optimal level of CLC for cryopreservation of boar sperm was 1.5 mg CLC per 1.2 × 108 sperm (Fig. 5), which is similar to that reported
Fig. 6. Effect of cholesterol-loaded-cyclodextrin (CLC), methyl-ˇcyclodextrin (MBCD) on CTC pattern of frozen–thawed boar sperm. Bars with different superscripts (a–c) for a particular pattern differ significantly (P < 0.05).
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for bull and stallion sperm (Moore et al., 2005; Purdy and Graham, 2004). However, when treated with CLC levels more than 1.5 mg per 1.2 × 108 sperm, the CLC becomes detrimental to sperm cell survival (Fig. 5). Probably, it may occur by affecting membrane fluidity and membrane function to the point that normal membrane function is affected that leads to sperm death during the freezing. Cholesterol plays an important role in stabilizing sperm membranes. Especially, during capacitation of sperm, the membrane lose cholesterol (Ehrenwald et al., 1988), making them more unstable thereby enabling the acrosome reaction. Sperm capacitation can induce cholesterol efflux from the sperm membrane (Choi and Toyoda, 1998; Cross, 1999; Iborra et al., 2000; Parinaud et al., 2000; Visconti et al., 1999). Similarly, when sperm are cooled or frozen, the membranes undergo a phase transition, which results in rearrangement of membrane lipids and proteins (Hammerstedt et al., 1990; Parks and Graham, 1992) and a loss of lipids from the membranes (DarinBennett et al., 1973), which in turn causes protein and lipid aggregation within the membrane, and a loss of membrane-selective permeability (Bailey and Buhr, 1994; Buhr et al., 1989), resulting in sperm that are prematurely capacitated (Cormier et al., 1997; Parrish et al., 1986). This premature capacitation could reduce fertilizing ability and lifespan of the sperm (Cormier et al., 1997; Parrish et al., 1986). In our results, micrographs of sperm cells treated with fluorescently labeled CLC (Fig. 3) indicated higher cholesterol incorporation in the acrosomal membrane. CLC reduced the premature acrosome reaction and capacitation of sperm by cryodamage, such as the level of fresh semen (Fig. 6), suggesting CLC is very useful for freezing spermatozoa than MBCD, and can inhibit the responding of acrosome reaction during cryopreservation. However, we need to more study about the difference of CLC and MBCD via analysis of chemical property. In order to comprehend how added cholesterol is affecting cells, it is important to know where this cholesterol is located within the cells. Micrographs of sperm cells treated with fluorescently labeled cholesterol indicated that the added CLC incorporated throughout the sperm plasma membrane and was not restricted to one membrane compartment exclusively. This observation is very important because it revealed that the added cholesterol incorporated in all plasma membrane compartments. Therefore, each membrane compartment (acrosomal, postacrosomal head region, midpiece, and principal piece) could be affected by the added cholesterol. It was also evident that different amounts of cholesterol incorporation occurred in the various membrane compartments. It is beyond the scope of these studies to determine how much cholesterol is incorporated in each membrane compartment. However, it appeared that higher cholesterol incorporation was noticed in the acrosomal membrane compartment when treated with CLC. This may have resulted in effectively protecting the sperm acrosomal membrane from cryodamage. In conclusion, CLC effectively incorporated cholesterol into the sperm membrane of boar spermatozoa from cryodamage prior to cryopreservation. Therefore, addition of CLC to boar sperm before cryopreservation could be improved post-thaw sperm survival.
