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Enrichment of Bovine Semen with X-Bearing Spermatozoa Using Percoll™ and Optiprep® Discontinuous Gradients

Received: 14 December 2014     Accepted: 12 January 2015     Published: 22 January 2015
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Abstract

In cattle, sex selection has a significant economic impact when it improves herd capacity of milk or meat production. Density gradient centrifugation might be an approach to sexing spermatozoa because of the additional DNA content and volume of X-bearing sperm head. In the present work, the accuracy of sperm sexing by Percoll™ and OptiPrep® gradients centrifugation was compared. Bovine semen from three breeds (Holstein, Gir, and Red Angus) was used. The sperm viability and sexing accuracy after sperm selection by centrifugation of low and high-density discontinuous gradient composed by three layers of Percoll™ or OptiPrep® were assessed by in vitro embryo production (IVP) and pregnancy rate following artificial insemination (AI). After IVP, cleavage rate was higher (P < 0.05) for unsexed (81.3%) than for sexed (75.5%) spermatozoa. A significantly lower cleavage rate was observed for sexed (76.1%) spermatozoa when compared to sexed Red Angus (81.2%) spermatozoa. Additionally, significantly lower blastocyst rate was observed using Holstein sexed sperm by OptiPrep® gradient (23.3%) when compared with Gir (41.9%) and Red Angus (48.7%) sexed sperm by Percoll™ gradient. Nevertheless, in all breeds analyzed, a significant sex ratio deviation to females was observed. Analyzing the pregnancy rate after AI with sexed sperm by Percoll™ and OptiPrep®, semen from Gir bulls presented lower (P <0.05) pregnancy/AI (61.3%)when compared to Red Angus (75.9%) and Holstein (77.4%) and bulls. From the results, it was concluded that the increase in female percentage after insemination and/or IVP with bovine sexed sperm by centrifugation of high density gradient composed by three layers of Percoll™ and OptiPrep® may allow this spermatozoa sexing technique to be used in large scale which would support genetic enhancement for milk and meat production as well as for progeny tests in cattle.

Published in Animal and Veterinary Sciences (Volume 3, Issue 1)
DOI 10.11648/j.avs.20150301.11
Page(s) 1-7
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2015. Published by Science Publishing Group

