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RESEARCH ARTICLE

Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation

Olga García-Álvarez A C , Alejandro Maroto-Morales A , Manuel Ramón B , Enrique del Olmo A , Pilar Jiménez-Rabadán B , M. Rocio Fernández-Santos A , Luis Anel-López A , J. Julián Garde A and Ana J. Soler A
+ Author Affiliations
- Author Affiliations

A SaBio IREC (Instituto de Investigacion en Recursos Cinegeticos), Campus Universitario s.n. 02071 Albacete, Spain.

B CERSYRA (Centro Regional de Seleccion y Reproduccion Animal) JCCM, Avda. Del Vino 6, 13300 Valdepeñas, Spain.

C Corresponding author. Email: garciaalvarez.olga@gmail.com

Reproduction, Fertility and Development 26(5) 725-732 https://doi.org/10.1071/RD13034
Submitted: 3 February 2013  Accepted: 28 April 2013   Published: 27 May 2013

Abstract

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm samples were incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2 h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. A significantly greater increase (P ≤ 0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as ‘hyperactivated like’, with major changes occurring after 1 h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place.

Additional keywords: hyperactivation, membrane fluidity.


References

Bailey, J. L., and Buhr, M. M. (1993). Ca2+ regulation by cryopreserved bull spermatozoa in response to A23187. Cryobiology 30, 470–481.
Ca2+ regulation by cryopreserved bull spermatozoa in response to A23187.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXhtVehurk%3D&md5=c028bb1acfffb0a1c369bc01166c0e9cCAS | 8252915PubMed |

Bailey, J. L., Bilodeau, J. F., and Cormier, N. (2000). Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J. Androl. 21, 1–7.
| 1:STN:280:DC%2BD3c7jtl2ktg%3D%3D&md5=0390e0d48a080baf0b64dece7e86e93dCAS | 10670514PubMed |

Berlinguer, F., Madeddu, M., Pasciu, V., Succu, S., Spezzigu, A., Satta, V., Mereu, P., Leoni, G. G., and Naitana, S. (2009). Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model. Reprod. Biol. Endocrinol. 7, 125.
Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model.Crossref | GoogleScholarGoogle Scholar | 19900288PubMed |

Buffone, M. G., Doncel, G. F., Marin Briggiler, C. I., Vazquez-Levin, M. H., and Calamera, J. C. (2004). Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation. Hum. Reprod. 19, 139–146.
Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2c%2FhsFarsQ%3D%3D&md5=bc71bb4087aeaedf787192dcfa1898e9CAS | 14688172PubMed |

Buffone, M. G., Doncel, G. F., Calamera, J. C., and Verstraeten, S. V. (2009). Capacitation-associated changes in membrane fluidity in asthenozoospermic human spermatozoa. Int. J. Androl. 32, 360–375.
Capacitation-associated changes in membrane fluidity in asthenozoospermic human spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVersrfN&md5=38916c83b104d20404eb0531b96e4fe6CAS | 18399983PubMed |

Cognié, Y., Baril, G., Poulin, N., and Mermillod, P. (2003). Current status of embryo technologies in sheep and goat. Theriogenology 59, 171–188.
Current status of embryo technologies in sheep and goat.Crossref | GoogleScholarGoogle Scholar | 12499027PubMed |

Gadella, B. M. (2013). Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation. Reprod. Fertil. Dev. 25, 26–37.
Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation.Crossref | GoogleScholarGoogle Scholar |

Gadella, B. M., and Van Gestel, R. A. (2004). Bicarbonate and its role in mammalian sperm function. Anim. Reprod. Sci. 82–83, 307–319.
Bicarbonate and its role in mammalian sperm function.Crossref | GoogleScholarGoogle Scholar | 15271462PubMed |

García-Álvarez, O., Maroto-Morales, A., Martinez-Pastor, F., Garde, J. J., Ramón, M., Fernández-Santos, M. R., Esteso, M. C., Pérez-Guzmán, M. D., and Soler, A. J. (2009). Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: electroejaculation and postmortem collection. Theriogenology 72, 160–168.
Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: electroejaculation and postmortem collection.Crossref | GoogleScholarGoogle Scholar | 19349069PubMed |

García-Álvarez, O., Maroto-Morales, A., Ramón, M., del Olmo, E., Montoso, V., Dominguez-Rebolledo, A. E., Bisbal, A., Jiménez-Rabadán, P., Pérez-Guzmán, M. D., and Soler, A. J. (2010). Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa. Theriogenology 74, 979–988.
Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa.Crossref | GoogleScholarGoogle Scholar | 20580077PubMed |