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Conflicts of interest The authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgments This research was supported by Agricultural Biotechnology Development Program (IPET31060-05-1-CG000), Food and Rural Affairs, Ministry of Agriculture, Republic of Korea. And this study was also supported partially by 2014 Research Grant from Kangwon National University, Republic of Korea (No. 120140209). References Amann, R., Pickett, B., 1987. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J. Equine Vet. Sci. 7, 145–173. Bailey, J., Buhr, M., 1994. Cryopreservation alters the Ca2+ flux of bovine spermatozoa. Can. J. Anim. Sci. 74, 45–51. Bailey, J.L., Lessard, C., Jacques, J., Brèque, C., Dobrinski, I., Zeng, W., Galantino-Homer, H.L., 2008. Cryopreservation of boar semen and its future importance to the industry. Theriogenology 70, 1251–1259. Buhr, M., Canvin, A., Bailey, J., 1989. Effects of semen preservation on boar spermatozoa head membranes. Gamete Res. 23, 441–449. Chakrabarty, J., Banerjee, D., Pal, D., De, J., Ghosh, A., Majumder, G.C., 2007. Shedding off specific lipid constituents from sperm cell membrane during cryopreservation. Cryobiology 54, 27–35. Choi, Y.H., Toyoda, Y., 1998. Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol. Reprod. 59, 1328–1333. Chiu, P.C., Chung, M.K., Tsang, H.Y., Koistinen, R., Koistinen, H., Seppala, M., Lee, K.F., Yeung, W.S., 2005. Glycodelin-S in human seminal plasma reduces cholesterol efflux and inhibits capacitation of spermatozoa. J. Biol. Chem. 280, 25580–25589. Cormier, N., Sirard, M.A., Bailey, J.L., 1997. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J. Androl. 18, 461–468. Cross, N.L., 1999. Effect of methyl-beta-cyclodextrin on the acrosomal responsiveness of human sperm. Mol. Reprod. Dev. 53, 92–98. Darin-Bennett, A., Poulos, A., White, I., 1973. The effect of cold shock and freeze-thawing on release of phospholipids by ram, bull, and boar spermatozoa. Aust. J. Biol. Sci. 26, 1409–1420. Darin-Bennett, A., White, I., 1977. Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology 14, 466–470. Ehrenwald, E., Parks, J.E., Foote, R.H., 1988. Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova. Gamete Res. 20, 413–420. Glazar, A.I., Mullen, S.F., Liu, J., Benson, J.D., Critser, J.K., Squires, E.L., Graham, J.K., 2009. Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol. Cryobiology 59, 201–206. Hammerstedt, R.H., Graham, J.K., Nolan, J.P., 1990. Cryopreservation of mammalian sperm: what we ask them to survive. J. Androl. 11, 73–88. Hartel, S., Diehl, H.A., Ojeda, F., 1998. Methyl-beta-cyclodextrins and liposomes as water-soluble carriers for cholesterol incorporation into membranes and its evaluation by a microenzymatic fluorescence assay and membrane fluidity-sensitive dyes. Anal. Biochem. 258, 277–284. Holt, W., 2000. Basic aspects of frozen storage of semen. Anim. Reprod. Sci. 62, 3–22. Iborra, A., Companyó, M., Martínez, P., Morros, A., 2000. Cholesterol efflux promotes acrosome reaction in goat spermatozoa. Biol. Reprod. 62, 378–383. Kim, S.K., Jang, H.Y., Park, D.H., Park, C.K., Cheong, H.T., Kim, C.I., Yang, B.K., 2006. Establishment of the convenient boar semen freezing method and assessment of viability on frozen/thawed boar semen. Reprod. Dev. Biol. 30, 59–64.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effect of cholesterol-loaded-cyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation Yong-Seung Lee a,1 , Seunghyung Lee a,b,∗,1 , Sang-Hee Lee a , Boo-Keun Yang a , Choon-Keun Park a a b
College of Animal Life Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea Institute of Animal Resources, Kangwon National University, Chuncheon 200-701, Republic of Korea
a r t i c l e
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Article history: Received 10 April 2015 Received in revised form 2 June 2015 Accepted 3 June 2015 Available online 9 June 2015 Keywords: Cholesterol-loaded-cyclodextrin Spermatozoa Viability Cryopreservation
