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RESEARCH ARTICLE

Effects of reduced glutathione on acrosin activity in frozen–thawed boar spermatozoa

Efrén Estrada A , Joan E. Rodríguez-Gil A , Maria M. Rivera del Álamo A , Alejandro Peña A and Marc Yeste A B C
+ Author Affiliations
- Author Affiliations

A Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain.

B Present address: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK.

C Corresponding author. Email: marc.yeste@obs-gyn.ox.ac.uk

Reproduction, Fertility and Development 29(2) 283-293 https://doi.org/10.1071/RD15118
Submitted: 26 March 2015  Accepted: 27 June 2015   Published: 21 July 2015

Abstract

In pigs, acrosin activity in extended semen is correlated with reproductive performance and has recently been identified as a freezability marker. Reduced glutathione (GSH) is known to decrease sperm cryodamage and increase the reproductive performance of frozen–thawed boar spermatozoa. However, the effects of GSH on the acrosin activity of good and poor freezability ejaculates (GFE and PFE, respectively) is yet to be examined. The present study investigated how supplementing cryopreservation media with GSH affected acrosin activity in GFE and PFE, as well as the relationship between acrosin activity and reproductive performance in frozen–thawed boar spermatozoa. In addition, we examined whether the increase in fertility rates and litter sizes observed after the addition of 2 mM GSH to cryopreservation extenders was related to acrosin activity. Supplementing freezing media with 2 mM GSH partially counteracted the cryopreservation-related decrease in acrosin activity in GFE but not PFE. Acrosin activity was found to be significantly correlated with in vivo reproductive performance of frozen–thawed boar semen. In conclusion, the effects of adding GSH to freezing extenders on the acrosin activity of frozen–thawed boar spermatozoa rely on the intrinsic freezability of the ejaculate. Furthermore, the maintenance of proper acrosin activity could contribute to the increase in reproductive performance mediated by GSH.

Additional keywords: cryopreservation, reproductive performance.


References

Almlid, T., and Hofmo, P. O. (1995). A brief review of frozen semen application under Norwegian AI service conditions. Reprod. Domest. Anim. 31, 169–173.
A brief review of frozen semen application under Norwegian AI service conditions.Crossref | GoogleScholarGoogle Scholar |

Benson, J. D., Woods, E. J., Walters, E. M., and Critser, J. K. (2012). The cryobiology of spermatozoa. Theriogenology 78, 1682–1699.
The cryobiology of spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhsFSlt7vK&md5=dfa8314911698acaaf7810e850c7bf81CAS | 23062722PubMed |

Berger, T., Anderson, D. L., and Penedo, M. C. T. (1996). Porcine sperm fertilizing potential in relationship to sperm functional capacities. Anim. Reprod. Sci. 44, 231–239.
Porcine sperm fertilizing potential in relationship to sperm functional capacities.Crossref | GoogleScholarGoogle Scholar |

Casas, I., and Flores, E. (2013). Gene banking, the freezing strategy. In ‘Boar Reproduction: Fundamentals and New Biotechnological Trends’. (Eds S. Bonet, I. Casas, W. V. Holt and M. Yeste.) pp. 551–588. (Springer: Berlin.)

Casas, I., Sancho, S., Briz, M., Pinart, E., Bussalleu, E., Yeste, M., and Bonet, S. (2009). Freezability prediction of boar ejaculates assessed by functional sperm parameters and sperm proteins. Theriogenology 72, 930–948.
Freezability prediction of boar ejaculates assessed by functional sperm parameters and sperm proteins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFGlsr3E&md5=126cc65c2ee548c12517e15caef70a75CAS | 19651432PubMed |

Casas, I., Sancho, S., Briz, M., Pinart, E., Bussalleu, E., Yeste, M., and Bonet, S. (2010). Fertility after post-cervical artificial insemination with cryopreserved sperm from boar ejaculates of good and poor freezability. Anim. Reprod. Sci. 118, 69–76.
Fertility after post-cervical artificial insemination with cryopreserved sperm from boar ejaculates of good and poor freezability.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1MfhsFyltw%3D%3D&md5=2f06a3ec0a0304499b1e8480d1eee93bCAS | 19577868PubMed |

Casas, I., Torner, E., Yeste, M., and Bonet, S. (2012). Boar sperm thawing practices: the number of straws does matter. Theriogenology 77, 1487–1494.
Boar sperm thawing practices: the number of straws does matter.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC38zpvFamtA%3D%3D&md5=af97ee0956f064077356447cb0a057dcCAS | 22225687PubMed |

