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

Vera Hossepian de Lima; Mariney Flavia Di-Tanno Ramalho; Beatriz Costa Aguiar Alves; Aline Costa Lucio; Leticia Zoccolario Oliveira; Carlos Alberto Moreira Filho; Luísa Cunha Carneiro

doi:doi:10.11648/j.avs.20150301.11

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

Vera Hossepian de Lima, Mariney Flavia Di-Tanno Ramalho, Beatriz Costa Aguiar Alves, Aline Costa Lucio, Leticia Zoccolario Oliveira, Carlos Alberto Moreira Filho, Luísa Cunha Carneiro

Published in Animal and Veterinary Sciences (Volume 3, Issue 1)

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

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[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.
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[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.
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[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.
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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

Vera Hossepian de Lima

Department of Preventive Veterinary Medicine and Animal Reproduction, Estadual University of Sao Paulo, UNESP, Jaboticabal, Brazil
Mariney Flavia Di-Tanno Ramalho

Research Center of Biotechnology, University of Sao Paulo, Sao Paulo, Brazil
Beatriz Costa Aguiar Alves

Department of Pediatrics, Medicine School of Estadual Universtiy of Sao Paulo, Sao Paulo, SP, Brazil
Aline Costa Lucio

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

Department of Preventive Veterinary Medicine and Animal Reproduction, Estadual University of Sao Paulo, UNESP, Jaboticabal, Brazil
Carlos Alberto Moreira Filho

Department of Pediatrics, Medicine School of Estadual Universtiy of Sao Paulo, Sao Paulo, SP, Brazil
Luísa Cunha Carneiro

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

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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

Copy | Download

@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}
}

Copy | Download

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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