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Vertebrate reproductive science and technology
RESEARCH ARTICLE

Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility

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

A SaBio IREC (CSIC – UCLM – JCCM), Campus Universitario s.n. 02071 Albacete, Spain.

B Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, 13300 Valdepeñas, Spain.

C Corresponding author. Email: MRocio.Fernandez@uclm.es

Reproduction, Fertility and Development 27(8) 1187-1196 https://doi.org/10.1071/RD14043
Submitted: 6 February 2014  Accepted: 27 April 2014   Published: 19 June 2014

Abstract

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2 h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2 h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r = –0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2 h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

Additional keywords: artificial insemination, flow cytometry, fluorescein (CM-H2DCFDA), frozen semen.


References

Aisen, E., Quintana, M., Medina, V., Morello, H., and Venturino, A. (2005). Ultramicroscopic and biochemical changes in ram spermatozoa cryopreserved with trehalose-based hypertonic extenders. Cryobiology 50, 239–249.
Ultramicroscopic and biochemical changes in ram spermatozoa cryopreserved with trehalose-based hypertonic extenders.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXltVOktrk%3D&md5=9e63803264cb2e1ebd716df629a2508eCAS | 15925576PubMed |

Aitken, R. J. (2011). The capacitation–apoptosis highway: oxysterols and mammalian sperm function. Biol. Reprod. 85, 9–12.
The capacitation–apoptosis highway: oxysterols and mammalian sperm function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXotFOlsro%3D&md5=7429b181914e420abd5dfe8a9b82162dCAS | 21490245PubMed |

Aitken, R. J., and De Iuliis, G. N. (2010). On the possible origins of DNA damage in human spermatozoa. Mol. Hum. Reprod. 16, 3–13.
On the possible origins of DNA damage in human spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsFOhtrrN&md5=d0966281bb1510d38b3686fc52e32d64CAS | 19648152PubMed |

Aitken, R. J., and Krausz, C. (2001). Oxidative stress, DNA damage and the Y chromosome. Reproduction 122, 497–506.
Oxidative stress, DNA damage and the Y chromosome.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXotFGltLc%3D&md5=27cd088e5fd1b16bcc8c3d97df786262CAS | 11570956PubMed |

Aitken, R. J., Clarkson, J. S., and Fishel, S. (1989). Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biol. Reprod. 41, 183–197.
Generation of reactive oxygen species, lipid peroxidation and human sperm function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1MXlslWksLs%3D&md5=0bded68484f81cb14840787ba0a3ccf8CAS | 2553141PubMed |

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=8b324da7320fcd5d46631b91e1639577CAS | 22879525PubMed |

Alvarez, J. G., and Storey, B. T. (1992). Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation. J. Androl. 13, 232–241.
| 1:CAS:528:DyaK38Xlt1yqs7o%3D&md5=7b9e2dd159130e08bb254ee059135fcdCAS | 1601742PubMed |

Amann, R. P., and Seidel, G. E. Amann, R. P., and Seidel, G. E. (2000). Fertilizing potential in vitro of semen from young beef bulls containing a high or low percentage of sperm with a proximal droplet. Theriogenology 54, 1499–1515.
| 1:STN:280:DC%2BD3M7isVGgtw%3D%3D&md5=308a3a94b850a0d870eaf6c0f7b80e7eCAS | 11191872PubMed |

Avendaño, C., and Oehninger, S. (2011). DNA fragmentation in morphologically normal spermatozoa: how much should we be concerned in the ICSI era? J. Androl. 32, 356–363.
DNA fragmentation in morphologically normal spermatozoa: how much should we be concerned in the ICSI era?Crossref | GoogleScholarGoogle Scholar | 21088229PubMed |

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=3f82f60e72bd0fbdba61e5da41bee564CAS | 10670514PubMed |

Benchaib, M., Lornage, J., Mazoyer, C., Lejeune, H., Salle, B., and François Guerin, J. (2007). Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome. Fertil. Steril. 87, 93–100.
Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhvVSlsb8%3D&md5=3455eaea7d5e78e192091d763ecf6c48CAS | 17074327PubMed |

Bennetts, L. E., and Aitken, R. J. (2005). A comparative study of oxidative DNA damage in mammalian spermatozoa. Mol. Reprod. Dev. 71, 77–87.
A comparative study of oxidative DNA damage in mammalian spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjtVOqsrk%3D&md5=0a6dc79575431e87d9b268e1bc9428dcCAS | 15736137PubMed |

