Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Differences in preservation of canine chilled semen using simple sperm washing, single-layer centrifugation and modified swim-up preparation techniques

J. Dorado A E , M. J. Gálvez A , S. Demyda-Peyrás A , I. Ortiz A , J. M. Morrell B , F. Crespo C , J. Gósalvez D and M. Hidalgo A
+ Author Affiliations
- Author Affiliations

A Animal Reproduction Group, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Cordoba, Campus de Rabanales (Edif. Hospital Clínico Veterinario), Ctra. Madrid-Cádiz, km 396, 14071 Córdoba, Spain.

B Department of Clinical Sciences, Division of Reproduction, Swedish University of Agricultural Sciences, Ullsväg 14C, Box 7054, SE-75007 Uppsala, Sweden.

C Veterinary Services of the Spanish Army (FESCCR-Ministry of Defense), C/Arsenio Guitiérrez Palacios s/n, 05005 Avila, Spain.

D Department of Biology, Genetics Unit, Autonomous University of Madrid, 20849 Madrid, Spain.

E Corresponding author. Email: jdorado@uco.es

Reproduction, Fertility and Development 28(10) 1545-1552 https://doi.org/10.1071/RD15071
Submitted: 21 November 2014  Accepted: 24 February 2015   Published: 17 April 2015

Abstract

This study compared the efficacy of simple sperm washing (SW), single-layer centrifugation (SLC) and modified swim-up (SU) techniques in the preparation of dog spermatozoa for cooling. Eighteen ejaculates, collected from three dogs (six per dog), were pooled (three ejaculates per pool) and divided into three aliquots: (1) one aliquot was washed and cooled at 5°C for 72 h, considered as control (SW-control), (2) the second aliquot was selected by SLC through Androcoll-C and subsequently cooled in the same way as the SW-control samples (SLC-AC) and (3) the last aliquot was selected by a modified SU method with Androcoll-C and cooled as mentioned above (SU-AC). Assessment of sperm motility, sperm morphology, sperm membrane integrity and acrosome integrity were performed on aliquots of fresh semen and chilled–rewarmed samples. Sperm membrane integrity and progressive motility were significantly (P < 0.05) improved by SU-AC compared with SW-control. Morphological sperm abnormalities decreased significantly (P < 0.001) in SLC-AC samples compared with SW-control samples. These sperm variables did not differ between SLC-AC and SU-AC methods (P > 0.05). The recovery rates were not significantly (P > 0.05) different between SW-control, SLC-AC and SU-AC samples. Our results confirm that SU-AC may be a successful method for the preparation of dog spermatozoa for cooling.

Additional keywords: Androcoll-C, chilled dog semen, colloid, sperm migration, sperm selection.


References

Aitken, R. J., and Clarkson, J. S. (1988). Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J. Androl. 9, 367–376.
| 1:CAS:528:DyaL1MXhvVyrs78%3D&md5=fe367758aab1e20d7183ae1361004dc5CAS | 3215823PubMed |

Aitken, R. J., Jones, K. T., and Robertson, S. A. (2012). Reactive oxygen species and sperm function – in sickness and in health. J. Androl. 33, 1096–1106.
Reactive oxygen species and sperm function – in sickness and in health.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXlvFaqsQ%3D%3D&md5=010ceeceed5ece4735d0c1e3f6b01b33CAS | 22879525PubMed |

Alvarez, J. G., Lasso, J. L., Blasco, L., Nunez, R. C., Heyner, S., Caballero, P. P., and Storey, B. T. (1993). Centrifugation of human spermatozoa induces sub-lethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime. Hum. Reprod. 8, 1087–1092.
| 1:STN:280:DyaK2c%2FhtleitQ%3D%3D&md5=45d2a9cd72f9b8a3591f1d70ef0cdb04CAS | 7691866PubMed |

Bukowska, D., Kempisty, B., Sikora, J., Jackowska, M., Wozna, M., Antosik, P., Piotrowska, H., Budna, J., and Jaskowski, J. M. (2011). The effect of swim-up purification and incubation of cells on sperm viability in dogs of different ages. Vet. Med. (Praha) 56, 248–254.