a b s t r a c t This study was undertaken to examine the effect of cholesterol-loaded-cyclodextrin (CLC) on boar sperm viability and spermatozoa cryosurvival during boar semen cryopreservation, and methyl-ˇ-cyclodextrin (MBCD) was treated for comparing with CLC. Boar semen treated with CLC and MBCD before freezing process to monitor the effect on survival and capacitation status by flow cytometry with appropriate fluorescent probes. Sperm viability was higher in 1.5 mg CLC-treated sperm (76.9 ± 1.01%, P < 0.05) than un-treated and MBCD-treated sperm before cryopreservation (58.7 ± 1.31% and 60.3 ± 0.31%, respectively). For CTC patterns, F-pattern was higher in CLC treated sperm than MBCD-treated sperm, for B-pattern was higher in CLC-treated sperm than fresh sperm (P < 0.05). For AR pattern (an acrosome-reacted sperm) was lower in CLC-treated sperm than MBCD-treated sperm (P < 0.05). Moreover, we examined in vitro development of porcine oocytes after in vitro fertilization using CLC-treated frozen–thawed semen, in which CLC treatment prior to freezing and thawing increased the development of oocytes to blastocyst stage in vitro. In conclusion, CLC could protect the viability of spermatozoa from cryodamage prior to cryopreservation in boar semen. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Semen cryopreservation extends the availability of sperm for research, artificial insemination (AI), and in vitro fertilization (IVF) irrespective of time or location. This technique provides the accessibility to sperm, despite of time and location, and permitted the successful development of AI and IVF techniques (Bailey et al., 2008). However,
∗ Corresponding author at: College of Animal Life Science & Institute of Animal Resource, Kangwon National University, Chuncheon 200-701, Republic of Korea. Tel.: +82 33 250 8689. E-mail address: [email protected] (S. Lee). 1 Both the authors contributed equally to this study. http://dx.doi.org/10.1016/j.anireprosci.2015.06.006 0378-4320/© 2015 Elsevier B.V. All rights reserved.
frozen–thawed sperm do not possess the same fertilizing potential as fresh sperm, since the cooling process induces lipid and protein rearrangements within the cell membranes when they are cooled from 22 to 1 ◦ C (Parks and Graham, 1992). This variation was induced by the membrane changing from the fluid to the gel-state at low temperature. Thermotropic phase transitions are considered to be one of the mains reasons for reduced viability and fertility of sperm during the cryopreservation. Especially, boar semen is extremely vulnerable to ‘cold shock’ by cooling during the cryopreservation compared to other species. Sperm sensitivity to cold shock damage is determined primarily by phospholipid constitution and the cholesterol to phospholipid ratio in membrane (Holt, 2000). The cholesterol to phospholipid ratio of the sperm membrane is
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widely considered to be a major determinant in membrane fluidity and stability during cryopreservation. Boar sperm differ from those species that have a high cholesterol to phospholipid ratio in the membrane, such as human and rabbit sperm do not experience this membrane damage when cooled (Darin-Bennett and White, 1977). Membrane damage is considered to be one of the major reasons for cell death (Watson, 1981). Membrane destabilization occurs when the membrane undergoes the phase transition, from the fluid phase to the gel phase, as temperature is decreased. During this phase transition, phospholipids are lost from the plasma membrane leading to increased membrane permeability, membrane disruption, and cell death (Darin-Bennett et al., 1973; Watson, 1981). If the temperature at which the membrane lipid phase transition occurs could be lowered, or if the membrane phase transition could be eliminated completely, membranes would remain fluid at low temperatures and the damage that occur to membranes may be reduced. Previous studies indicated that the cholesterol/phospholipid ratio of the plasma membrane is a major determinant in plasma membrane fluidity and stability during the cryopreservation (Darin-Bennett and White, 1977; Watson, 1981). Cholesterol, a major structural constituent of the membrane, plays an important role as a regulator of membrane function (Yeagle, 1985). Furthermore, cholesterol in the cell membrane has been shown to be an important determinant of membrane fluidity (Hartel et al., 1998) and permeability (McGrath, 1988). Also, cholesterol reduces the phase transition temperature of membrane, and maintains it in a fluid state at reduced temperature thereby reducing the membrane damage that occurs at low temperatures (Amann and Pickett, 1987). Cholesterol can be incorporated readily into or extracted from the membrane of cells using cyclodextrins (Purdy and Graham, 2004; Zeng and Terada, 2001). Cyclodextrins are cyclic heptasaccharides consisting of ˇ (1–4) glucopyranose