Boe-Hansen, G. B., Christensen, P., Vibjerg, D., Nielsen, M. B. F., and Hedeboe, A. M. (2008). Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility. Theriogenology 69, 728–736.
Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXjtVWhsL0%3D&md5=c34b73b499424534c32e610c42b713b4CAS | 18242673PubMed |

Breininger, E., Beorlegui, N. B., O’Flaherty, C. M., and Beconi, M. T. (2005). Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen. Theriogenology 63, 2126–2135.
Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjtFyrtLc%3D&md5=45cc06fba38c9f336c36b37b5dae57e9CAS | 15826678PubMed |

Bucak, M. N., Tuncer, P. B., Sarıözkan, S., Başpınar, N., Taşpınar, M., Coyan, K., Bilgili, A., Akalın, P. P., Büyükleblebici, S., Aydos, S., Ilgaz, S., Sunguroğlu, A., and Oztuna, D. (2010). Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage. Cryobiology 61, 248–253.
Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhsVOrsr%2FM&md5=6da4f70762f0c4909a1e1ac3870c8cebCAS | 20833164PubMed |

Bungum, M., Humaidan, P., Axmon, A., Spano, M., Bungum, L., Erenpreiss, J., and Giwercman, A. (2007). Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum. Reprod. 22, 174–179.
Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtlChtbzN&md5=d3ec1036a31b197df4c79009333886b6CAS | 16921163PubMed |

Carrell, D. T., Liu, L., Peterson, C. M., Jones, K. P., Hatasaka, H. H., Erickson, L., and Campbell, B. (2003). Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch. Androl. 49, 49–55.
Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xpt1Kqtrk%3D&md5=8f8ca9a8cf5ff608a4ffa834e2c14a59CAS | 12647778PubMed |

Chatterjee, S., and Gagnon, C. (2001). Production of reactive oxygen species by spermatozoa undergoing cooling, freezing and thawing. Mol. Reprod. Dev. 59, 451–458.
Production of reactive oxygen species by spermatozoa undergoing cooling, freezing and thawing.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXltVWmsb4%3D&md5=d4d07d337d2337fb0717396eb621edfbCAS | 11468782PubMed |

de Lamirande, D. E., and Gagnon, C. (1992). Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J. Androl. 13, 368–378.
| 1:CAS:528:DyaK3sXkslSj&md5=1b8c6600774eadb2bbb2fa5b52ed2e94CAS |

de Lamirande, D. E., and Gagnon, C. (1993). Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Radic. Biol. Med. 14, 157–166.
Human sperm hyperactivation and capacitation as parts of an oxidative process.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXitFSisb4%3D&md5=5b6f9cbb8b8aec0e3c37345aa787be80CAS |

Del Olmo, E., Bisbal, A., Maroto-Morales, A., García-Alvare, O., Ramon, M., Jimenez-Rabadan, P., Martínez-Pastor, F., Soler, A. J., Garde, J. J., and Fernandez-Santos, M. R. (2013). Fertility of cryopreserved ovine semen is determined by sperm velocity. Anim. Reprod. Sci. 138, 102–109.
Fertility of cryopreserved ovine semen is determined by sperm velocity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXktlGhtL0%3D&md5=8bfe4e7207e6f0def7859fe18a2e5317CAS |

Domínguez-Rebolledo, A. E., Fernández-Santos, M. R., Bisbal, A., Ros-Santaella, J. L., Ramón, M., Carmona, M., Martínez-Pastor, F., and Garde, J. J. (2010). Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments. Reprod. Fertil. Dev. 22, 856–870.
Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments.Crossref | GoogleScholarGoogle Scholar | 20450838PubMed |

Evenson, D. P., and Wixon, R. (2006). Predictive value of the sperm chromatin assay in different populations. Fertil. Steril. 85, 810–811.
Predictive value of the sperm chromatin assay in different populations.Crossref | GoogleScholarGoogle Scholar | 16500374PubMed |

Evenson, D. P., Larson, K. L., and Jost, L. K. (2002). Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J. Androl. 23, 25–43.
| 11780920PubMed |