Keywords

Artificial Insemination, Bovine Sexed Semen, In Vitro Production

References
[1] R.K. Splan, L.V. Cundiff, L. D. Van Vleck, “Genetic parameters for sex-specific traits in beef cattle,” J. Anim. Sci., v. 76, pp.2272-2278, 1998.
[2] W.D. Hohenboken, “Applications of sexed semen in cattle production”, Theriogenology, v. 52, pp.14, 1999.
[3] A. De Vries, M. Overton, J. Fetrow, K. Leslie, S. Eicker, G. Rogers. “Exploring the impact of sexed semen on the structure of the dairy industry”, J. Dairy Sci., v.91, pp.847–56, 2008.
[4] D. L Garner Hoechst 33342: The dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm. Theriogenology 71, 11-21. 2009
[5] J.C. Sharpe, K.M .Evans, “Advances in flow cytometry for sexing sperm,” Theriogenology, v.71, pp.4-10, 2009.
[6] E.B. Van Munster, J .Stap, R. A. Hoebe, G.J. Te Meerman, J.A Aten, “ Difference in volume of X- and Y-chromosome bearing bovine sperm heads matches difference in DNA content,” Citometry, v. 35,pp. 125-128. 1999a.
[7] E.B .Van Munster, J. Stap, R. A. Hoebe, G.J. Te Meerman, J.A. Aten, “Difference in sperm head volume as a theorical basis for a sorting X- and Y- bearing spermatozoa: a potencials and limitation”, Theriogenology, v. 52, pp.1281-1293, 1999b.
[8] S. Blottner, M. Schwerin, M. Böttcher, C. Pitra, “Selective enrichment of bovine X- and Y-spermatozoa by Percoll™ density gradient”, Arch. Tierz, v.36, pp.153-162, 1993.
[9] W. Lange, H. Pemsel, S .Blottner, R. Roselius, J. Pfeilsticker, P. Rommel, “Über de einflu des bullen auf die selective anreicherung der X-und Y- spermienmit der Percoll™-dichtegradienten-zentrifugation”, Arch. Tierz. v.38, pp.155-161, 1995.
[10] V.F.M. Hossepian de Lima, M.F.P.D-T Ramalho, L.H. Rodrigues, E. B. Malheiros, C. A. Moreira-Filho, “Separation of X- and Y- bearing bovine spermatozoa by Percoll™ density gradient centrifugation”, Theriogenology , v.53 pp. 280, 2000.
[11] V.F.M. Hossepian de Lima, “Methodological advances on spermatozoa sexing for using in genetic improvement and animal production.” Braz. J. Anim. Sci., v. 36, pp.219-228, 2007.
[12] E. Curry, S.L. Pratt, D.R. Lapin, J.R. Gibbons,“Efficacy of a commercially available post-thaw bovine semen sexing kit in both single-ovulating and hyperstimuled cows”, Anim. Reprod. Sci., v. 116, p.376-380, 2009.
[13] F. Aleahmad, H .Gourabi, B. Zeinali, S. Ashtiani,H. Baharvand., “Separation of X- and Y-bearing human spermatozoa by sperm isolation medium gradients evaluated by FISH”, Reprod. Biom. OnLine, v. 18, pp.475-478, 2009.
[14] D.P. Windsor, G. Evans, I.G, “White Sex predetermination by separation of X and Y chromossome- bearing sperm: A review”, Reprod. Fert. Develop. v. 5, p.155-171, 1993.
[15] H. Schwiderski, S. Blottner, M. Schwerin, “Mikroinjektion und in vitro befruchtung mit spermien aus versuchen zur anreicherung der geschlechtss pezifischen spermientypen beim rind”, Reprod. Dom. Anim., v.26, pp. 143-144, 1991.
[16] C.Y. Andersen, A.G. Byskov,” Enhanced separation of X and Y bearing sperm cells by a combined density gradient centrifugation evaluated by fluorescence in situ hybridization of the Y-chromosome”, Acta Obstet. Gyn. Scan,v. 76, pp.131-134, 1997.
[17] G. Vajta, N. Rindom, T.T. Peura, P. Holm, T. Greve, H. Callesen , “The effect of media, serum and temperature on in vitro survival of bovine blastocysts after open pulled straw (OPS) vitrification”, Theriogenologyv.52, pp.939-948, 1999.
[18] A. Gutiérrez-Adán, J. Granados, B. Pintado, J. De La Fuente, “ Influence of glucose on the sex ratio of bovine IVM/IVF embryos cultured in vitro”, Reprod. Fertil. Dev., v. 13, pp.1-5. 2001.
[19] K. R. Bondioli, S. B. Ellis, J. H. Pryor, M. W. Williams, M. M. Harpold, “The use of male-specific chromosomal DNA fragments to determine the sex of bovine preimplantation embryos” Theriogenology, v. 31, pp. 95-104, 1989.
[20] M. Schwerin, S.Blottner, P.D. Thomsen, D. Roschlau, G. Brockmann, “Quantification of Y chromosome bearing spermatozoa of cattle using in situ hybridization”, Mol. Reprod. Dev., v. 30, pp.39-43, 1991.
[21] S. B. Ellis, K.R. Bondioli, M. E. Williams, J. H. Pryor, M. M. Harpold Sex determination of bovine embryos using male-specific DNA probes. Theriogenology 29, 242. 1988.
[22] S. Verberckmoes, A. Van Soom, I. Depauw, J. Dewulf, T. Rijsselaere, A. Kruif , “Effect of hole blood and serum on bovine sperm quality and in vitro fertilization capacity”, Theriogenology, v. 61,pp. 25-33, 2004.
[23] S .Curran, J. P. Kastelic, O. J. Ginther, “Determining sex of the bovine fetus by ultrasonic assessment of the relative location of the genital tubercle”, Anim. Reprod. Sci. v. 19, pp.217-227, 1989.
[24] Kaneko S, Oshio S, Kobayashi T, Mohri H, Iizuka R 1984.Selective isolation of human X- bearing sperm by differential velocity sedimentation in Percoll™ density gradients. Biomedical Research 5, 187-194.
[25] R. Iizuka, S .Kaneko, R. Aoki, T. Kobayashi, “Sexing of human sperm by discontinuos Percoll™ density gradient and its clinical application”, Hum. Reprod. v. 2,pp.573-575, 1987.
[26] M. A. Larson, K. Kimura, H. M. Kubisch, R. M. Roberts, “Sexual dimorphism among bovine embryos in their ability to make the transition to expanded blastocyst and in the expression of the signaling molecule IFN-τ”, Proc.Natl.Acad. Sci.v.98, pp.9677–9682, 2001.
[27] K. Kimura, H. Iwata, G. J. Thompson, “The effect of glucosamine concentration on the development and sex ratio of bovine embryos”, Anim. Reprod. Sci., v.103, pp.228–238, 2008.
Cite This Article
  • APA Style

