Free Standard AU & NZ Shipping For All Book Orders Over $80!
Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

In vitro characterisation of fresh and frozen sex-sorted bull spermatozoa

Shauna A. Holden A , Craig Murphy A , Juan F. Moreno B , Stephen T. Butler C , Andrew R. Cromie D , Patrick Lonergan E and Sean Fair A F
+ Author Affiliations
- Author Affiliations

A Laboratory of Animal Reproduction, Department of Life Sciences, Faculty of Science and Engineering, University of Limerick, Castletroy, Limerick, Ireland.

B Sexing Technologies, Navasota, 22575 Highway 6 South, TX 77868, USA.

C Teagasc Animal and Grassland Research and Innovation Centre, Moorepark, Fermoy, Co. Cork, Ireland.

D Irish Cattle Breeding Federation, Highfield House, Shinagh, Bandon, Co. Cork, Ireland.

E School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Dublin, Ireland.

F Corresponding author. Email: sean.fair@ul.ie

Reproduction, Fertility and Development 29(7) 1415-1425 https://doi.org/10.1071/RD16086
Submitted: 4 November 2015  Accepted: 17 May 2016   Published: 4 July 2016

Abstract

This study sought to compare the in vitro characteristics of fresh and frozen non-sorted (NS) and sex-sorted (SS) bull spermatozoa. Experiment 1: Holstein–Friesian ejaculates (n = 10 bulls) were split across four treatments and processed: (1) NS fresh at 3 × 106 spermatozoa, (2) X-SS frozen at 2 × 106 spermatozoa, (3) X-SS fresh at 2 × 106 spermatozoa and (4) X-SS fresh at 1 × 106 spermatozoa. NS frozen controls of 20 × 106 spermatozoa per straw were sourced from previously frozen ejaculates (n = 3 bulls). Experiment 2: Aberdeen Angus ejaculates (n = 4 bulls) were split across four treatments and processed as: (1) NS fresh 3 × 106 spermatozoa, (2) Y-SS fresh at 1 × 106 spermatozoa, (3) Y-SS fresh at 2 × 106 spermatozoa and (4) X-SS fresh at 2 × 106 spermatozoa. Controls were sourced as per Experiment 1. In vitro assessments for progressive linear motility, acrosomal status and oxidative stress were carried out on Days 1, 2 and 3 after sorting (Day 0 = day of sorting. In both experiments SS fresh treatments had higher levels of agglutination in comparison to the NS fresh (P < 0.001), NS frozen treatments had the greatest PLM (P < 0.05) and NS spermatozoa exhibited higher levels of superoxide anion production compared with SS spermatozoa (P < 0.05). Experiment 1 found both fresh and frozen SS treatments had higher levels of viable acrosome-intact spermatozoa compared with the NS frozen treatments (P < 0.01).

Additional keywords: agglutination, bovine, gender bias, oxidative stress, sexed semen.


References

Agarwal, A., Saleh, R. A., and Bedaiwy, M. A. (2003). Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil. Steril. 79, 829–843.
Role of reactive oxygen species in the pathophysiology of human reproduction.Crossref | GoogleScholarGoogle Scholar | 12749418PubMed |

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=668369c26335f232861c4c306de7f315CAS | 22879525PubMed |

Al-Makhzoomi, A., Lundeheim, N., Håård, M., and Rodríguez-Martínez, H. (2008). Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden. Theriogenology 70, 682–691.
Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cvovVeiuw%3D%3D&md5=67a9ecdc3209fd9c5d449c902a31ddd3CAS | 18539320PubMed |

Al Naib, A., Ward, F., Kelly, A. K., Wade, M., Marti, J. I., and Lonergan, P. (2011). Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm. Theriogenology 76, 1070–1075.
Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3MfgvFanug%3D%3D&md5=ee93d6312d051a396bf37f91f9fd7aa2CAS | 21719089PubMed |