Coloma, M. A., Toledano-Díaz, A., López-Sebastián, A., and Santiago-Moreno, J. (2010). The influence of washing Spanish ibex (Capra pyrenaica) spermatozoa on the effects of cryopreservation in dependency of the photoperiod. Theriogenology 73, 900–908.
The influence of washing Spanish ibex (Capra pyrenaica) spermatozoa on the effects of cryopreservation in dependency of the photoperiod.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3c3gsVSktA%3D%3D&md5=b0592a52be650fb060af76dd1bfa1034CAS | 20092881PubMed |

Correa, J. R., and Zavos, P. M. (1996). Preparation and recovery of frozen–thawed bovine spermatozoa via various sperm selection techniques employed in assisted reproductive technologies. Theriogenology 46, 1225–1232.
Preparation and recovery of frozen–thawed bovine spermatozoa via various sperm selection techniques employed in assisted reproductive technologies.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28zgtVOguw%3D%3D&md5=3e48632d6ee6bcf9ed357347348b0a84CAS | 16727985PubMed |

Dorado, J., Rodríguez, I., and Hidalgo, M. (2007). Cryopreservation of goat spermatozoa: comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination. Theriogenology 68, 168–177.
Cryopreservation of goat spermatozoa: comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXmsFKgsLw%3D&md5=1fd26d7ba1d555fe68f9d221554386abCAS | 17532460PubMed |

Dorado, J., Alcaráz, L., Duarte, N., Portero, J., Acha, D., Demyda, S., Muñoz-Serrano, A., and Hidalgo, M. (2011a). Centrifugation on PureSperm density gradient improved quality of spermatozoa from frozen–thawed dog semen. Theriogenology 76, 381–385.
Centrifugation on PureSperm density gradient improved quality of spermatozoa from frozen–thawed dog semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3MngtVSiug%3D%3D&md5=56db2d9f3294ed8659c58b38f295682cCAS | 21497393PubMed |

Dorado, J., Alcaráz, L., Duarte, N., Portero, J., Acha, D., and Hidalgo, M. (2011b). Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm gradient. Anim. Reprod. Sci. 125, 211–218.
Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm gradient.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3MrpsVOjtg%3D%3D&md5=e09e871fbf89cbab4f4010e786fd7238CAS | 21530113PubMed |

Dorado, J., Galvez, M. J., Murabito, M. R., Munoz-Serrano, A., and Hidalgo, M. (2011c). Identification of sperm subpopulations in canine ejaculates: effects of cold storage and egg-yolk concentration. Anim. Reprod. Sci. 127, 106–113.
Identification of sperm subpopulations in canine ejaculates: effects of cold storage and egg-yolk concentration.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3MfjtVGlsA%3D%3D&md5=103d3e16928c3576062e4e4fbfc52a4fCAS | 21820824PubMed |

Dorado, J., Alcaraz, L., Galvez, M. J., Acha, D., Ortiz, I., Urbano, M., and Hidalgo, M. (2013a). Single-layer centrifugation through PureSperm 80 selects improved-quality spermatozoa from frozen–thawed dog semen. Anim. Reprod. Sci. 140, 232–240.
Single-layer centrifugation through PureSperm 80 selects improved-quality spermatozoa from frozen–thawed dog semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhtF2hsrvJ&md5=6fa2868fd12da67a15851e53da268645CAS | 23896392PubMed |

Dorado, J., Gálvez, M., Morrell, J., Alcaráz, L., and Hidalgo, M. (2013b). Use of single-layer centrifugation with Androcoll-C to enhance sperm quality in frozen–thawed dog semen. Theriogenology 80, 955–962.
Use of single-layer centrifugation with Androcoll-C to enhance sperm quality in frozen–thawed dog semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhtlektrbL&md5=0541f65f6571084d059d9292f581daf7CAS | 23987984PubMed |

England, G. C. W., and Ponzio, P. (1996). Comparison of the quality of frozen–thawed and cooled–rewarmed dog semen. Theriogenology 46, 165–171.
Comparison of the quality of frozen–thawed and cooled–rewarmed dog semen.Crossref | GoogleScholarGoogle Scholar |

García-López, N., Ollero, M., Muino-Blanco, T., and Cebrián-Pérez, J. (1996). A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma. Theriogenology 46, 141–151.
A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma.Crossref | GoogleScholarGoogle Scholar |