Chelucci, S., Pasciu, V., Succu, S., Addis, D., Leoni, G. G., Manca, M. E., Naitana, S., and Berlinguer, F. (2015). Soybean lecithin-based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation. Theriogenology 83, 1064–1074.
Soybean lecithin-based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXosFWlug%3D%3D&md5=79833ef389c940649c4fb9e18c62c72dCAS | 25595356PubMed |

Cross, N. L., and Hanks, S. E. (1991). Effects of cryopreservation on human sperm acrosomes. Hum. Reprod. 6, 1279–1283.
| 1:STN:280:DyaK38%2FptFCksw%3D%3D&md5=faefb46b5b5c29c68dd956688d9b1135CAS | 1752931PubMed |

Estrada, E., Rodríguez-Gil, J. E., Rocha, L. G., Balasch, S., Bonet, S., and Yeste, M. (2014). Supplementing cryopreservation media with reduced glutathione increases fertility and prolificacy of sows inseminated with frozen–thawed boar semen. Andrology 2, 88–99.
Supplementing cryopreservation media with reduced glutathione increases fertility and prolificacy of sows inseminated with frozen–thawed boar semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXitVSqtL7L&md5=80f8ea1c7acfa6a1bb38fffd7a90d5a9CAS | 24123940PubMed |

Froman, D. P., Amann, R. P., Riek, P. M., and Olar, T. T. (1984). Acrosin activity of canine spermatozoa as an index of cellular damage. J. Reprod. Fertil. 70, 301–308.
Acrosin activity of canine spermatozoa as an index of cellular damage.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2cXotlCntg%3D%3D&md5=9ebdcd8bede6c9322e80de405dfeb810CAS | 6420555PubMed |

Gadea, J., Sellés, E., Marco, M. A., Coy, P., Matás, C., Romar, R., and Ruiz, S. (2004). Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology 62, 690–701.
Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXltF2rs78%3D&md5=54afa844b0beaf4556e558641b7b1ef6CAS | 15226023PubMed |

Gadea, J., García-Vázquez, F. A., Matás, C., Gardón, J. C., Cánovas, S., and Gumbao, D. (2005). Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function. J. Androl. 26, 396–404.
Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXkvVWjsrg%3D&md5=8541039235b15d416d2cd8a818599cbaCAS | 15867008PubMed |

Garner, D. L., and Johnson, L. A. (1995). Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol. Reprod. 53, 276–284.
Viability assessment of mammalian sperm using SYBR-14 and propidium iodide.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXntVSmtbc%3D&md5=0f6a789b563b0e235afb8865e5d855dbCAS | 7492679PubMed |

Giaretta, E., Estrada, E., Bucci, D., Spinaci, M., Rodríguez-Gil, J. E., and Yeste, M. (2015). Combining reduced glutathione and ascorbic acid has supplementary beneficial effects on boar sperm cryotolerance. Theriogenology 83, 399–407.
Combining reduced glutathione and ascorbic acid has supplementary beneficial effects on boar sperm cryotolerance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXhslyrtbrJ&md5=4aee94780fb81641ad295db2f51a3e57CAS | 25459422PubMed |

Guthrie, H. D., and Welch, G. R. (2006). Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J. Anim. Sci. 84, 2089–2100.
Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xot1Onur8%3D&md5=97fdb32528aa5edb10bd8c6f2e16d339CAS | 16864869PubMed |

Hammitt, D. G., and Martin, P. A. (1989). Correlations among assays of porcine semen quality following cryopreservation. Theriogenology 32, 369–384.
Correlations among assays of porcine semen quality following cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD283pvFCntQ%3D%3D&md5=768cf1af8de64041745a1686f0ece805CAS | 16726684PubMed |

Hancock, J. L., and Howell, G. J. L. (1959). The collection of boar semen. Vet. Rec. 71, 664–665.