García-Álvarez, O., Maroto-Morales, A., Berlinguer, F., Fernández-Santos, M. R., Esteso, M. C., Mermillod, P., Ortiz, J. A., Ramon, M., Pérez-Guzmán, M. D., Garde, J. J., and Soler, A. J. (2011). Effect of storage temperature during transport of ovaries on in vitro embryo production in Iberian red deer (Cervus elaphus hispanicus). Theriogenology 75, 65–72.
Effect of storage temperature during transport of ovaries on in vitro embryo production in Iberian red deer (Cervus elaphus hispanicus).Crossref | GoogleScholarGoogle Scholar | 21040958PubMed |

Gardner, D. K., Lane, M., Spitzer, A., and Batt, P. A. (1994). Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development. Biol. Reprod. 50, 390–400.
Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK2c7ptFWisA%3D%3D&md5=b462f69c71f9cc1d1475615c400df9c0CAS | 8142556PubMed |

Goodson, S. G., Zhang, Z., Tsuruta, J. K., Wang, W., and O’Brien, D. A. (2011). Classification of mouse sperm motility pattern using an automated multiclass support vector machines model. Biol. Reprod. 84, 1207–1215.
Classification of mouse sperm motility pattern using an automated multiclass support vector machines model.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXmvFemu74%3D&md5=412fd7f26b12dae7542b577adafdbe90CAS | 21349820PubMed |

Green, C. E., and Watson, P. F. (2001). Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation. Reproduction 122, 889–898.
Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XhvVWjug%3D%3D&md5=499e78f76f873cf5a889f89318289a8eCAS | 11732984PubMed |

Harrison, R. A. P. (1996). Capacitation mechanism, and the role of capacitation as seen in eutherian mammals. Reprod. Fertil. Dev. 8, 581–594.
Capacitation mechanism, and the role of capacitation as seen in eutherian mammals.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK2s%2FitlOnsQ%3D%3D&md5=a30f4e336d42d020cf4260d03d883ca7CAS |

Harrison, R. A. P., and Gadella, B. M. (2005). Bicarbonate-induced membrane processing in sperm capacitation. Theriogenology 63, 342–351.
Bicarbonate-induced membrane processing in sperm capacitation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjsVSg&md5=c1040b46693e34c213975ccdd4cf4ef2CAS |

Ho, H. C., and Suarez, S. S. (2001). An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility. Biol. Reprod. 65, 1606–1615.
An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXnvVersb0%3D&md5=0b562692ceb607a74bf41e10108843cfCAS | 11673282PubMed |

Holt, W. V., and Van Look, K. J. (2004). Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality. Reproduction 127, 527–535.
Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXkvFOqurk%3D&md5=d7b8bc8ea2536c6b41cd9c570b68c722CAS | 15129008PubMed |

Huneau, D., Crozet, N., and Ahmed-Ali, M. (1994). Estrous sheep serum as a potent agent for ovine IVF: effect on cholesterol efflux from spermatozoa and the acrosome reaction. Theriogenology 42, 1017–1028.
Estrous sheep serum as a potent agent for ovine IVF: effect on cholesterol efflux from spermatozoa and the acrosome reaction.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXis1Sms78%3D&md5=65be8159c2c3e8119daa77f18585d339CAS | 16727605PubMed |

Maroto-Morales, A., Ramón, M., García-Álvarez, O., Soler, A. J., Fernández-Santos, M. R., Roldan, E. R., Gomendio, M., Pérez-Guzmán, M. D., and Garde, J. J. (2012). Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and interindividual variation. Theriogenology 77, 1529–1539.
Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and interindividual variation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC38vkt1GmtA%3D%3D&md5=5651a41f5cd859f9a085ba0624b9bf85CAS | 22365694PubMed |

Marquez, B., and Suarez, S. S. (2007). Bovine sperm hyperactivation is promoted by alkaline-stimulated Ca2+ influx. Biol. Reprod. 76, 660–665.
Bovine sperm hyperactivation is promoted by alkaline-stimulated Ca2+ influx.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXjsFCmsr8%3D&md5=0649a20e5a2e5ce22179fa2f5b9f87e9CAS | 17182893PubMed |

Martinez-Pastor, F., Garcia-Macias, V., Alvarez, M., Herraez, P., Anel, L., and de Paz, P. (2005). Sperm subpopulations in Iberian red deer epididymal sperm and their changes through the cryopreservation process. Biol. Reprod. 72, 316–327.
Sperm subpopulations in Iberian red deer epididymal sperm and their changes through the cryopreservation process.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXptFGmtA%3D%3D&md5=199fb1e483c1955bac99d349b4043231CAS | 15385419PubMed |

Martinez-Pastor, F., Garcia-Macias, V., Alvarez, M., Chamorro, C., Herraez, P., de Paz, P., and Anel, L. (2006). Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer. Theriogenology 65, 471–485.
Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer.Crossref | GoogleScholarGoogle Scholar | 15996726PubMed |