Fernández-Santos, M. R., Martínez-Pastor, F., García-Macías, V., Esteso, M. C., Soler, A. J., Paz, P., Anel, L., and Garde, J. J. (2007). Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and non-enzymatic antioxidants. J. Androl. 28, 294–305.
Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and non-enzymatic antioxidants.Crossref | GoogleScholarGoogle Scholar | 17079744PubMed |

Fernández-Santos, M. R., Domínguez-Rebolledo, A. E., Esteso, M. C., Garde, J. J., and Martínez-Pastor, F. (2009). Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity. Int. J. Androl. 32, 353–359.
Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity.Crossref | GoogleScholarGoogle Scholar | 18298570PubMed |

Fernández-Santos, M. R., Soler, A. J., Ramón, M., Ros-Santaella, J. L., Maroto-Morales, A., García-Álvarez, O., Bisbal, A., Garde, J. J., Coloma, M. A., and Santiago-Moreno, J. (2011). Effect of post-mortem time on post-thaw characteristics of Spanish ibex (Capra pyrenaica) spermatozoa. Anim. Reprod. Sci. 129, 56–66.
| 22014412PubMed |

Fiser, P. S., Ainsworth, L., and Fairfull, R. W. (1987). Evaluation of a new diluent and different processing procedures for cryopreservation of ram semen. Theriogenology 28, 599–607.
Evaluation of a new diluent and different processing procedures for cryopreservation of ram semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD283pvFWjug%3D%3D&md5=9f226d80eeca66ee4b0f7335f933d7c6CAS | 16726342PubMed |

Foote, R. H., Brockett, C. C., and Kaproth, M. T. (2002). Motility and fertility of bull sperm in whole-milk extender containing antioxidants. Anim. Reprod. Sci. 71, 13–23.
Motility and fertility of bull sperm in whole-milk extender containing antioxidants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xjt1WisL8%3D&md5=01cb5cd8150d73d752fa809f8c6befa2CAS | 11988368PubMed |

García-Álvarez, O., Maroto-Morales, A., Martínez-Pastor, F., Fernández-Santos, M. R., Esteso, M. C., Pérez-Guzmán, M. D., and Soler, A. J. (2009). Heterologous in vitro fertilisation is a good procedure to assess the fertility of thawed ram spermatozoa. Theriogenology 71, 643–650.
Heterologous in vitro fertilisation is a good procedure to assess the fertility of thawed ram spermatozoa.Crossref | GoogleScholarGoogle Scholar | 18996578PubMed |

García-Macías, V., de Paz, P., Martínez-pastor, F., Alvarez, M., Gomes-Alves, S., Bernardo, J., Anel, E., and Anel, L. (2007). DNA fragmentation assessment by flow cytometry and Sperm-Bos-Halomax (bright-field microscopy and fluorescence microscopy) in bull sperm. Int. J. Androl. 30, 88–98.
DNA fragmentation assessment by flow cytometry and Sperm-Bos-Halomax (bright-field microscopy and fluorescence microscopy) in bull sperm.Crossref | GoogleScholarGoogle Scholar | 17166172PubMed |

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=5ed8d866fac8675105903e17acf5f861CAS | 8142556PubMed |

Gil-Guzman, E., Ollero, M., Lopez, M. C., Sharma, R. K., Alvarez, J. G., and Thomas, A. J. Gil-Guzman, E., Ollero, M., Lopez, M. C., Sharma, R. K., Alvarez, J. G., and Thomas, A. J. (2001). Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Hum. Reprod. 16, 1922–1930.
Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXntlahu70%3D&md5=a9994f02e3b817270e8a53d0f09e1ba9CAS | 11527899PubMed |

Gillan, L., Evans, G., and Maxwell, W. M. C. (2005). Flow cytometric evaluation of sperm parameters in relation to fertility potential. Theriogenology 63, 445–457.
Flow cytometric evaluation of sperm parameters in relation to fertility potential.Crossref | GoogleScholarGoogle Scholar | 15626410PubMed |

Hinshaw, D. B., Sklar, L. A., Bohl, B., Schraufstatter, I. U., Hyslop, P. A., Rossi, M. W., Spragg, R. G., and Cochrane, C. G. (1986). Cytoskeletal and morphologic impact of cellular oxidant injury. Am. J. Pathol. 123, 454–464.
| 1:STN:280:DyaL283jvFGnsg%3D%3D&md5=60b56388772f59808baf9ad141619735CAS | 3717299PubMed |