Holt, W. V. (2000). Basics aspects of frozen storage of semen. Anim. Reprod. Sci. 62, 3–22.
Basics aspects of frozen storage of semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXlt1Kgsbo%3D&md5=ccec61cde0e440d46733b4ff64686a9dCAS | 10924818PubMed |

Johnson, L. A., Weitze, K. F., Fiser, P., and Maxwell, W. M. C. (2000). Storage of boar semen. Anim. Reprod. Sci. 62, 143–172.
Storage of boar semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXlt1Kgsbc%3D&md5=f3b823924653c634152a21a25f2bfc6bCAS | 10924823PubMed |

Knox, R. V. (2011). The current value of frozen–thawed boar semen for commercial companies. Reprod. Domest. Anim. 46, 4–6.
The current value of frozen–thawed boar semen for commercial companies.Crossref | GoogleScholarGoogle Scholar | 21884269PubMed |

Langlois, M. R., Oorlynck, L., Vanderkerckhove, F., Criel, A., Bernard, D., and Blaton, V. (2005). Discrepancy between sperm acrosin activity and sperm morphology: significance for fertilization in vitro. Clin. Chim. Acta 351, 121–129.
Discrepancy between sperm acrosin activity and sperm morphology: significance for fertilization in vitro.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhtVant73I&md5=5b22738cd1e6678667d7bb2a80abbf22CAS | 15563880PubMed |

Lo Leggio, L., Williams, R. M., and Jones, R. (1994). Some effects of zona pellucida glycoproteins and sulfated polymers on the autoactivation of boar sperm proacrosin and activity of beta-acrosin. J. Reprod. Fertil. 100, 177–185.
Some effects of zona pellucida glycoproteins and sulfated polymers on the autoactivation of boar sperm proacrosin and activity of beta-acrosin.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK2c3jvVGiuw%3D%3D&md5=1552bccbd8c85c3eb33d1eeeeee27e1dCAS | 8182587PubMed |

Mack, S. R., and Zaneveld, L. J. (1987). Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa. Gamete Res. 18, 375–383.
Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1cXltlGnsw%3D%3D&md5=0c6e163912e3f7706fe4e441dd7607efCAS | 3507383PubMed |

Nagy, S., Jansen, J., Topper, E. K., and Gadella, B. M. (2003). A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles. Biol. Reprod. 68, 1828–1835.
A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjt12ltLk%3D&md5=70c3b51ddaae47f3cc682cf974a0307fCAS | 12606354PubMed |

Paudel, K. P., Kumar, S., Meur, S. K., and Kumaresan, A. (2010). Ascorbic acid, catalase and chlorpromazine reduce cryopreservation-induced damages to crossbred bull spermatozoa. Reprod. Domest. Anim. 45, 256–262.
Ascorbic acid, catalase and chlorpromazine reduce cryopreservation-induced damages to crossbred bull spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXltV2htLg%3D&md5=01accc3d39814512d034b401bbeac1a8CAS | 19032433PubMed |

Petrunkina, A. M., Waberski, D., Bollwein, H., and Sieme, H. (2010). Identifying non-sperm particles during flow cytometric physiological assessment: a simple approach. Theriogenology 73, 995–1000.
Identifying non-sperm particles during flow cytometric physiological assessment: a simple approach.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3c3gsVejtQ%3D%3D&md5=697102a69a3354ddb1f1eaf5a17bda5aCAS | 20171719PubMed |

Pinart, E., Yeste, M., Puigmulé, M., Barrera, X., and Bonet, S. (2013). Acrosin activity is a suitable indicator of boar sperm preservation at 17°C when increasing environmental temperature and radiation. Theriogenology 80, 234–247.
Acrosin activity is a suitable indicator of boar sperm preservation at 17°C when increasing environmental temperature and radiation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXns1Kmsb8%3D&md5=64d89f37aa99be63b6d7bbb2e9831323CAS | 23669168PubMed |

Pinart, E., Yeste, M., and Bonet, S. (2015). Acrosin activity is a good predictor of boar sperm freezability. Theriogenology , .
Acrosin activity is a good predictor of boar sperm freezability.Crossref | GoogleScholarGoogle Scholar | 25796288PubMed |

Polge, C., Salamon, S., and Wilmut, I. (1970). Fertilizing capacity of frozen boar semen following surgical insemination. Vet. Rec. 87, 424–429.
Fertilizing capacity of frozen boar semen following surgical insemination.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaE3M%2FltlClsw%3D%3D&md5=954c64003e9577a509db0c22ff951e11CAS | 5485635PubMed |

Puigmulé, M., Fàbrega, A., Yeste, M., Bonet, S., and Pinart, E. (2011). Study of the proacrosin–acrosin activity system in epididymal, ejaculated and in vitro capacitated boar spermatozoa. Reprod. Fertil. Dev. 23, 837–845.
Study of the proacrosin–acrosin activity system in epididymal, ejaculated and in vitro capacitated boar spermatozoa.Crossref | GoogleScholarGoogle Scholar | 21871203PubMed |