    Vera Hossepian de Lima, Mariney Flavia Di-Tanno Ramalho, Beatriz Costa Aguiar Alves, Aline Costa Lucio, Leticia Zoccolario Oliveira, et al. (2015). Enrichment of Bovine Semen with X-Bearing Spermatozoa Using Percoll™ and Optiprep® Discontinuous Gradients. Animal and Veterinary Sciences, 3(1), 1-7. https://doi.org/10.11648/j.avs.20150301.11

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    ACS Style

    Vera Hossepian de Lima; Mariney Flavia Di-Tanno Ramalho; Beatriz Costa Aguiar Alves; Aline Costa Lucio; Leticia Zoccolario Oliveira, et al. Enrichment of Bovine Semen with X-Bearing Spermatozoa Using Percoll™ and Optiprep® Discontinuous Gradients. Anim. Vet. Sci. 2015, 3(1), 1-7. doi: 10.11648/j.avs.20150301.11

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    AMA Style

    Vera Hossepian de Lima, Mariney Flavia Di-Tanno Ramalho, Beatriz Costa Aguiar Alves, Aline Costa Lucio, Leticia Zoccolario Oliveira, et al. Enrichment of Bovine Semen with X-Bearing Spermatozoa Using Percoll™ and Optiprep® Discontinuous Gradients. Anim Vet Sci. 2015;3(1):1-7. doi: 10.11648/j.avs.20150301.11

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  • @article{10.11648/j.avs.20150301.11,
      author = {Vera Hossepian de Lima and Mariney Flavia Di-Tanno Ramalho and Beatriz Costa Aguiar Alves and Aline Costa Lucio and Leticia Zoccolario Oliveira and Carlos Alberto Moreira Filho and Luísa Cunha Carneiro},
      title = {Enrichment of Bovine Semen with X-Bearing Spermatozoa Using Percoll™ and Optiprep® Discontinuous Gradients},
      journal = {Animal and Veterinary Sciences},
      volume = {3},
      number = {1},
      pages = {1-7},
      doi = {10.11648/j.avs.20150301.11},
      url = {https://doi.org/10.11648/j.avs.20150301.11},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.avs.20150301.11},
      abstract = {In cattle, sex selection has a significant economic impact when it improves herd capacity of milk or meat production. Density gradient centrifugation might be an approach to sexing spermatozoa because of the additional DNA content and volume of X-bearing sperm head. In the present work, the accuracy of sperm sexing by Percoll™ and OptiPrep® gradients centrifugation was compared. Bovine semen from three breeds (Holstein, Gir, and Red Angus) was used. The sperm viability and sexing accuracy after sperm selection by centrifugation of low and high-density discontinuous gradient composed by three layers of Percoll™ or OptiPrep® were assessed by in vitro embryo production (IVP) and pregnancy rate following artificial insemination (AI). After IVP, cleavage rate was higher (P < 0.05) for unsexed (81.3%) than for sexed (75.5%) spermatozoa. A significantly lower cleavage rate was observed for sexed (76.1%) spermatozoa when compared to sexed Red Angus (81.2%) spermatozoa. Additionally, significantly lower blastocyst rate was observed using Holstein sexed sperm by OptiPrep® gradient (23.3%) when compared with Gir (41.9%) and Red Angus (48.7%) sexed sperm by Percoll™ gradient. Nevertheless, in all breeds analyzed, a significant sex ratio deviation to females was observed. Analyzing the pregnancy rate after AI with sexed sperm by Percoll™ and OptiPrep®, semen from Gir bulls presented lower (P <0.05) pregnancy/AI (61.3%)when compared to Red Angus (75.9%) and Holstein (77.4%) and bulls. From the results, it was concluded that the increase in female percentage after insemination and/or IVP with bovine sexed sperm by centrifugation of high density gradient composed by three layers of Percoll™ and OptiPrep® may allow this spermatozoa sexing technique to be used in large scale which would support genetic enhancement for milk and meat production as well as for progeny tests in cattle.},
     year = {2015}
    }
    