units, are water soluble but have a hydrophobic center (Purdy and Graham, 2004), and can transport cholesterol in or out of membranes down a concentration gradient (Klein et al., 1995). When cholesterol is loaded into bull sperm membrane prior to cryopreservation with cholesterol-loaded cyclodextrin (CLC), a higher percent sperm motility and membrane were recovered after thawing compare to un-loaded sperm (Purdy and Graham, 2004). However, this procedure has not yet been optimized for boar sperm, nor is it known how the cholesterol regulates the cryosurvival of boar sperm and the function of physiology of the membrane. Additionally, we also used methyl-ˇ-cyclodextrin (MBCD) for this study, since MBCD induces the capacitation and efflux of human sperm (Chiu et al., 2005). Thus, understanding of the regulation of the cholesterol and the protection of sperm membrane and viability are important to the overall understanding cryosurvival during sperm cryopreservation. In this study, we evaluated the effect of cholesterol modification on the cryosurvival of boar sperm during a conventional semen freezing procedure. Further, using cyclodextrin, we investigated in vitro development of porcine oocytes after in vitro fertilization using CLC-treated frozen–thawed semen.
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2. Materials and methods 2.1. Preparation of CLC We prepared cholesterol-loaded-cyclodextrins (CLC) such as method of Purdy and Graham (2004). Briefly, 1 g methyl-ˇ-cyclodextrin (Sigma, St. Louis, MO, USA) was dissolved in 2 mL methanol. 200 mg cholesterol (Sigma) was dissolved in 1 mL chloroform in a separate glass tube. Then a 0.45 mL CLC solution was added to the cyclodextrin solution, and mixed and stirred. The solvents removed using a stream of N2 gas. Finally, the resulting crystals were stored in a glass container at 22 ◦ C, after dry. The working solution was prepared by adding 100 mg of CLC to 1 mL m-Modena B (Lee et al., 2005) at 37 ◦ C. 2.2. Semen collection and treatment with CLC Semen was collected from six miniature pigs (PWG Company, Seoul, Korea) by gloved-hand technique and filtered through cotton gauze to remove the gel particles. All procedures that involved the use of animals were approved by the Kangwon National University Institutional Animal Care and Use Committee (KIACUC-09-0139). The ejaculated sperm were transported to the laboratory at 25 ◦ C within 1 h. The ejaculated sperm were diluted with same volume of extender (m-Modena B). After maintenance at room temperature for 10 min, semen was diluted to 1.2 × 108 sperm in 1 mL of m-Modena B, and incubated with different levels of CLC (0, 0.75, 1.5, 3.0, and 6.0 mg/mL) and 1.5 mg/mL MBCD (negative control) for 15 min at 25 ◦ C. The control group (0 mg/mL) was considered to be a positive control. The incubated semen samples were later centrifuged (400 × g, 10 min) to remove CLC and MBCD from sperm before freezing. 2.3. Semen freezing and thawing Semen treated with CLC and MBCD were processed using the straw freezing procedure described with minor modifications (Kim et al., 2006). The incubated semen was re-suspended with first lactose-egg yolk (LEY) extender (80 mL of 11% lactose and 20 mL egg yolk) to provide 5 × 108 sperm/mL and later centrifuged (400 × g, 10 min, 22 ◦ C) to remove CLC and MBCD from sperm, and were cooled to 5 ◦ C for 3 h. After cooling the semen were diluted 2:1 (v:v) with second LEY extender (LEY extender with 1.5% Orvus Es Paste and 9% glycerol). The sperm were packaged into 0.5 mL straws cooled to −120 ◦ C for 10 min before being plunged into liquid nitrogen for storage using static nitrogen vapor. Frozen-sperm was thawed in a water bath at 50 ◦ C for 10 s. 2.4. Cholesterol incorporation in sperm membrane Semen was extended to 1.2 × 108 sperm/mL in m-Modena B and incubated with 0.75 and 1.5 mg CLC/1.2 × 108 sperm for 15 min at 25 ◦ C. In this experiment, the cyclodextrin has been pre-loaded with cholesterol in which the cholesterol was labeled with the fluorescent molecule 22-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
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Fig. 1. Analysis of sperm viability by flow cytometry. (A) It is the created region of sperm for forward (FSC, relative cell size) and side (SSC, cell internal complexity) scatters. (B) It shows the fluorescence intensity of PI (dead cell stain, a), SYBR-14 + PI (double stained cell stain, b), and SYBR-14 (live cell stain, c) in region of sperm (A).