Januskauskas, A., Johannisson, A., and Rodríguez-Martínez, H. (2001). Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen–thawed semen from Swedish AI bulls. Theriogenology 55, 947–961.
Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen–thawed semen from Swedish AI bulls.Crossref | GoogleScholarGoogle Scholar |

Kathiravan, P., Kalatharan, J., Edwin, M. J., and Veerapandian, C. (2008). Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes. Anim. Reprod. Sci. 104, 9–17.
| 1:STN:280:DC%2BD1c%2FgvFSgtg%3D%3D&md5=341a20092affde4c834f6d9aee36e35bCAS | 17254723PubMed |

Lewis, S. E., and Aitken, R. J. (2005). DNA damage to spermatozoa has impacts on fertilisation and pregnancy. Cell Tissue Res. 322, 33–41.
DNA damage to spermatozoa has impacts on fertilisation and pregnancy.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2Mnksl2ruw%3D%3D&md5=ed48aa5d3ecd4a5a55ad9f8a6a141f0eCAS | 15912407PubMed |

Lopes, S., Jurisicova, A., Sun, J. G., and Casper, R. F. (1998). Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum. Reprod. 13, 896–900.
Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXjs12gtbo%3D&md5=65a66c596a92346398bda823134e4907CAS | 9619544PubMed |

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 subpopulations. Reprod. Fertil. Dev. 20, 547–556.
Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulations.Crossref | GoogleScholarGoogle Scholar | 18577351PubMed |

Martínez-Pastor, F., Aisen, E., Fernández-Santos, M. R., Esteso, M. C., Maroto-Morales, A., García-Alvarez, O., and Garde, J. J. (2009). Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa. Reproduction 137, 225–235.
Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa.Crossref | GoogleScholarGoogle Scholar | 19028926PubMed |

O’Flaherty, C., Beconi, M., and Beorlegui, N. (1997). Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen–thawed bull spermatozoa. Andrologia 29, 269–275.
Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen–thawed bull spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXmslyhs70%3D&md5=23cc12b9f3f753bad966d584709c289dCAS | 9350327PubMed |

O’Meara, C. M., Hanrahan, J. P., Prathalingam, N. S., Owen, J. S., Donovan, A., Fair, S., Ward, F., Wade, M., Evans, A. C., and Lonergan, P. (2008). Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen–thawed semen. Theriogenology 69, 513–522.
Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen–thawed semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1c%2FptFWktg%3D%3D&md5=d0d8e05588fed5616da6081b368abaa8CAS |

Papadopoulos, S., Hanrahan, J. P., Donovan, A., Duffy, P., Boland, M. P., and Lonergan, P. (2005). In vitro fertilisation as a predictor of fertility from cervical insemination of sheep. Theriogenology 63, 150–159.
In vitro fertilisation as a predictor of fertility from cervical insemination of sheep.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2cnhvFektQ%3D%3D&md5=f5b6053082683fbca7d178ce07167a2eCAS | 15589281PubMed |

Peris, S. I., Bilodeau, J. F., Dufour, M., and Bailey, J. L. (2007). Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation and functional parameters in ram sperm. Mol. Reprod. Dev. 74, 878–892.
Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation and functional parameters in ram sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXmtlKlsbc%3D&md5=3c529df2f8c595b054d4babfecc163b2CAS | 17186553PubMed |

Petrunkina, A. M., Waberski, D., Günzel-Apel, A. R., and Töpfer-Petersen, E. (2007). Determinants of sperm quality and fertility in domestic species. Reproduction 134, 3–17.
Determinants of sperm quality and fertility in domestic species.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXpvFGisrs%3D&md5=be54e8aea7195333ef0e6e2026bed4b2CAS | 17641084PubMed |

Roca, J., Rodríguez, M. J., Gil, M. A., Carvajal, G., Garcia, E. M., Cuello, C., Vazquez, J. M., and Martinez, E. A. (2005). Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase. J. Androl. 26, 15–24.
| 1:CAS:528:DC%2BD2MXmsFWgtQ%3D%3D&md5=d5cd1cea2dac03860570505d80ac6a8bCAS | 15611562PubMed |

Rodríguez-Martínez, H. (2003). Laboratory semen assessment and prediction of fertility: still utopia? Reprod. Domest. Anim. 38, 312–318.
Laboratory semen assessment and prediction of fertility: still utopia?Crossref | GoogleScholarGoogle Scholar | 12887570PubMed |