Martínez-Pastor, F., Fernández-Santos, M. R., del Olmo, E., Domínguez-Rebolledo, A. E., Esteso, M. C., Montoro, V., and Garde, J. J. (2008). Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulation. Reprod. Fertil. Dev. 20, 547–556.
Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulation.Crossref | GoogleScholarGoogle Scholar | 18577351PubMed |

McPartlin, L. A., Suarez, S. S., Czaya, C. A., Hinrichs, K., and Bedford-Guaus, S. J. (2009). Hyperactivation of stallion sperm is required for succesful in vitro fertilization of equine oocytes. Biol. Reprod. 81, 199–206.
Hyperactivation of stallion sperm is required for succesful in vitro fertilization of equine oocytes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXnslaqs7w%3D&md5=3b089cbab3af6cf5ed911981979de382CAS | 19208544PubMed |

Mortimer, S. T., and Maxwell, W. M. (1999). Kinematic definition of ram sperm hyperactivation. Reprod. Fertil. Dev. 11, 25–30.
Kinematic definition of ram sperm hyperactivation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7ktlagtg%3D%3D&md5=32f5b154eae77c1457675f0ebe6e955cCAS | 10680999PubMed |

Mortimer, S. T., and Mortimer, D. (1990). Kinematics of human spermatozoa incubated under capacitating conditions. J. Androl. 11, 195–203.
| 1:STN:280:DyaK3czks1Kguw%3D%3D&md5=0b3345af2946f461e0cc73a4aadd8702CAS | 2384341PubMed |

O’Meara, C. M., Hanrahan, J. P., Donovan, A., Fair, S., Rizos, D., Wade, M., Boland, M. P., Evans, A. C., and Lonergan, P. (2005). Relationship between in vitro fertilization of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen–thawed ram semen. Theriogenology 64, 1797–1808.
Relationship between in vitro fertilization of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen–thawed ram semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2Mrlt1Cmug%3D%3D&md5=56cb661029789f4445a2c36f4129ed91CAS | 15936809PubMed |

Papadopoulos, S., Hanrahan, J. P., Donovan, A., Duffy, P., Boland, M. P., and Lonergan, P. (2005). In vitro fertilization as a predictor of fertility from cervical insemination of sheep. Theriogenology 63, 150–159.
In vitro fertilization as a predictor of fertility from cervical insemination of sheep.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2cnhvFektQ%3D%3D&md5=6781337a6acc96f4d01ff7240af859d4CAS | 15589281PubMed |

Parrish, J. J. (1991). Application of in vitro fertilization to domestic animals. In ‘Elements of Mammalian Fertilization’. (Ed. P. M. Wassarman.) pp. 111–132. (CRC Press: Boca Raton.)

Pérez-Pé, R., Grasa, P., Fernández-Juan, M., Peleato, M. L., Cebrián-Pérez, J. A., and Muiño-Blanco, T. (2002). Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa. Mol. Reprod. Dev. 61, 226–233.
Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.Crossref | GoogleScholarGoogle Scholar | 11803559PubMed |

Pons-Rejraji, H., Bailey, J. L., and Leclerc, P. (2009). Cryopreservation affects bovine sperm intracellular parameters associated with capacitation and acrosome exocytosis. Reprod. Fertil. Dev. 21, 525–537.
Cryopreservation affects bovine sperm intracellular parameters associated with capacitation and acrosome exocytosis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXksF2ru7w%3D&md5=6e947b52998462125b7bc9666e34489cCAS | 19383259PubMed |

R Development Core Team (2012). ‘R: A Language and Environmental for Statistical Computing.’ (R Foundation for Statistical Computing: Vienna.) Available from http://www-R-project.org/ [Verified 8 May 2013].

Ramió, L., Rivera, M. M., Ramírez, A., Concha, I. I., Peña, A., Rigau, T., and Rodríguez-Gil, J. E. (2008). Dynamics of motile-sperm subpopulation structure in boar ejaculates subjected to ‘in vitro’ capacitation and further ‘in vitro’ acrosome reaction. Theriogenology 69, 501–512.
Dynamics of motile-sperm subpopulation structure in boar ejaculates subjected to ‘in vitro’ capacitation and further ‘in vitro’ acrosome reaction.Crossref | GoogleScholarGoogle Scholar | 18068222PubMed |

Rathi, R., Colenbrander, B., Bevers, M. M., and Gadella, B. M. (2001). Evaluation of in vitro capacitation of stallion spermatozoa. Biol. Reprod. 65, 462–470.
Evaluation of in vitro capacitation of stallion spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXls1Wnsr4%3D&md5=2cc9bd9f65494db765686f5bfed5914cCAS | 11466214PubMed |

Robertson, L., Wolf, D. P., and Tash, J. S. (1988). Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm. Biol. Reprod. 39, 797–805.
Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL1M%2FovVemug%3D%3D&md5=716c122a0e1b2b47c29a26086194a05cCAS | 3207805PubMed |