aminl-23,24-bisnor-5-cholen-3B-ol (NBD cholesterol, Invitrogen, Korea). The incubated semen samples were centrifuged (400 × g, 10 min) to remove CLC and MBCD from sperm, then resuspended sperm with phosphate buffer saline. The amount of NBD-labeled cholesterol was measured using flow cytometry. NBD was excited using an argon laser tuned to 488 nm. A total of 10,000 cells were analyzed per sample. The fluorescent image of cholesterol incorporation in the membrane of sperm was monitored by fluorescence microscope. After suspension of semen, 5 L drops of sample were placed onto pre-heated glass slides (37 ◦ C) and examined using a microscope (OLYMPUS LX70 Tokyo, Japan) with an argon laser tuned to 488 nm, a 505 nm dichroic mirror and a 520 nm long-pass filter, to determine sperm membrane that contained NBD fluorescence. 2.5. Assessment of sperm viability and acrosome state Frozen–thawed sperm (1.2 × 108 sperm/mL) were stained with 5 L SYBR-14 (20 M solution in DMSO), and 5 L propidium iodide (2.4 mM solution in water; Molecular Probes, Eugene, OR) for flow cytometric viability analysis. The samples were incubated for 10 min at 37 ◦ C before being analyzed (10,000 cells per sample) using a flow cytometer equipped with an argon laser tuned to 488 nm (Fig. 1). The acrosomal state and capacitation of sperm were assessed using a cyclotetracyclin (CTC) staining method (Abeydeera et al., 1997). Each cell was observed under ultraviolet (UV) illumination (emission at 420 nm). The three fluorescent staining patterns identified were: F pattern (an uncapacitated sperm), with uniform fluorescence over the whole sperm head; B pattern (a capacitated sperm), with a fluorescence-free band in the postacrosome region; and AR pattern (an acrosome-reacted
sperm), with almost no fluorescence over the sperm head. 2.6. In vitro fertilization (IVF) Oocytes were selected and cultured, and used for IVF as described (Lee and Park, 2015). Briefly, we collected ovaries from prepubertal gilts at a slaughter house (Pocheonfarm, Pochoen, Korea) and moved to our laboratory within 2 h. Oocytes were cultured in modified TCM-199 with EGF, hCG, pFF, LH, and FSH in a humidified, 5% CO2 atmosphere at 38.5 ◦ C for 44 h. Oocytes were separated from the enclosing cumulus cells in maturation medium with 0.1% hyaluronidase after 44 h for IVF. After fertilization, extra spermatozoa were removed from the oocytes by repetitive pipetting action and oocytes were washed three times in culture medium (porcine zygote medium: PZM-3 with 0.3% BSA) and incubated at 38.5 ◦ C for 144 h under 5% CO2 in humidified air. The oocyte cleavage and blastocyst rates were counted using microscope. 2.7. Statistical analysis The results were analyzed using Statistical Analysis System (SAS) software version 8.01. Treatment groups were compared for differences using of Duncan’s modified multiple range tests. Data were presented as mean ± SEM, and a 5% probability was considered significant. 3. Results 3.1. Cholesterol incorporation in sperm membrane Flow cytometric analysis of sperm treated with 0.75 mg and 1.5 mg of NBD-labeled CLC indicated that the fluorescence of NBD was increased by cholesterol level (Fig. 2).