Alvarez, J. G., Touchstone, J. C., Blasco, L., and Storey, B. T. (1987). Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: superoxide dismutase as major enzyme protectant against oxygen toxicity. J. Androl. 8, 338–348.
Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: superoxide dismutase as major enzyme protectant against oxygen toxicity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2sXmtFGhtb4%3D&md5=6454ac07b802bad44f3dd131995a1190CAS | 2822642PubMed |

Balao da Silva, C. M., Ortega Ferrusola, C., Morillo Rodriguez, A., Gallardo Bolanos, J. M., Plaza Davila, M., Morrell, J. M., Rodriguez Martinez, H., Tapia, J. A., Aparicio, I. M., and Pena, F. J. (2013). Sex sorting increases the permeability of the membrane of stallion spermatozoa. Anim. Reprod. Sci. 138, 241–251.
Sex sorting increases the permeability of the membrane of stallion spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXlsVKgsLg%3D&md5=f662f1c9cf231ec30f938e50f423c46cCAS | 23567220PubMed |

Bansal, A. K., and Bilaspuri, G. S. (2011). Impacts of oxidative stress and antioxidants on semen functions. Vet. Med. Int. 2011, 686137.
Impacts of oxidative stress and antioxidants on semen functions.Crossref | GoogleScholarGoogle Scholar |

Blondin, P., Beaulieu, M., Fournier, V., Morin, N., Crawford, L., Madan, P., and King, W. A. (2009). Analysis of bovine sexed sperm for IVF from sorting to the embryo. Theriogenology 71, 30–38.
Analysis of bovine sexed sperm for IVF from sorting to the embryo.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cjnt1Gmuw%3D%3D&md5=d06c0854d41c74e277e4a7b899915e36CAS | 19004490PubMed |

Borchersen, S., and Peacock, M. (2009). Danish A.I. field data with sexed semen. Theriogenology 71, 59–63.
Danish A.I. field data with sexed semen.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cjnt1Cmug%3D%3D&md5=6d1abe65b7a42e2c1aa10a4be05d2862CAS | 18992932PubMed |

Breininger, E., Cetica, P. D., and Beconi, M. T. (2010). Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm. Theriogenology 74, 1036–1049.
Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtFWqtLnE&md5=89eea839bc3270e1514723d709415e2bCAS | 20580081PubMed |

Bucci, D., Galeati, G., Tamanini, C., Vallorani, C., Rodriguez-Gil, J. E., and Spinaci, M. (2012). Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa. Theriogenology 77, 1206–1216.
Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xjs1altrs%3D&md5=b4b44f45dfeb815bfeb06408ec6d37a3CAS | 22192401PubMed |

Burroughs, C. A., Graham, J. K., Lenz, R. W., and Seidel, G. E. (2013). Seminal plasma effects on sex-sorting bovine sperm. Theriogenology 79, 551–557.
Seminal plasma effects on sex-sorting bovine sperm.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3s3isVKhtQ%3D%3D&md5=3184ed5faf86582eb20212f640040e37CAS | 23244767PubMed |

Butler, S. T., Hutchinson, I. A., Cromie, A. R., and Shalloo, L. (2014a). Applications and cost benefits of sexed semen in pasture-based dairy production systems. Animal 8, 165–172.
Applications and cost benefits of sexed semen in pasture-based dairy production systems.Crossref | GoogleScholarGoogle Scholar | 24679704PubMed |

Butler, S. T., Hutchinson, I. A., and Cromie, A. R. (2014b). Preliminary results from a field trial to evaluate sexed semen in dairy cows and heifers. In ‘Agricultural Research Forum’. (Ed. M. Diskin.) p. 115. (Tullamore: Ireland.)

