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Vertebrate reproductive science and technology
RESEARCH ARTICLE

Pretreatment of Asian elephant (Elephas maximus) spermatozoa with cholesterol-loaded cyclodextrins and glycerol addition at 4°C improves cryosurvival

Wendy K. Kiso A F , Atsushi Asano B , Alexander J. Travis B , Dennis L. Schmitt C D , Janine L. Brown E and Budhan S. Pukazhenthi E
+ Author Affiliations
- Author Affiliations

A Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC 20008, USA.

B Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

C The William H. Darr School of Agriculture, Missouri State University, Springfield, MO 65897, USA.

D The Ringling Brothers Center for Elephant Conservation, Polk City, FL 33868, USA.

E Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA 22630, USA.

F Corresponding author. Email: wkiso@feldinc.com

Reproduction, Fertility and Development 24(8) 1134-1142 https://doi.org/10.1071/RD11266
Submitted: 15 October 2011  Accepted: 4 April 2012   Published: 3 May 2012

Abstract

Asian elephant spermatozoa are sensitive to chilling and do not respond well to cryopreservation. The objectives of the present study were to: (1) determine whether cholesterol content can be modified by preincubation of Asian elephant spermatozoa with cholesterol-loaded cyclodextrin (CLC); and (2) assess the effects of CLC concentration(s), temperature at time of glycerol addition (22°C vs 4°C) and dilution medium on post-thaw sperm survival. Spermatozoa incubated with ≥1.5 mg CLC exhibited increased (P < 0.05) cholesterol concentrations. Pretreatment of spermatozoa with 1.5 mg CLC resulted in improvements (P < 0.05) in all post-thaw parameters. Glycerol addition at 4°C also improved all post-thaw parameters compared with 22°C. Dilution of thawed spermatozoa in an egg yolk-based medium improved (P < 0.05) motility compared with Ham’s F-10 culture medium. In summary, our findings indicate that modifying cholesterol content within the plasma membrane improves the cryosurvival of Asian elephant spermatozoa. The development of an improved cryopreservation method that includes modification of membrane cholesterol and the addition of glycerol at 4°C, as reported in the present study, is an important step towards utilisation of cryopreserved spermatozoa in captive management of this species.

Additional keywords: gamete, genome resource bank, storage.


References

Aksoy, M., Akman, O., Lehimcioglu, N. C., and Erdem, H. (2010). Cholesterol-loaded cyclodextrin enhances osmotic tolerance and inhibits the acrosome reaction in rabbit spermatozoa. Anim. Reprod. Sci. 120, 166–172.
Cholesterol-loaded cyclodextrin enhances osmotic tolerance and inhibits the acrosome reaction in rabbit spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXmtFWrtro%3D&md5=c8a523271578d3058b9f582cfa1c9830CAS | 20304567PubMed |

Amann, R. P., and Pickett, B. W. (1987). Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J. Equine Vet. Sci. 7, 145–173.
Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa.Crossref | GoogleScholarGoogle Scholar |

Amidi, F., Farshad, A., and Khor, A. K. (2010). Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa. Cryobiology 61, 94–99.
Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXpvVGkt7Y%3D&md5=ceb7316c9be497da57f8f67b65902c05CAS | 20561514PubMed |

Amorim, E. A. M., Graham, J. K., Spizziri, B., Meyers, M., and Torres, C. A. A. (2009). Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm. Cryobiology 58, 210–214.
Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisFWgs78%3D&md5=7d143b227ceec2d2393a43b2c9f5ac22CAS |

Bartlett, F. D., and Van Demark, N. L. (1962). Effect of temperature on the survival of bovine spermatozoa stored in carbonated diluents. J. Dairy Sci. 45, 368–374.
Effect of temperature on the survival of bovine spermatozoa stored in carbonated diluents.Crossref | GoogleScholarGoogle Scholar |

Blackshaw, A. W., Salisbury, G. W., and Van Demark, N. L. (1957). Factors influencing metabolic activity of bull spermatozoa. I. 37, 21, and 5°C. J. Dairy Sci. 40, 1093–1098.
Factors influencing metabolic activity of bull spermatozoa. I. 37, 21, and 5°C.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaG1cXht1Sluw%3D%3D&md5=70430e3ce88bdb0db527adcf12261c57CAS |

Bligh, E. G., and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can. J. Physiol. Pharmacol. 37, 911–917.
A rapid method of total lipid extraction and purification.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaG1MXhtVSgt70%3D&md5=557aa864e3ea314dcf30622b35c1e188CAS |

Brown, J. L., Goritz, F., Pratt-Hawkes, N., Hermes, R., Galloway, M., Graham, L. H., Gray, C., Walker, S. L., Gomez, A., Moreland, R., Murray, S., Schmitt, D. L., Howard, J. G., Lehnhardt, J., Beck, B., Bellem, A., Montali, R., and Hildebrandt, T. B. (2004). Successful artificial insemination of an Asian elephant at the National Zoological Park. Zoo Biol. 23, 45–63.
Successful artificial insemination of an Asian elephant at the National Zoological Park.Crossref | GoogleScholarGoogle Scholar |