Hallap, T., Håård, M., Jaakma, Ü., Larsson, B., and Rodriguez-Martinez, H. (2004). Does cleansing of frozen–thawed bull semen before assessment provide samples that relate better to potential fertility? Theriogenology 62, 702–713.
Does cleansing of frozen–thawed bull semen before assessment provide samples that relate better to potential fertility?Crossref | GoogleScholarGoogle Scholar | 15226024PubMed |

Iguer-ouada, M., and Verstegen, J. P. (2001). Long-term preservation of chilled canine semen: effect of commercial and laboratory prepared extenders. Theriogenology 55, 671–684.
Long-term preservation of chilled canine semen: effect of commercial and laboratory prepared extenders.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3M3gsFKlug%3D%3D&md5=4fd1c53abbe1994ae77a71f81678839bCAS | 11233792PubMed |

Jiménez-Rabadán, P., Morrell, J., Johannisson, A., Ramón, M., García-Álvarez, O., Maroto-Morales, A., Álvaro-García, P., Pérez-Guzmán, M., Fernández-Santos, M., and Garde, J. (2012). Single-layer centrifugation (SLC) improves sperm quality of cryopreserved Blanca-Celtibérica buck semen. Anim. Reprod. Sci. 136, 47–54.
Single-layer centrifugation (SLC) improves sperm quality of cryopreserved Blanca-Celtibérica buck semen.Crossref | GoogleScholarGoogle Scholar | 23084569PubMed |

Johannisson, A., Morrell, J. M., Thoren, J., Jonsson, M., Dalin, A. M., and Rodriguez-Martinez, H. (2009). Colloidal centrifugation with Androcoll-E prolongs stallion sperm motility, viability and chromatin integrity. Anim. Reprod. Sci. 116, 119–128.
Colloidal centrifugation with Androcoll-E prolongs stallion sperm motility, viability and chromatin integrity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVKkt7vK&md5=55e4a0b580c44cf5083c8fcd64f1264fCAS | 19217226PubMed |

Kim, S.-H., Yu, D.-H., and Kim, Y.-J. (2010). Apoptosis-like change, ROS and DNA status in cryopreserved canine spermatozoa recovered by glass-wool filtration and Percoll-gradient centrifugation techniques. Anim. Reprod. Sci. 119, 106–114.
Apoptosis-like change, ROS and DNA status in cryopreserved canine spermatozoa recovered by glass-wool filtration and Percoll-gradient centrifugation techniques.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXit1Kjsrc%3D&md5=9a6030b2fa548b9175fbbaf912676074CAS | 19942382PubMed |

Linde-Forsberg, C. (1991). Achieving canine pregnancy by using frozen or chilled extended semen. Vet. Clin. North Am. Small Anim. Pract. 21, 467–485.
Achieving canine pregnancy by using frozen or chilled extended semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK3MzhtlOksQ%3D%3D&md5=d87869a713d9cdf32c244f5c0ea85c50CAS | 1858243PubMed |

Linde-Forsberg, C. (2000). Fertility data from 2041 controlled artificial inseminations in dogs. In ‘Advances in Dog, Cat and Exotic Carnivore Reproduction’. p. 120. (European Veterinary Society for Small Animal Reproduction: Oslo.)

Liu, B., Cui, Y., and Yu, S. J. (2013). Effect of swim-up and Percoll treatment on sperm quality and in vitro embryo development in yak. J. Integr. Agr. 12, 2235–2242.
Effect of swim-up and Percoll treatment on sperm quality and in vitro embryo development in yak.Crossref | GoogleScholarGoogle Scholar |

Martí, E., Pérez-Pé, R., Muiño-Blanco, T., and Cebrián-Pérez, J. A. (2006). Comparative study of four different sperm washing methods using apoptotic markers in ram spermatozoa. J. Androl. 27, 746–753.
Comparative study of four different sperm washing methods using apoptotic markers in ram spermatozoa.Crossref | GoogleScholarGoogle Scholar | 16809277PubMed |

Martinez-Alborcia, M. J., Morrell, J. M., Parrilla, I., Barranco, I., Vazquez, J. M., Martinez, E. A., and Roca, J. (2012). Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing. Theriogenology 78, 1117–1125.
Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC38jnsVejtw%3D%3D&md5=87955f0602357a7eed748103d9799d16CAS | 22763074PubMed |