Pursel, V. G., and Johnson, L. A. (1975). Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40, 99–102.
| 1:STN:280:DyaE2M%2FnslCjtw%3D%3D&md5=135a765f5f592ff091b81f4c622c34cfCAS | 1110222PubMed |

Rao, M., Zhao, X. L., Yang, J., Hu, S. F., Lei, H., Xia, W., and Zhu, C. H. (2015). Effect of transient scrotal hyperthermia on sperm parameters, seminal plasma biochemical markers, and oxidative stress in men. Asian J. Androl. 17, 668–675.
Effect of transient scrotal hyperthermia on sperm parameters, seminal plasma biochemical markers, and oxidative stress in men.Crossref | GoogleScholarGoogle Scholar | 25652627PubMed |

Rath, D. (2002). Low dose insemination in the sow: a review. Reprod. Domest. Anim. 37, 201–205.
Low dose insemination in the sow: a review.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD38vhvFOluw%3D%3D&md5=a93fcd4c733403fe05cf718076ea97bcCAS | 12173984PubMed |

Roca, J., Parrilla, I., Rodriguez-Martinez, H., Gil, M. A., Cuello, C., Vázquez, J. M., and Martínez, E. A. (2011). Approaches towards efficient use of boar semen in the pig industry. Reprod. Domest. Anim. 46, 79–83.
Approaches towards efficient use of boar semen in the pig industry.Crossref | GoogleScholarGoogle Scholar | 21884284PubMed |

Rodríguez-Gil, J. E., and Estrada, E. (2013). Artificial insemination in boar reproduction. In ‘Boar Reproduction: Fundamentals and New Biotechnological Trends’. (Eds S. Bonet, I. Casas, W. V. Holt and M. Yeste.) pp. 589–608. (Springer: Berlin.)

Rosatti, M. I., Beconi, M. T., and Córdoba, M. (2004). Proacrosin-acrosin activity in capacitated and acrosome reacted sperm from cryopreserved bovine semen. Biocell 28, 311–316.
| 1:CAS:528:DC%2BD2MXls1Sktg%3D%3D&md5=c8b7faa6c69204137da7cd5f4d668f28CAS | 15633454PubMed |

Sirat, M. P., Sinha, A. K., Singh, B. K., and Prasad, R. L. (1996). Effect of cryoprotectants on release of various enzymes from buck spermatozoa during freezing. Theriogenology 45, 405–416.
Effect of cryoprotectants on release of various enzymes from buck spermatozoa during freezing.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28zgtVGlsw%3D%3D&md5=0c180fc1ea60c5308aa2ef12b5b4096cCAS | 16727804PubMed |

Thurston, L. M., Watson, P. F., Mileham, A. J., and Holt, W. V. (2001). Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation. J. Androl. 22, 382–394.
| 1:STN:280:DC%2BD3MrgsVOltA%3D%3D&md5=436b82b0b9d39537184c87a72533c615CAS | 11330638PubMed |

Thurston, L. M., Siggins, K., Mileham, A. J., Watson, P. F., and Holt, W. V. (2002). Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation. Biol. Reprod. 66, 545–554.
Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XhvVeit7Y%3D&md5=f86f83fe21e7335810d224ed49a6fa0aCAS | 11870056PubMed |

Töpfer-Petersen, E., and Cechová, D. (1990). Zona pellucida induces conversion of proacrosin to acrosin. Int. J. Androl. 13, 190–196.
Zona pellucida induces conversion of proacrosin to acrosin.Crossref | GoogleScholarGoogle Scholar | 2117585PubMed |

Waberski, D., Weitze, K. F., Gleumes, T., Schwarz, M., Willmen, T., and Petzoldt, R. (1994). Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen. Theriogenology 42, 831–840.
Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28zgtVentw%3D%3D&md5=cb9a921d5463e9f1803479035a5985a2CAS | 16727588PubMed |

Watson, P. F., and Behan, J. R. (2002). Intrauterine insemination of sows with reduced sperm numbers: results of a commercially based field trial. Theriogenology 57, 1683–1693.
Intrauterine insemination of sows with reduced sperm numbers: results of a commercially based field trial.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD383psFyntw%3D%3D&md5=bc9ccf44290ea1e0b407fbc3b7d8d25cCAS | 12035978PubMed |