Cerolini, S., Maldjian, A., Pizzi, F., and Gliozzi, T. (2001). Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction 121, 395–401.
Changes in sperm quality and lipid composition during cryopreservation of boar semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXisFSqu7s%3D&md5=e231fecdcc146c95983722b8718da8fdCAS | 11226065PubMed |

Combes, G. B., Varner, D. D., Schroeder, F., Burghardt, R. C., and Blanchard, T. L. (2000). Effect of cholesterol on the motility and plasma membrane integrity of frozen equine spermatozoa after thawing. J. Reprod. Fertil. Suppl. 56, 127–132.
| 20681124PubMed |

Crockett, E. L. (1998). Cholesterol function in plasma membranes from ectotherms: membrane-specific roles in adaptation to temperature. Am. Zool. 38, 291–304.
| 1:CAS:528:DyaK1cXjt1yksb4%3D&md5=6c7f242cb7ca5f95031fabba64138212CAS |

Cross, N. L. (1996). Effect of cholesterol and other sterols on human sperm acrosomal responsiveness. Mol. Reprod. Dev. 45, 212–217.
Effect of cholesterol and other sterols on human sperm acrosomal responsiveness.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XmsFGmtbo%3D&md5=93cafed8b4b9daa224b08a1eafc37077CAS | 8914079PubMed |

Darin-Bennett, A., and White, I. G. (1977). Influence of cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology 14, 466–470.
Influence of cholesterol content of mammalian spermatozoa on susceptibility to cold-shock.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE2sXltlWmtr4%3D&md5=0119d8a32cd0d7ef54b476e8bb8dc105CAS | 560945PubMed |

Holt, W. V. (2000). Basic aspects of frozen storage of semen. Anim. Reprod. Sci. 62, 3–22.
Basic aspects of frozen storage of semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXlt1Kgsbo%3D&md5=552ad066bbaf27f21d8decb382931fe7CAS | 10924818PubMed |

Johnson, L. A., and Welch, G. R. (1999). Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency. Theriogenology 52, 1323–1341.
Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7pvFamtg%3D%3D&md5=7e4eb96768db2465b6d5992f303523ceCAS | 10735079PubMed |

Kiso, W. K., and Schmitt, D. L. (2004). Cryopreservation of Asian elephant (Elephas maximus) semen in conjunction with the use of Spermac® stain to evaluate acrosome status in both Asian and African (Loxodonta africana) elephants. Masters Thesis, Missouri State University, USA.

Kiso, W. K., Brown, J. L., Siewerdt, F., Schmitt, D. L., Olson, D., Crichton, E. G., and Pukazhenthi, B. S. (2011). Liquid semen storage in elephants (Elephas maximus and Loxodonta africana): species differences and storage optimization. J. Androl. 32, 420–431.
Liquid semen storage in elephants (Elephas maximus and Loxodonta africana): species differences and storage optimization.Crossref | GoogleScholarGoogle Scholar | 21127305PubMed |

Larson, J. L., and Miller, D. J. (1999). Simple histochemical stain for acrosome on sperm from several species. Mol. Reprod. Dev. 52, 445–449.
Simple histochemical stain for acrosome on sperm from several species.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXhsl2hsrk%3D&md5=1b6c849d653baff021ea820361d2e98aCAS | 10092125PubMed |

Medeiros, C. M. O., Forell, F., Oliveira, A. T. D., and Rodrigues, J. L. (2002). Current status of sperm cryopreservation: why isn’t it better? Theriogenology 57, 327–344.
Current status of sperm cryopreservation: why isn’t it better?Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XktFyltA%3D%3D&md5=b29896184762c9895e7f28f8cfb609f8CAS |

Mocé, E., and Graham, J. K. (2006). Cholesterol-loaded cyclodextrins added to fresh bull ejaculates improve sperm cryosurvival. J. Anim. Sci. 84, 826–833.
| 16543559PubMed |

Mocé, E., Purdy, P. H., and Graham, J. K. (2010). Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival. Anim. Reprod. Sci. 118, 236–247.
Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival.Crossref | GoogleScholarGoogle Scholar | 19615833PubMed |

Moore, A. I., Squires, E. L., and Graham, J. K. (2005). Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival. Cryobiology 51, 241–249.
Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtlShsLbP&md5=6dbfc404d64930888234c12fb51437ecCAS | 16122725PubMed |

Moraes, E. A., Graham, J. K., Torres, C. A. A., Meyers, M., and Spizziri, B. (2010). Delivering cholesterol or cholestanol to bull sperm membranes improves cryosurvival. Anim. Reprod. Sci. 118, 148–154.
Delivering cholesterol or cholestanol to bull sperm membranes improves cryosurvival.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtVGltbs%3D&md5=2caadca9bd39dde6c321b0b4fce9d87aCAS | 19733986PubMed |

Parks, J. E., and Graham, J. K. (1992). Effects of cryopreservation procedures on sperm membrane. Theriogenology 38, 209–222.
Effects of cryopreservation procedures on sperm membrane.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD283pvFyqtg%3D%3D&md5=8e21d102b86bc890c8fab5619639012eCAS | 16727131PubMed |