Sidhu, K. S., Mate, K. E., Gunasekera, T., Veal, D., Hetherington, L., Baker, M. A., Aitken, R. J., and Rodger, J. C. (2004). A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula). Reproduction 127, 95–103.
A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhvVOiurc%3D&md5=eb1f143f6f2643058075d3d2bfd6d8f0CAS | 15056774PubMed |

Suarez, S. S. (2008). Control of hyperactivation in sperm. Hum. Reprod. Update 14, 647–657.
Control of hyperactivation in sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXht1Ois7%2FK&md5=c9d6b095775fe2c65142f01c6067a6a9CAS | 18653675PubMed |

Takahashi, Y., and First, N. L. (1992). In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 37, 963–978.
In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXpvVOmtg%3D%3D&md5=2e75cafe1e37db9a99ff32d405842fc7CAS | 16727096PubMed |

Tamayo-Canul, J., Alvarez, M., López-Urueña, E., Nicolas, M., Martinez-Pastor, F., Anel, E., Anel, L., and de Paz, P. (2011). Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes. Anim. Reprod. Sci. 126, 76–82.
Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3MnltVOjtQ%3D%3D&md5=f8fc98b6e0f83150feac6dda47c70e42CAS | 21601384PubMed |

Topper, E. K., Killian, G. J., Way, A., Engel, B., and Woelders, H. (1999). Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa. J. Reprod. Fertil. 115, 175–183.
Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXhs12isLo%3D&md5=c25f5a98dcd0d99472754d82f400c077CAS | 10341736PubMed |

Tsai, P. S., Garcia-Gil, N., van Haeften, T., and Gadella, B. M. (2010). How pig sperm prepares to fertilize: stable acrosome docking to the plasma membrane. PLoS One 5, e11204.
How pig sperm prepares to fertilize: stable acrosome docking to the plasma membrane.Crossref | GoogleScholarGoogle Scholar | 20585455PubMed |

Visconti, P. E., and Kopf, G. S. (1998). Regulation of protein phosphorylation during sperm capacitation. Biol. Reprod. 59, 1–6.
Regulation of protein phosphorylation during sperm capacitation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXktFaksLw%3D&md5=dabded89a0bf24a8d6ecb8c520214529CAS | 9674985PubMed |

Visconti, P. E., Galantino-Homer, H., Ning, X., Moore, G. D., Valenzuela, J. P., Jorgez, C. J., Alvarez, J. G., and Kopf, G. S. (1999). Cholesterol efflux-mediated signal transduction in mammalian sperm. Beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation. J. Biol. Chem. 274, 3235–3242.
Cholesterol efflux-mediated signal transduction in mammalian sperm. Beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXpsVCisQ%3D%3D&md5=ae4c7da66a007acac2d12ebbb5746968CAS | 9915865PubMed |

Visconti, P. E., Westbrook, V. A., Chertihin, O., Demarco, I., Sleight, S., and Diekman, A. B. (2002). Novel signaling pathways involved in sperm acquisition of fertilizing capacity. J. Reprod. Immunol. 53, 133–150.
Novel signaling pathways involved in sperm acquisition of fertilizing capacity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXos1Kjtrw%3D&md5=41bbfa1dc75b7ea6490f1ed1ca56a456CAS | 11730911PubMed |

Vulcano, G. J., Moses, D. F., Valcárcel, A., and de las Heras, M. A. (1998). A lineal equation for the classification of progressive and hyperactive spermatozoa. Math. Biosci. 149, 77–93.
A lineal equation for the classification of progressive and hyperactive spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK1c3ntlSgsA%3D%3D&md5=6404fa125570e88765ae9d382ad5f03cCAS | 9610112PubMed |

Watson, P. F. (1995). Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7, 871–891.
Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK283ns1alsQ%3D%3D&md5=8f96510fb51d101b2064e6aa8f7569c0CAS | 8711221PubMed |

Watson, P. F. (2000). The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60–61, 481–492.
The causes of reduced fertility with cryopreserved semen.Crossref | GoogleScholarGoogle Scholar | 10844218PubMed |

Yanagimachi, R. (1970). The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fertil. 23, 193–196.
The movement of golden hamster spermatozoa before and after capacitation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaE3M%2FhtFCrtQ%3D%3D&md5=e15c3ebd39937073e89de835535dc90eCAS | 5472441PubMed |

Yanagimachi, R. (1994). Mammalian fertilization. In ‘The Physiology of Reproduction’, Volume I. (Eds E. Knobil, J. D. Neill, L. L. Edwing, C. L. Markert, G. S. Greenwald and D. W. Pfaff.) pp. 135–185. (Raven Press: New York.)