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  • TY  - JOUR
    T1  - Enrichment of Bovine Semen with X-Bearing Spermatozoa Using Percoll™ and Optiprep® Discontinuous Gradients
    AU  - Vera Hossepian de Lima
    AU  - Mariney Flavia Di-Tanno Ramalho
    AU  - Beatriz Costa Aguiar Alves
    AU  - Aline Costa Lucio
    AU  - Leticia Zoccolario Oliveira
    AU  - Carlos Alberto Moreira Filho
    AU  - Luísa Cunha Carneiro
    Y1  - 2015/01/22
    PY  - 2015
    N1  - https://doi.org/10.11648/j.avs.20150301.11
    DO  - 10.11648/j.avs.20150301.11
    T2  - Animal and Veterinary Sciences
    JF  - Animal and Veterinary Sciences
    JO  - Animal and Veterinary Sciences
    SP  - 1
    EP  - 7
    PB  - Science Publishing Group
    SN  - 2328-5850
    UR  - https://doi.org/10.11648/j.avs.20150301.11
    AB  - In cattle, sex selection has a significant economic impact when it improves herd capacity of milk or meat production. Density gradient centrifugation might be an approach to sexing spermatozoa because of the additional DNA content and volume of X-bearing sperm head. In the present work, the accuracy of sperm sexing by Percoll™ and OptiPrep® gradients centrifugation was compared. Bovine semen from three breeds (Holstein, Gir, and Red Angus) was used. The sperm viability and sexing accuracy after sperm selection by centrifugation of low and high-density discontinuous gradient composed by three layers of Percoll™ or OptiPrep® were assessed by in vitro embryo production (IVP) and pregnancy rate following artificial insemination (AI). After IVP, cleavage rate was higher (P < 0.05) for unsexed (81.3%) than for sexed (75.5%) spermatozoa. A significantly lower cleavage rate was observed for sexed (76.1%) spermatozoa when compared to sexed Red Angus (81.2%) spermatozoa. Additionally, significantly lower blastocyst rate was observed using Holstein sexed sperm by OptiPrep® gradient (23.3%) when compared with Gir (41.9%) and Red Angus (48.7%) sexed sperm by Percoll™ gradient. Nevertheless, in all breeds analyzed, a significant sex ratio deviation to females was observed. Analyzing the pregnancy rate after AI with sexed sperm by Percoll™ and OptiPrep®, semen from Gir bulls presented lower (P <0.05) pregnancy/AI (61.3%)when compared to Red Angus (75.9%) and Holstein (77.4%) and bulls. From the results, it was concluded that the increase in female percentage after insemination and/or IVP with bovine sexed sperm by centrifugation of high density gradient composed by three layers of Percoll™ and OptiPrep® may allow this spermatozoa sexing technique to be used in large scale which would support genetic enhancement for milk and meat production as well as for progeny tests in cattle.
    VL  - 3
    IS  - 1
    ER  - 

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Author Information
  • Department of Preventive Veterinary Medicine and Animal Reproduction, Estadual University of Sao Paulo, UNESP, Jaboticabal, Brazil

  • Research Center of Biotechnology, University of Sao Paulo, Sao Paulo, Brazil

  • Department of Pediatrics, Medicine School of Estadual Universtiy of Sao Paulo, Sao Paulo, SP, Brazil

  • Department of Preventive Veterinary Medicine and Animal Reproduction, Estadual University of Sao Paulo, UNESP, Jaboticabal, Brazil

  • Department of Preventive Veterinary Medicine and Animal Reproduction, Estadual University of Sao Paulo, UNESP, Jaboticabal, Brazil

  • Department of Pediatrics, Medicine School of Estadual Universtiy of Sao Paulo, Sao Paulo, SP, Brazil

  • Department of Preventive Veterinary Medicine and Animal Reproduction, Estadual University of Sao Paulo, UNESP, Jaboticabal, Brazil

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