Pitha, J., Irie, T., Sklar, P. B., and Nye, J. S. (1988). Drug solubilizers to aid pharmacologists: amorphous cyclodextrin derivatives. Life Sci. 43, 493–502.
Drug solubilizers to aid pharmacologists: amorphous cyclodextrin derivatives.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1cXkvFyksbw%3D&md5=223923fe7a8a09747bd2a474ec36c570CAS | 2841549PubMed |

Polge, C., Smith, A. U., and Parkes, A. S. (1949). Revival of spermatozoa after vitrification and dehydration at low temperature. Nature 164, 666.
Revival of spermatozoa after vitrification and dehydration at low temperature.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaH1M%2FksV2gtQ%3D%3D&md5=4843bdfbde9689ef9e6766398506b1c8CAS | 18143360PubMed |

Purdy, P. H., and Graham, J. K. (2004a). Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility. Biol. Reprod. 71, 522–527.
Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmtFWgtbk%3D&md5=930e0290a30d66b75fddedccf92bfbd6CAS | 15070825PubMed |

Purdy, P. H., and Graham, J. K. (2004b). Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm. Cryobiology 48, 36–45.
Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXht1yiur8%3D&md5=43f3d3c7e56b63af0d8d329d06be6fbfCAS | 14969680PubMed |

Saenger, W. (1980). Cyclodextrin inclusion compounds in research and industry. Angew. Chem. Int. Ed. 19, 344–362.
Cyclodextrin inclusion compounds in research and industry.Crossref | GoogleScholarGoogle Scholar |

Saragusty, J., Hildebrandt, T. B., Natan, Y., Hermes, R., Yavin, S., Goeritz, F., and Arav, A. (2005). Effect of egg-phosphatidylcholine on the chilling sensitivity and lipid phase transition of Asian elephant (Elephas maximus) spermatozoa. Zoo Biol. 24, 233–245.
Effect of egg-phosphatidylcholine on the chilling sensitivity and lipid phase transition of Asian elephant (Elephas maximus) spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXnsl2gtb8%3D&md5=25a12e11294903ac721f610c8957b36dCAS |

Saragusty, J., Hildebrandt, T. B., Behr, B., Knieriem, A., Kruse, J., and Hermes, R. (2009). Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa. Anim. Reprod. Sci. 115, 255–266.
Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXptl2iu78%3D&md5=8645f5e0665d2d6afd1cc2f0d52ce3e8CAS | 19111407PubMed |

Schmitt, D. L., and Hildebrandt, T. B. (1998). Manual collection and characterization of semen from Asian elephants (Elephas maximus). Anim. Reprod. Sci. 53, 309–314.
Manual collection and characterization of semen from Asian elephants (Elephas maximus).Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK1M%2FltFSnug%3D%3D&md5=ede37b2baf7df4b598f7686cc703a829CAS | 9835384PubMed |

Spizziri, B. E., Fox, M. H., Bruemmer, J. E., Squires, E. L., and Graham, J. K. (2010). Cholesterol-loaded-cyclodextrins and fertility potential of stallions spermatozoa. Anim. Reprod. Sci. 118, 255–264.
Cholesterol-loaded-cyclodextrins and fertility potential of stallions spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtVGlurs%3D&md5=f93fb92057349d7514a9813b4f24ce26CAS | 19762177PubMed |

Thongtip, N., Saikhun, J., Damyang, M., Mahasawangkul, S., Suthunmapinata, P., Yindee, M., Kongsila, A., Angkawanish, T., Jansittiwate, S., Wongkalasin, W., Wajjwalkul, W., Kitiyanant, Y., Pavasuthipaisit, K., and Pinyopummin, A. (2004). Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa of extender and cryoprotectant. Theriogenology 62, 748–760.
Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa of extender and cryoprotectant.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXltF2rs70%3D&md5=725fbe67d2ba7a55c9038e89ad5884feCAS | 15226027PubMed |

Watson, P. F. (1981). The effects of cold shock on sperm cell membranes. In ‘Effects of Low Temperatures on Biological Membranes’. (Eds G. J. Morris and A. Clarke.) pp. 189–218. (Academic Press: New York, NY.)

White, I. G. (1993). Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reprod. Fertil. Dev. 5, 639–658.
Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXksl2jsbk%3D&md5=046ea48672127f2384226ec1de822633CAS | 9627725PubMed |

Wildt, D. E., Rall, W. F., Critser, J. K., Monfort, S. L., and Seal, U. S. (1997). Genome resource banks: living collections for biodiversity conservation. Bioscience 47, 689–698.
Genome resource banks: living collections for biodiversity conservation.Crossref | GoogleScholarGoogle Scholar |

Woods, E. J., Benson, J. D., Agca, Y., and Critser, J. K. (2004). Fundamental cryobiology of reproductive cells and tissues. Cryobiology 48, 146–156.
Fundamental cryobiology of reproductive cells and tissues.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXjt1amurw%3D&md5=99bf1cc5fd38c1dee818495a6ebedec4CAS | 15094091PubMed |