Carvalho, J. O., Sartori, R., Machado, G. M., Mourão, G. B., and Dode, M. A. N. (2010). Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production. Theriogenology 74, 1521–1530.
Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3cbjtFWnsg%3D%3D&md5=ed27b6cf3ed1485bd6e987c35180c4caCAS | 20728930PubMed |

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=d8d1459a0651eef5d9c9d78aaae63d27CAS | 11468782PubMed |

Cromie, A. R., Kearney, F., Evans, R., and Berry, D. P. (2014). Genomics for pedigree and cross-bred beef cattle populations; some experiences from Ireland. In ‘10th World Congress of Genetics Applied to Livestock Production, Vancouver’. Available at https://asas.org/docs/default-source/wcgalp-proceedings-oral/260_paper_10495_manuscript_1361_0.pdf?sfvrsn=2 [Verified 15 June 2016]

de Graaf, S. P., Leahy, T., and Vishwanath, R. (2014). Biological and practical lessons associated with the use of sexed semen. In ‘9th International Symposium on Reproduction in Domestic Ruminants’. (Eds J. L. Juengel, A. Miyamoto, C. Price, L. P. Reynolds, M. F. Smith and R. Webb.) pp. 125–140. (Ashby-de-la-Zouch)

DeJarnette, J. M., Nebel, R. L., and Marshall, C. E. (2009). Evaluating the success of sex-sorted semen in US dairy herds from on farm records. Theriogenology 71, 49–58.
Evaluating the success of sex-sorted semen in US dairy herds from on farm records.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cjnt1CrtQ%3D%3D&md5=e01744f22664fa24bb5f2359382dcc96CAS | 18996579PubMed |

DeJarnette, J. M., Leach, M. A., Nebel, R. L., Marshall, C. E., McCleary, C. R., and Moreno, J. F. (2011). Effects of sex-sorting and sperm dosage on conception rates of Holstein heifers: is comparable fertility of sex-sorted and conventional semen plausible? J. Dairy Sci. 94, 3477–3483.
Effects of sex-sorting and sperm dosage on conception rates of Holstein heifers: is comparable fertility of sex-sorted and conventional semen plausible?Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXnvFahs7c%3D&md5=d55c723c806fe50e10132d8dc1708da9CAS | 21700034PubMed |

de Lamirande, E., Jiang, H., Zini, A., Kodama, H., and Gagnon, C. (1997). Reactive oxygen species and sperm physiology. Rev. Reprod. 2, 48–54.
Reactive oxygen species and sperm physiology.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXhs1Kmsrk%3D&md5=1df874979edd42f7f3e98863dfd86c05CAS | 9414465PubMed |

del Olmo, D., Parrilla, I., Gil, M. A., Maside, C., Tarantini, T., Angel, M. A., Roca, J., Martinez, E. A., and Vazquez, J. M. (2013). Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability. Theriogenology 80, 350–356.
Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3sjhslSltw%3D%3D&md5=f5beb00147380bb4a7db3e9ad8bdb973CAS | 23746874PubMed |

De Vos, A., Van De Velde, H., Joris, H., Verheyen, G., Devroey, P., and Van Steirteghem, A. (2003). Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection. Fertil. Steril. 79, 42–48.
Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 12524062PubMed |

Dott, H. M., and Walton, A. (1960). Effects of dilution and washing on ram spermatozoa studied by the flow dialysis technique. J. Reprod. Fertil. 1, 350–367.
Effects of dilution and washing on ram spermatozoa studied by the flow dialysis technique.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaF3c%2FktlKitA%3D%3D&md5=ef9536601eee082f19558f5be427cdb7CAS | 13724017PubMed |

Espinosa-Cervantes, R., and Cordova-Izquierdo, A. (2012). Sexing sperm of domestic animals. Trop. Anim. Health Prod. 45, 1–8.
Sexing sperm of domestic animals.Crossref | GoogleScholarGoogle Scholar | 22829354PubMed |

Frijters, A. C., Mullaart, E., Roelofs, R. M., van Hoorne, R. P., Moreno, J. F., Moreno, O., and Merton, J. S. (2009). What affects fertility of sexed bull semen more, low sperm dosage or the sorting process? Theriogenology 71, 64–67.
What affects fertility of sexed bull semen more, low sperm dosage or the sorting process?Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cjnt1Gmug%3D%3D&md5=5acd715faba5bc1c5201bc72ab193890CAS | 19004486PubMed |