Maxwell, W. M. C., Parrilla, I., Caballero, I., Garcia, E., Roca, J., Martinez, E. A., Vazquez, J. M., and Rath, D. (2007). Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSperm density-gradient centrifugation. Reprod. Domest. Anim. 42, 489–494.
Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSperm density-gradient centrifugation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2srjtF2qtQ%3D%3D&md5=4451a3c92a499d0f685ef09c7a03ff72CAS |

Mehmood, A., Anwar, M., and Naqvi, S. M. S. (2009). Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of spermatozoa separated by swim-up or Percoll-gradient method from frozen–thawed buffalo semen. Anim. Reprod. Sci. 111, 141–148.
Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of spermatozoa separated by swim-up or Percoll-gradient method from frozen–thawed buffalo semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1M7it1yrtQ%3D%3D&md5=920f78def78b985f37e7598cd663381dCAS | 18378413PubMed |

Morales, E. C. L., Aragon, M. A., Pescador, S. N., and Salazar, G. F. (2012). Swim-up procedure in boar semen improves motility and viability but recovered spermatozoa could carry active caspases and chromatin damage. J. Anim. Vet. Adv. 11, 431–437.
Swim-up procedure in boar semen improves motility and viability but recovered spermatozoa could carry active caspases and chromatin damage.Crossref | GoogleScholarGoogle Scholar |

Morrell, J. M., and Rodriguez-Martinez, H. (2009). Biomimetic techniques for improving sperm quality in animal breeding: a review. T. O. Androl. J. 1, 1–9.

Morrell, J. M., and Rodriguez-Martinez, H. (2011). Practical applications of sperm selection techniques as a tool for improving reproductive efficiency. Vet. Med. Int. 2011, 894767.
Practical applications of sperm selection techniques as a tool for improving reproductive efficiency.Crossref | GoogleScholarGoogle Scholar |

Morrell, J. M., Rodriguez-Martinez, H., and Linde-Forsberg, C. (2008). Single-layer centrifugation on a colloid selects motile and morphologically normal spermatozoa from dog semen: preliminary results. Reprod. Domest. Anim. 43, 61.

Morrell, J. M., Rodriguez-Martinez, H., and Johannisson, A. (2010). Single-layer centrifugation of stallion spermatozoa improves sperm quality compared with sperm washing. Reprod. Biomed. Online 21, 429–436.
Single-layer centrifugation of stallion spermatozoa improves sperm quality compared with sperm washing.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3cfgtVGgug%3D%3D&md5=44e65bea2cd20b524f5c2272ee265fdfCAS | 20674501PubMed |

Morrell, J. M., Garcia, B. M., Pena, F. J., and Johannisson, A. (2011). Processing stored stallion semen doses by single-layer centrifugation. Theriogenology 76, 1424–1432.
Processing stored stallion semen doses by single-layer centrifugation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3MfptFOktw%3D%3D&md5=3c883cbda49a4279d83f1d6e79c9de21CAS | 21798585PubMed |

Morrell, J., Karlsson, J., and Axner, E. (2013). Single-layer centrifugation can be scaled up to process larger volumes of dog semen. Reprod. Domest. Anim. 48, 64–65.

Nicolas, M., Alvarez, M., Borragán, S., Martinez-Pastor, F., Chamorro, C., Alvarez-Rodriguez, M., de Paz, P., and Anel, L. (2012). Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine and PureSperm 100) in preparation of fresh or frozen–thawed brown bear spermatozoa. Theriogenology 77, 1119–1128.
Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine and PureSperm 100) in preparation of fresh or frozen–thawed brown bear spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC383otVeksg%3D%3D&md5=e872fda8273d2c42d1def01bd65da940CAS | 22154477PubMed |

Núñez-Martínez, I., Moran, J., and Peña, F. (2006). A three-step statistical procedure to identify sperm kinematic subpopulations in canine ejaculates: changes after cryopreservation. Reprod. Domest. Anim. 41, 408–415.
A three-step statistical procedure to identify sperm kinematic subpopulations in canine ejaculates: changes after cryopreservation.Crossref | GoogleScholarGoogle Scholar | 16984346PubMed |

Parrish, J. J., Krogenaes, A., and Susko-Parrish, J. L. (1995). Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilisation and early embryonic development. Theriogenology 44, 859–869.
Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilisation and early embryonic development.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28zgtVGgug%3D%3D&md5=6fe6a3e35453d4b0df24bd49bbb6b2f3CAS | 16727781PubMed |