Westendorf, P., Richter, L., and Treu, H. (1975). Zur Tiefgefrierung von Ebersperma Labor und Besamungsergebnisse mit dem Hülsenberger Pailetten-Verfahren [Deep freezing of boar sperm. Laboratory and insemination results using the Hülsenberger paillete method]. Dtsch. Tierarztl. Wochenschr. 82, 261–267.
| 1:STN:280:DyaE28%2FmsFyntg%3D%3D&md5=dad310176eea33a9ee08e6e29b7d5663CAS | 1104331PubMed |

Yeste, M. (2013). Boar spermatozoa within the oviductal environment (III): Fertilisation. In ‘Boar Reproduction: Fundamentals and New Biotechnological Trends’. (Eds S. Bonet, I. Casas, W. V. Holt and M. Yeste.) pp. 343–406. (Springer: Berlin.)

Yeste, M., Briz, M., Pinart, E., Sancho, S., Garcia-Gil, N., Badia, E., Bassols, J., Pruneda, A., Bussalleu, E., Casas, I., and Bonet, S. (2008a). Boar spermatozoa and prostaglandin F2alfa. Quality of boar sperm after addition of prostaglandin F2alfa to the short-term extender over cooling time. Anim. Reprod. Sci. 108, 180–195.
Boar spermatozoa and prostaglandin F2alfa. Quality of boar sperm after addition of prostaglandin F2alfa to the short-term extender over cooling time.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtVejtLnP&md5=586bc07bba55e39907a8750d1ea0ca78CAS | 17897798PubMed |

Yeste, M., Briz, M., Pinart, E., Sancho, S., Garcia-Gil, N., Badia, E., Bassols, J., Pruneda, A., Bussalleu, E., Casas, I., and Bonet, S. (2008b). Hyaluronic acid delays boar sperm capacitation after 3 days of storage at 15 degrees C. Anim. Reprod. Sci. 109, 236–250.
Hyaluronic acid delays boar sperm capacitation after 3 days of storage at 15 degrees C.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXht12gu7zI&md5=7234d09abceaa326f6f0d3edf65ef367CAS | 18162335PubMed |

Yeste, M., Briz, M., Pinart, E., Sancho, S., Bussalleu, E., and Bonet, S. (2010). The osmotic tolerance of boar spermatozoa and its usefulness as sperm quality parameter. Anim. Reprod. Sci. 119, 265–274.
The osmotic tolerance of boar spermatozoa and its usefulness as sperm quality parameter.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXktVait74%3D&md5=ef4b99987c45c6fc4388c89037fbded7CAS | 20227204PubMed |

Yeste, M., Flores, E., Estrada, E., Bonet, S., Rigau, T., and Rodríguez-Gil, J. E. (2013a). Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze-thawing by stabilising disulphide bonds. Reprod. Fertil. Dev. 25, 1036–1050.
Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze-thawing by stabilising disulphide bonds.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXht1Gms7rM&md5=e44123104a78e5dfe583f66498ffda37CAS | 23089308PubMed |

Yeste, M., Estrada, E., Casas, I., Bonet, S., and Rodríguez-Gil, J. E. (2013b). Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze–thawing but not in ROS levels. Theriogenology 79, 929–939.
Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze–thawing but not in ROS levels.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXisVGgtrY%3D&md5=9bc93eaab1fb5a684fe6d9d9bd739f98CAS | 23398739PubMed |

Yeste, M., Estrada, E., Pinart, E., Bonet, S., Miró, J., and Rodríguez-Gil, J. E. (2014a). The improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability. Cryobiology 68, 251–261.
The improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXivFCgs7s%3D&md5=e83bc3ff96c66da11c9b83bdb1f36cf2CAS | 24530509PubMed |

Yeste, M., Estrada, E., Rivera, M. M., Bonet, S., Rigau, T., and Rodríguez-Gil, J. E. (2014b). The increase in phosphorylation levels of serine residues of protein HSP70 during holding time at 17°C is concomitant with a higher cryotolerance of boar spermatozoa. PLoS One 9, e90887.
The increase in phosphorylation levels of serine residues of protein HSP70 during holding time at 17°C is concomitant with a higher cryotolerance of boar spermatozoa.Crossref | GoogleScholarGoogle Scholar | 24603527PubMed |