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Table 1 In vitro development of porcine oocytes fertilized with addition of CLC frozen–thawed semen at 144 h after in vitro fertilization. Treatments
No. of oocytes in IVF
Cleavage (%)
Control CLC
266 249
209 (79.5 ± 2.6) 199 (80.2 ± 4.3)
No. of embryo development to (%) 2 cell ∼ morula
Blastocytes
172 (80.5 ± 5.7) 144 (75.3 ± 3.1)
37 (17.0 ± 1.8)b 55 (28.3 ± 3.8)a
Values in the same column with different superscripts letters (a, b) differ significantly (P < 0.05). Data are presented as the mean ± SEM (n = 3).
CLC compared to untreated or treated with 1.5 mg MBCD (Fig. 4). The higher percent sperm viability (76.9 ± 1.01%, P < 0.05) was revealed (Fig. 5) after thawing, when 1.5 mg CLC was added to sperm before freezing compared to other CLC levels (3.0 and 6.0 mg) and sperm treated with 1.5 mg MBCD (60.3 ± 0.31%) and untreated sperm (58.7 ± 1.31%). Also, 1.5 mg CLC-added sperm was higher percentages of capacitated acrosome-intact spermatozoa (B pattern) and significant (P < 0.05) lower percentage of acrosome-reacted spermatozoa (AR pattern) than untreated sperm (control) and MBCD-treated samples (Fig. 6). In F pattern (uncapacitated acrosome-intact spermatozoa), CLC-treated sample (P < 0.05) was higher than MBCD-treated sample, not than fresh semen. 3.3. Oocyte cleavage and blastocyst formation rates Fig. 2. Single parameter histogram for the log green fluorescence of boar sperm. The sperms treated with 0.5 and 2 mg cholesterol-loadedcyclodextrin (CLC) per 1.2 × 108 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Micrographs of sperm cells treated with 0.75 mg and 1.5 mg of fluorescently labeled cholesterol indicated that cholesterol was incorporated into the membrane compartment (Fig. 3). The sperm cells treated with 0.75 mg NBD labeled CLC showed acrosomal compartment to be more heavily labeled, but cells treated with 1.5 mg NBD labeled CLC showed fluorescence in the acrosomal and mitochondrial compartments. 3.2. Effect of cholesterol on viability and acrosome state of frozen–thawed sperm Flow cytometric dotplots data for the viability assay of frozen–thawed sperm showed higher sperm viability in the live cell zone (blue quadrant) when treated with 1.5 mg
The embryo cleavage and blastocyst formation rates at 144 h after IVF are summarized in Table 1. 226 oocytes used for control and 249 for CLC-treated samples, respectively. The oocyte cleavage rate in the CLC-treated sperm did not significantly differ, but the blastocyst formation rate was significantly higher in CLC-treated samples (28.3 ± 3.8%) than without CLC (17.0 ± 1.8%, P < 0.05). 4. Discussion Boar spermatozoa are more sensitive to events associated with cryopreservation than other species such as sheep, human, and bull (Medeiros et al., 2002; Padilha et al., 2012). Cryopreservation induces many forms of stress on sperm, including destabilizing the membrane, dilution, storage condition, heat stress, osmotic stress, cold shock, and intracellular ice formation. Cell death is observed during the freezing process especially by formation of intracellular ice crystals and membrane damage
Fig. 3. Fluorescent imaging of cholesterol incorporation in the membrane of sperm as observed by fluorescent microscope; non-treated cholesterol-loadedcyclodextrin (CLC); 0.75 mg CLC treated; 1.5 mg CLC treated.
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Fig. 4. Flow cytometric dot plots show fluorescence signals in boar sperms. The sperms stained with PI and SYBR-14 for viability assay, and control was unstained with PI and SYBR-14, and treated with non-treated cholesterol-loaded-cyclodextrin (CLC), 1.5 mg CLC and 1.5 mg methyl-ˇ-cyclodextrin (MBCD) during the freezing process.