Peña, A. I., Lugilde, L. L., Barrio, M., Herradón, P. G., and Quintela, L. A. (2003). Effects of Equex from different sources on post-thaw survival, longevity and intracellular Ca2+ concentration of dog spermatozoa. Theriogenology 59, 1725–1739.
Effects of Equex from different sources on post-thaw survival, longevity and intracellular Ca2+ concentration of dog spermatozoa.Crossref | GoogleScholarGoogle Scholar | 12566147PubMed |

Phillips, T. C., Dhaliwal, G. K., Verstegen-Onclin, K. M., and Verstegen, J. P. (2012). Efficacy of four density-gradient separation media to remove erythrocytes and nonviable spermatozoa from canine semen. Theriogenology 77, 39–45.
Efficacy of four density-gradient separation media to remove erythrocytes and nonviable spermatozoa from canine semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC38%2FlvVGrtA%3D%3D&md5=eab6bb460c03c9d9a409b5d3995c531fCAS | 21803408PubMed |

Ponglowhapan, S., Essén-Gustavsson, B., and Linde Forsberg, C. (2004). Influence of glucose and fructose in the extender during long-term storage of chilled canine semen. Theriogenology 62, 1498–1517.
Influence of glucose and fructose in the extender during long-term storage of chilled canine semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXnvV2rtr4%3D&md5=fff611153550e43c0eabf7075045e867CAS | 15451258PubMed |

Rho, G. J., Hahnel, A. C., and Betteridge, K. J. (2001). Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro. Theriogenology 56, 503–516.
Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3MvntFCktg%3D%3D&md5=0c9330db6b21963283c606c113ea48b9CAS | 11516129PubMed |

Rijsselaere, T., Van Soom, A., Maes, D., and de Kruif, A. (2002). Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa. Theriogenology 57, 1669–1681.
Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD383psFyntg%3D%3D&md5=1311ed92d5bcc8cdc10e3446dd4f91b0CAS | 12035977PubMed |

Rodriguez-Martinez, H., Larsson, B., and Pertoft, H. (1997). Evaluation of sperm damage and techniques for sperm clean-up. Reprod. Fertil. Dev. 9, 297–308.
Evaluation of sperm damage and techniques for sperm clean-up.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK2svgsFajug%3D%3D&md5=bc4244939c6c6ae41551e97b83bf75cdCAS | 9261878PubMed |

Rota, A., Milani, C., and Romagnoli, S. (2007). Effect of post-thaw dilution with autologous prostatic fluid on dog semen motility and sperm acrosome status. Theriogenology 67, 520–525.
Effect of post-thaw dilution with autologous prostatic fluid on dog semen motility and sperm acrosome status.Crossref | GoogleScholarGoogle Scholar | 17010416PubMed |

Sánchez, R., Risopatrón, J., Schulz, M., Villegas, J., Isachenko, V., Kreinberg, R., and Isachenko, E. (2011). Canine sperm vitrification with sucrose: effect on sperm function. Andrologia 43, 233–241.
Canine sperm vitrification with sucrose: effect on sperm function.Crossref | GoogleScholarGoogle Scholar | 21486402PubMed |

Sharma, R. K., Vemulapalli, S., Kohn, S., and Agarwal, A. (1997). Effect of centrifuge speed, refrigeration medium and sperm washing medium on cryopreserved sperm quality after thawing. Arch. Androl. 39, 33–38.
Effect of centrifuge speed, refrigeration medium and sperm washing medium on cryopreserved sperm quality after thawing.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK2szks1Citg%3D%3D&md5=1ff07997156bf5ff2c965c64b69809a6CAS | 9202831PubMed |

Somfai, T., Bodó, S., Nagy, S., Papp, A., Ivancsics, J., Baranyai, B., Gocza, E., and Kovacs, A. (2002). Effect of swim-up and Percoll treatment on viability and acrosome integrity of frozen–thawed bull spermatozoa. Reprod. Domest. Anim. 37, 285–290.
Effect of swim-up and Percoll treatment on viability and acrosome integrity of frozen–thawed bull spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD38njtlWgug%3D%3D&md5=0040e4cea96ea61c1275fe91caa6bbd5CAS | 12354181PubMed |