Garner, D. L. (2006). Flow cytometric sexing of mammalian sperm. Theriogenology 65, 943–957.
Flow cytometric sexing of mammalian sperm.Crossref | GoogleScholarGoogle Scholar | 16242764PubMed |

Garner, D. L., and Seidel, G. E. (2008). History of commercializing sexed semen for cattle. Theriogenology 69, 886–895.
History of commercializing sexed semen for cattle.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1c3hsFertw%3D%3D&md5=0ca1c83cdab67ae9d781866a75d7a2edCAS | 18343491PubMed |

Garner, D. L., Evans, K. M., and Seidel, G. E. (2013) Sex-sorting sperm using flow cytometry/cell sorting. ‘Spermatogenesis: Methods and Protocols’. (Eds D. T. Carrell and K. I. Aston.) pp. 279–295. (Springer: New York.)

Gosálvez, J., Ramirez, M. A., López-Fernández, C., Crespo, F., Evans, K. M., Kjelland, M. E., and Moreno, J. F. (2011). Sex-sorted bovine spermatozoa and DNA damage: II. Dynamic features. Theriogenology 75, 206–211.
Sex-sorted bovine spermatozoa and DNA damage: II. Dynamic features.Crossref | GoogleScholarGoogle Scholar | 21040960PubMed |

Guthrie, H. D., and Welch, G. R. (2012). Effects of reactive oxygen species on sperm function. Theriogenology 78, 1700–1708.
Effects of reactive oxygen species on sperm function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XosFCgsLw%3D&md5=c0ef589d3bd0342bbf74f20959890252CAS | 22704396PubMed |

Harayama, H., Liao, P. C., Gage, D. A., Miyake, M., Kato, S., and Hammerstedt, R. H. (2000). Biochemical characterization of sialoprotein “anti-agglutinin” purified from boar epididymal and seminal plasma. Mol. Reprod. Dev. 55, 96–103.
Biochemical characterization of sialoprotein “anti-agglutinin” purified from boar epididymal and seminal plasma.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXnvFynt74%3D&md5=9e6dac673288652906e3be1e32cc7e05CAS | 10602279PubMed |

Healy, A. A., House, J. K., and Thomson, P. C. (2013). Artificial insemination field data on the use of sexed and conventional semen in nulliparous Holstein heifers. J. Dairy Sci. 96, 1905–1914.
Artificial insemination field data on the use of sexed and conventional semen in nulliparous Holstein heifers.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhsFCnu7w%3D&md5=7645462c8fb24c1c5cbd17be1dce9045CAS | 23357013PubMed |

Hohenboken, W. D. (1999). Applications of sexed semen in cattle production. Theriogenology 52, 1421–1433.
Applications of sexed semen in cattle production.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7pvFalsw%3D%3D&md5=644bf9d2bfc616b2ef1d08121c969099CAS | 10735086PubMed |

Kiernan, M., Fahey, A., and Fair, S. (2013). The effect of the in vitro supplementation of exogenous long-chain fatty acids on bovine sperm cell function. Reprod. Fertil. Dev. 25, 947–954.
The effect of the in vitro supplementation of exogenous long-chain fatty acids on bovine sperm cell function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhtFSrtrzI&md5=1c5f0eb00e6499139e34addf34e75dafCAS | 23036717PubMed |

Klinc, P., Frese, D., Osmers, H., and Rath, D. (2007). Insemination with sex sorted fresh bovine spermatozoa processed in the presence of antioxidative substances. Reprod. Domest. Anim. 42, 58–62.
Insemination with sex sorted fresh bovine spermatozoa processed in the presence of antioxidative substances.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2s%2FitVaqsQ%3D%3D&md5=af21f67270b44dc0df5fac6174d8b1bfCAS | 17214775PubMed |