(Hammerstedt et al., 1990; Leeuw et al., 1993; Mazur, 1977; Maxwell and Johnson, 1997; Steponkus et al., 1983). Cholesterol reduces the transition temperature of membranes, and maintains them in a fluid state at reduced temperatures thereby reducing the membrane damage that occurs at low temperatures (Amann and Pickett, 1987; Chakrabarty et al., 2007; Glazar et al., 2009). Cholesterol can easily be incorporated into or extracted from the plasma membranes of cells using cyclodextrins. Li et al. (2006) and Purdy (2004) reported that intact sperm membrane was recovered in frozen–thawed sperm with cholesterol.
Fig. 5. Effect of cholesterol-loaded-cyclodextrin (CLC), methyl-ˇcyclodextrin (MBCD) on viability of frozen–thawed boar sperm. Bars with different superscripts (a–c) within the CLC levels category differ significantly (P < 0.05). Asterisks (*) shows significant difference between CLC- and MBCD-treated groups (P < 0.05).
Similar results have also been reported for stallion sperm treated with CLCs (Moore et al., 2005). In this study, higher proportion of viability and acrosome state of boar sperm treated with CLC was observed than sperm untreated or treated with MBCD (Figs. 5 and 6). The optimal level of CLC for cryopreservation of boar sperm was 1.5 mg CLC per 1.2 × 108 sperm (Fig. 5), which is similar to that reported
Fig. 6. Effect of cholesterol-loaded-cyclodextrin (CLC), methyl-ˇcyclodextrin (MBCD) on CTC pattern of frozen–thawed boar sperm. Bars with different superscripts (a–c) for a particular pattern differ significantly (P < 0.05).
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for bull and stallion sperm (Moore et al., 2005; Purdy and Graham, 2004). However, when treated with CLC levels more than 1.5 mg per 1.2 × 108 sperm, the CLC becomes detrimental to sperm cell survival (Fig. 5). Probably, it may occur by affecting membrane fluidity and membrane function to the point that normal membrane function is affected that leads to sperm death during the freezing. Cholesterol plays an important role in stabilizing sperm membranes. Especially, during capacitation of sperm, the membrane lose cholesterol (Ehrenwald et al., 1988), making them more unstable thereby enabling the acrosome reaction. Sperm capacitation can induce cholesterol efflux from the sperm membrane (Choi and Toyoda, 1998; Cross, 1999; Iborra et al., 2000; Parinaud et al., 2000; Visconti et al., 1999). Similarly, when sperm are cooled or frozen, the membranes undergo a phase transition, which results in rearrangement of membrane lipids and proteins (Hammerstedt et al., 1990; Parks and Graham, 1992) and a loss of lipids from the membranes (DarinBennett et al., 1973), which in turn causes protein and lipid aggregation within the membrane, and a loss of membrane-selective permeability (Bailey and Buhr, 1994; Buhr et al., 1989), resulting in sperm that are prematurely capacitated (Cormier et al., 1997; Parrish et al., 1986). This premature capacitation could reduce fertilizing ability and lifespan of the sperm (Cormier et al., 1997; Parrish et al., 1986). In our results, micrographs of sperm cells treated with fluorescently labeled CLC (Fig. 3) indicated higher cholesterol incorporation in the acrosomal membrane. CLC reduced the premature acrosome reaction and capacitation of sperm by cryodamage, such as the level of fresh semen (Fig. 6), suggesting CLC is very useful for freezing spermatozoa than MBCD, and can inhibit the responding of acrosome reaction during cryopreservation. However, we need to more study about the difference of CLC and MBCD via analysis of chemical property. In order to comprehend how added cholesterol is affecting cells, it is important to know where this cholesterol is located within the cells. Micrographs of sperm cells treated with fluorescently labeled cholesterol indicated that the added CLC incorporated throughout the sperm plasma membrane and was not restricted to one membrane compartment exclusively. This observation is very important because it revealed that the added cholesterol incorporated in all plasma membrane compartments. Therefore, each membrane compartment (acrosomal, postacrosomal head region, midpiece, and principal piece) could be affected by the added cholesterol. It was also evident that different amounts of cholesterol incorporation occurred in the various membrane compartments. It is beyond the scope of these studies to determine how much cholesterol is incorporated in each membrane compartment. However, it appeared that higher cholesterol incorporation was noticed in the acrosomal membrane compartment when treated with CLC. This may have resulted in effectively protecting the sperm acrosomal membrane from cryodamage. In conclusion, CLC effectively incorporated cholesterol into the sperm membrane of boar spermatozoa from cryodamage prior to cryopreservation. Therefore, addition of CLC to boar sperm before cryopreservation could be improved post-thaw sperm survival.