Rodríguez-Martínez, H. (2007). State of the art in farm-animal sperm evaluation. Reprod. Fertil. Dev. 19, 91–101.
State of the art in farm-animal sperm evaluation.Crossref | GoogleScholarGoogle Scholar | 17389138PubMed |

Schneider, C. S., Ellington, J. E., and Wright, R. W. (1999). Relationship between bull field fertility and in vitro embryo production using sperm preparation methods with and without somatic cell co-culture. Theriogenology 51, 1085–1098.
Relationship between bull field fertility and in vitro embryo production using sperm preparation methods with and without somatic cell co-culture.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7ptFeksA%3D%3D&md5=02f63ddd57ed9e63868048e095a08801CAS | 10729028PubMed |

Silva, P. F., Gadella, B. M., Colenbrander, B., Roelen, B. A., Peris, S. I., Morrier, A., and Dufour, M. (2007). Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage. Theriogenology 67, 609–619.
Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXitlyqug%3D%3D&md5=df2b74bef87d3d1225bdead3f55d8fc5CAS | 17056104PubMed |

Thomson, L. K., Zieschang, J. A., and Clark, A. M. (2011). Oxidative deoxyribonucleic acid damage in sperm has a negative impact on clinical pregnancy rate in intrauterine insemination but not intracytoplasmic sperm injection cycles. Fertil. Steril. 96, 843–847.
Oxidative deoxyribonucleic acid damage in sperm has a negative impact on clinical pregnancy rate in intrauterine insemination but not intracytoplasmic sperm injection cycles.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXht1GntLvL&md5=2e6e15684f4c1d12f8057867619dc64bCAS | 21839433PubMed |

Tremellen, K. (2008). Oxidative stress and male infertility – a clinical perspective. Hum. Reprod. Update 14, 243–258.
Oxidative stress and male infertility – a clinical perspective.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXkvFOqtbc%3D&md5=0579ba9eacb9c772572607499a3df8f7CAS | 18281241PubMed |

Virro, M. R., Larson-Cook, K. L., and Evenson, D. P. (2004). Sperm chromatin structure assay (SCSA) parameters are related to fertilisation, blastocyst development and ongoing pregnancy in in vitro fertilisation and intracytoplasmic sperm injection cycles. Fertil. Steril. 81, 1289–1295.
Sperm chromatin structure assay (SCSA) parameters are related to fertilisation, blastocyst development and ongoing pregnancy in in vitro fertilisation and intracytoplasmic sperm injection cycles.Crossref | GoogleScholarGoogle Scholar | 15136092PubMed |

Waterhouse, K. E., Haugan, T., Kommisrud, E., Tverdal, A., Flatberg, G., Farstad, W., Evenson, D. P., and De Angelis, P. M. (2006). Sperm DNA damage is related to field fertility of semen from young Norwegian red bulls. Reprod. Fertil. Dev. 18, 781–788.
Sperm DNA damage is related to field fertility of semen from young Norwegian red bulls.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XpsFWlurc%3D&md5=e11649779e1cedfa0c2133b3ae6305b6CAS | 17032587PubMed |

Zini, A., and Sigman, M. (2009). Are tests of sperm DNA damage clinically useful? Pros and cons. J. Androl. 30, 219–229.
Are tests of sperm DNA damage clinically useful? Pros and cons.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXlvFOjtbw%3D&md5=23be7f8458cb72eb6f268a4ce49f1ed3CAS | 19059901PubMed |

Zini, A., Blumenfeld, A., Libman, J., and Willis, J. (2005). Beneficial effect of microsurgical varicocelectomy on human sperm DNA integrity. Hum. Reprod. 20, 1018–1021.
Beneficial effect of microsurgical varicocelectomy on human sperm DNA integrity.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2M3lt1Wqtg%3D%3D&md5=27db8f214ae7075d5a93db3d02a5465dCAS | 15608026PubMed |

Zubkova, E. V., Wade, M., and Robaire, B. (2005). Changes in spermatozoal chromatin packaging and susceptibility to oxidative challenge during ageing. Fertil. Steril. 84, 1191–1198.
Changes in spermatozoal chromatin packaging and susceptibility to oxidative challenge during ageing.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtFOqtLfK&md5=18e17939314f419c347011de24fb90d0CAS | 16210011PubMed |