Kodama, H., Kuribayashi, Y., and Gagnon, C. (1996). Effect of sperm lipid peroxidation on fertilization. J. Androl. 17, 151–157.
| 1:CAS:528:DyaK28Xjt1WlsL8%3D&md5=5729fd2d58465d086d7b91d87da00923CAS | 8723439PubMed |

Koppers, A. J., De Iuliis, G. N., Finnie, J. M., McLaughlin, E. A., and Aitken, R. J. (2008). Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. J. Clin. Endocrinol. Metab. 93, 3199–3207.
Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXpvVOlsLw%3D&md5=52b0d0c6557bfb8c7646fd7ea0c1ed54CAS | 18492763PubMed |

Lindahl, P. E., and Sjoblom, P. (1981). On mechanisms of head-to-head association in bovine spermatozoa. Biol. Reprod. 25, 29–43.
On mechanisms of head-to-head association in bovine spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL3MXltlSgtb8%3D&md5=256e9405f55097e0ff59ef4663077418CAS | 6269665PubMed |

Maxwell, W. M. C., and Johnson, L. A. (1997). Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation. Mol. Reprod. Dev. 46, 408–418.
Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXhsFKhsr0%3D&md5=1bd3ce1df93866a2790e74120b5c5675CAS |

Mocé, E., Graham, J. K., and Schenk, J. L. (2006). Effect of sex-sorting on the ability of fresh and cryopreserved bull sperm to undergo an acrosome reaction. Theriogenology 66, 929–936.
Effect of sex-sorting on the ability of fresh and cryopreserved bull sperm to undergo an acrosome reaction.Crossref | GoogleScholarGoogle Scholar | 16564078PubMed |

Morado, S., Cetica, P., Beconi, M., Thompson, J., and Dalvit, G. (2013). Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation. Reproduction 145, 471–478.
Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXpsVejsLY%3D&md5=e30c0e3a7140ac2e73acfceee1a0bcf6CAS | 23630331PubMed |

Murphy, C., Fahey, A. G., Shafat, A., and Fair, S. (2013). Reducing sperm concentration is critical to limiting the oxidative stress challenge in liquid bull semen. J. Dairy Sci. 96, 4447–4454.
Reducing sperm concentration is critical to limiting the oxidative stress challenge in liquid bull semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXnt1aks7c%3D&md5=cac24b888a7bff895f8678d9820c580fCAS | 23660140PubMed |

Murphy, C., Holden, S. A., Murphy, E. M., Cromie, A. R., Lonergan, P., and Fair, S. (2015). The impact of storage temperature and sperm number on the fertility of liquid-stored bull semen. Reprod. Fertil. Dev. , .
The impact of storage temperature and sperm number on the fertility of liquid-stored bull semen.Crossref | GoogleScholarGoogle Scholar | 25765074PubMed |

O’Flaherty, C., Beorlegui, N., and Beconi, M. T. (2003). Participation of superoxide anion in the capacitation of cryopreserved bovine sperm. Int. J. Androl. 26, 109–114.
Participation of superoxide anion in the capacitation of cryopreserved bovine sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjtlKlsb8%3D&md5=a4445cb75033bcbd9a9eb234ec833ebbCAS | 12641829PubMed |

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=6128f795b189def21fb981f84e4bb02bCAS | 19383259PubMed |

Rivlin, J., Mendel, J., Rubinstein, S., Etkovitz, N., and Breitbart, H. (2004). Role of hydrogen peroxide in sperm capacitation and acrosome reaction. Biol. Reprod. 70, 518–522.
Role of hydrogen peroxide in sperm capacitation and acrosome reaction.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXnsl2itA%3D%3D&md5=deb2f1c9825ce263effe6ac5611bbd7cCAS | 14561655PubMed |