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Conflicts of interest The authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgments This research was supported by Agricultural Biotechnology Development Program (IPET31060-05-1-CG000), Food and Rural Affairs, Ministry of Agriculture, Republic of Korea. And this study was also supported partially by 2014 Research Grant from Kangwon National University, Republic of Korea (No. 120140209). References Amann, R., Pickett, B., 1987. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J. Equine Vet. Sci. 7, 145–173. Bailey, J., Buhr, M., 1994. Cryopreservation alters the Ca2+ flux of bovine spermatozoa. Can. J. Anim. Sci. 74, 45–51. Bailey, J.L., Lessard, C., Jacques, J., Brèque, C., Dobrinski, I., Zeng, W., Galantino-Homer, H.L., 2008. Cryopreservation of boar semen and its future importance to the industry. Theriogenology 70, 1251–1259. Buhr, M., Canvin, A., Bailey, J., 1989. Effects of semen preservation on boar spermatozoa head membranes. Gamete Res. 23, 441–449. Chakrabarty, J., Banerjee, D., Pal, D., De, J., Ghosh, A., Majumder, G.C., 2007. Shedding off specific lipid constituents from sperm cell membrane during cryopreservation. Cryobiology 54, 27–35. Choi, Y.H., Toyoda, Y., 1998. Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol. Reprod. 59, 1328–1333. Chiu, P.C., Chung, M.K., Tsang, H.Y., Koistinen, R., Koistinen, H., Seppala, M., Lee, K.F., Yeung, W.S., 2005. Glycodelin-S in human seminal plasma reduces cholesterol efflux and inhibits capacitation of spermatozoa. J. Biol. Chem. 280, 25580–25589. Cormier, N., Sirard, M.A., Bailey, J.L., 1997. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J. Androl. 18, 461–468. Cross, N.L., 1999. Effect of methyl-beta-cyclodextrin on the acrosomal responsiveness of human sperm. Mol. Reprod. Dev. 53, 92–98. Darin-Bennett, A., Poulos, A., White, I., 1973. The effect of cold shock and freeze-thawing on release of phospholipids by ram, bull, and boar spermatozoa. Aust. J. Biol. Sci. 26, 1409–1420. Darin-Bennett, A., White, I., 1977. Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology 14, 466–470. Ehrenwald, E., Parks, J.E., Foote, R.H., 1988. Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova. Gamete Res. 20, 413–420. Glazar, A.I., Mullen, S.F., Liu, J., Benson, J.D., Critser, J.K., Squires, E.L., Graham, J.K., 2009. Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol. Cryobiology 59, 201–206. Hammerstedt, R.H., Graham, J.K., Nolan, J.P., 1990. Cryopreservation of mammalian sperm: what we ask them to survive. J. Androl. 11, 73–88. Hartel, S., Diehl, H.A., Ojeda, F., 1998. Methyl-beta-cyclodextrins and liposomes as water-soluble carriers for cholesterol incorporation into membranes and its evaluation by a microenzymatic fluorescence assay and membrane fluidity-sensitive dyes. Anal. Biochem. 258, 277–284. Holt, W., 2000. Basic aspects of frozen storage of semen. Anim. Reprod. Sci. 62, 3–22. Iborra, A., Companyó, M., Martínez, P., Morros, A., 2000. Cholesterol efflux promotes acrosome reaction in goat spermatozoa. Biol. Reprod. 62, 378–383. Kim, S.K., Jang, H.Y., Park, D.H., Park, C.K., Cheong, H.T., Kim, C.I., Yang, B.K., 2006. Establishment of the convenient boar semen freezing method and assessment of viability on frozen/thawed boar semen. Reprod. Dev. Biol. 30, 59–64.
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