Seidel, G. E. J. (2014). Update on sexed semen technology in cattle. Animal 8, 160–164.
Update on sexed semen technology in cattle.Crossref | GoogleScholarGoogle Scholar |

Seidel, G. E., and Schenk, J. L. (2008). Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition. Anim. Reprod. Sci. 105, 129–138.
Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition.Crossref | GoogleScholarGoogle Scholar | 18178041PubMed |

Shekarriz, M., DeWire, D. M., Thomas, A. J., and Agarwal, A. (1995). A method of human semen centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species. Eur. Urol. 28, 31–35.
| 1:STN:280:DyaK287isV2gtA%3D%3D&md5=b676f3cd1a71e07c8745f44d76b740d2CAS | 8521891PubMed |

Shojaei Saadi, H. A., van Riemsdijk, E., Dance, A. L., Rajamanickam, G. D., Kastelic, J. P., and Thundathil, J. C. (2013). Proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation. J. Proteomics 82, 64–80.
Proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXmslehurs%3D&md5=176e5d66d6e9618a816ee6c5e5a0de9cCAS | 23500133PubMed |

Sikka, S. C. (1996). Oxidative stress and role of antioxidants in normal and abnormal sperm function. Front. Biosci. 1, e78–e86.
Oxidative stress and role of antioxidants in normal and abnormal sperm function.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2czktVCrtA%3D%3D&md5=bbe52b33b5fcc79a8f990b16a093267aCAS | 9159248PubMed |

Suh, T. K., Schenk, J. L., and Seidel, G. E. (2005). High pressure flow cytometric sorting damages sperm. Theriogenology 64, 1035–1048.
High pressure flow cytometric sorting damages sperm.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2MvlvF2mtA%3D%3D&md5=53921411c541dd53a729dda03c9d055dCAS | 16125550PubMed |

Suzuki, K., and Nagai, T. (2003). In vitro fertility and motility characteristics of frozen–thawed boar epididymal spermatozoa separated by Percoll. Theriogenology 60, 1481–1494.
In vitro fertility and motility characteristics of frozen–thawed boar epididymal spermatozoa separated by Percoll.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3svlvFSqtg%3D%3D&md5=9104bc0c43f68b05b8a5e9e826f9173aCAS | 14519469PubMed |

Vishwanath, R. (2014). SexedULTRA™ – raising the fertility bar of sexed sorted semen. In ‘Natl Assoc Artif Breeders’. Available at http://dairyknowledge.in/sites/default/files/vish_session_3_nddb_meeting_oct_2014.pdf [Verified 15 June 2016]

Vishwanath, R., and Shannon, P. (2000). Storage of bovine semen in liquid and frozen state. Anim. Reprod. Sci. 62, 23–53.
Storage of bovine semen in liquid and frozen state.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXlt1Kgsbs%3D&md5=99fbd24eabcb977f349abba34ec6d3dfCAS | 10924819PubMed |

Walczak-Jedrzejowska, R., Wolski, J. K., and Slowikowska-Hilczer, J. (2013). The role of oxidative stress and antioxidants in male fertility. Cent. European J. Urol. 66, 60–67.
The role of oxidative stress and antioxidants in male fertility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXotVKgurY%3D&md5=ba8b55caef609250754aecf059159b35CAS | 24578993PubMed |

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 |

Xu, Z. Z. (2014). Application of liquid semen technology improves conception rate of sex-sorted semen in lactating dairy cows. J. Dairy Sci. 97, 7298–7304.
Application of liquid semen technology improves conception rate of sex-sorted semen in lactating dairy cows.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXhslyrurvK&md5=660e0410c0115fd09f5b5a3414d5895aCAS | 25218757PubMed |

Yang, D. H., McMillan, A. G., Standley, N. T., Shannon, P., and Xu, Z. Z. (2012). Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm. Theriogenology 78, 1476–1486.
Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xht1Kju7zP&md5=6188bf0418153a2b72fd6a0c9928a53bCAS | 22925643PubMed |