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Research Article

volume 54 issue 10 (october 2020) : 1181-1187,   Doi: 10.18805/ijar.B-3823

X-sperm Enrichment of Bovine Semen by Percoll Density Gradient Method and Its Effect on Semen Quality, Sex Ratio and Conception Rate

Y
Y. Bhat
M
M. Sharma
1Department of Veterinary Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, G.B Pant University of Agriculture and Technology, Pantnagar-263 145, Uttarakhand, India.
Cite article:- Bhat Y., Sharma M. (2020). X-sperm Enrichment of Bovine Semen by Percoll Density Gradient Method and Its Effect on Semen Quality, Sex Ratio and Conception Rate. Indian Journal of Animal Research. 54(10): 1181-1187. doi: 10.18805/ijar.B-3823.

ABSTRACT

In cattle, sex selection has a significant economic impact when it improves herd capacity of milk or meat production and is commonly used for the production of calves of the desired gender.  New separation techniques which present both better accuracy and low costs are necessary. Density gradient centrifugation might be an approach to sexing spermatozoa because of the additional DNA content and volume of X-bearing sperm head. The present study was designed to apply the modified percoll discontinuous density gradient for X-sperm enrichment in cross bred bovine semen. We attempted to amplify the level of X-sperm enrichment by using double centrifugation in percoll gradient. In addition, effect of percoll density gradient on fertility of semen was also evaluated at three stages; before centrifugation (stage I), after 1st centrifugation (stage II) and after 2nd centrifugation (stage III) respectively and ultrasonography was carried out to confirm the sex of fetus at 60-90 days post AI. Semen was collected from two cross bred bovine bulls and 34 cows were inseminated with liquid X-sperm enriched semen after double centrifugation in percoll gradient. 22 cows were inseminated with non-sexed fresh liquid semen (control). Results showed that the volume, progressive motility, live sperms, concentration and HOST reactive sperms significantly (P<0.05) decreased at stage II and stage III compared to stage I. However, no significant (P>0.05) change was observed in pH and mass motility of semen at all three stages. There was non-significant effect on per cent spermatozoa with intact acrosomal membrane and fully damaged acrosomal membrane at all three stages however, partially damaged acrosomal membrane sperms were significantly (P<0.05) decreased at Stage II and Stage III compared to Stage I. The conception rate significantly (P<0.05) decreased with X-enriched semen (41.17%) compared to non-sexed liquid semen (59.09%). The female sex ratio of calves significantly (P<0.05) increased to 66.66% with X- sperm enriched semen compared to the male percentage of 33.33% with sexed semen and female percentage of 46.15% with non-sexed semen.

REFERENCES

  1. Blottner, S., Schwerin, M., Bottcher, M. Pitra, C. (1993). Selective enrichment of bovine X- and Y-spermatozoa by Percoll™ density gradient. Arch. Tierz. 36: 153-162.
  2. Buranaamnuay, K., Sangsuwan, P., Changsangfa, C., Faisaikarm, T. and Kaeoket, K. (2015) Influence of Discontinuous PureSperm and OptiPrep Gradient Centrifugations on Bovine Sperm Quality and the Sex Ratio of in vitro Produced Embryos. Chiang Mai J. Sci. 42(3): 637-649.
  3. Garner, D.L. (2006). Flow cytometric sexing of mammalian sperm. Theriogenology, Stoneham, 65: 943-957.
  4. Garner, D. L. (2009): The dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm. Theriogenology. 71: 11-21.
  5. Hossepian de Lima, V.F.M., Ramalho, M.F.P.D-T., Rodrigues, L.H., Malheiros, E.B., Moreira-Filho, C. A. (2000). Separation of X- and Y- bearing bovine spermatozoa by Percoll™ density gradient centrifugation. Theriogenology. 53: 280.
  6. Hossepian De Lima, V.F.M., Ramalho, M.D.T., Alves, B.C.A., Lucio, A.C., Oliveira, L.Z., Filho, A.M., Carneiro, L.C. (2015). Enrichment of Bovine Semen with X-Bearing Sperma- tozoa Using Percoll™ and Optiprep® Discontinuous Gradients. Animal and Veterinary Sciences. 3(1): 1-7.
  7. Lange, W., Pemsel, H., Blottner, S., Roselius, R., Pfeilsticker, J. and Rommel, P. (1995). Uber de einflu des bullen auf die selective anreicherung der X-und Y- spermienmit der Percoll™-dichtegradienten-zentrifugation. Arch. Tierz. 38: 155-161.
  8. Iizuka, R., Kaneko, S., Aoki, R. and Kobayashi, T. (1987). Sexing of human sperm by discontinuous percoll density gradient and its clinical application. Human Reproduction, Oxford. 2: 573-575.
  9. Lucio, A.C., Resende, M.V., Dernowseck, J.A., Perini1, A.P., Oliveira, L.Z., Miguel, M.C.V., et al. (2012). Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production. Arq. Bras. Med. Vet. Zootec. 64(3): 525-532.
  10. Malik, A., Haron, A.W., Yusoff, R., Bukar, M., Kasim, A. and Sabri, M. (2011). Verification of X- and Y-spermatozoa separation by nested polymerase chain reaction (PCR), motility and membrane integrity in bovine. African Journal of Biotech- -nology. 10(85): 19796-19801.
  11. Morton, K.M., Herrmann, D., Sieg, B., Struckmann, C., MaxwellL, W.M.C., Rath, D., et al. (2007). Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm. Molecular Reproduction and Development, New York.74: 931-940. 
  12. Oliveira, L. Z., Arruda, R. P. and Celeghini, E. C C. (2011). Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association. Andrologia. 44: 9-15. 
  13. Promthep, K., Satitmanwiwat, S., Kitiyanant1, N., Tantiwattanakul, P., Jirajaroenrat, K., et al. (2016). Practical use of percoll density gradient centrifugation on sperm sex determination in commercial dairy farm in Thailand. Indian J. Anim. Res. 50(3): 310-313.
  14. Resende, M.V., Bezerra, M.B., Perecin, F., AlMeida, A.O., Lucio, A.C. and De Lima, V.F.M.H. (2009). Separation of X bearing bovine sperm by centrifugation in continuous percoll and optiprep density gradient: Effect in sperm viability and in vitro embryo production. Ciência Animal Brasileira. 10(2): 581-587.\
  15. Resende, M.V., Lucio, A.C., Perini, A.P., Oliveira, L.Z., Almeida, A.O., Alves, B.C.A., et al. (2011). Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient. Arq. Bras. Med. Vet. Zootec. 63(3): 544-551.
  16. Seidel, G.E., Schenk, J.L., Herickhoff, L.A., Doyle, S.P., Brink, Z. and Green, R.D. (1999). Insemination of heifers with sexed sperm. Theriogenology. 52: 1407–20.
  17. Snedecor, G.W. and Cochran, W.G. (1989) Statistical Methods. 8th Edition, Iowa State University Press, Ames
  18. Stap, J., Hoebe, R.A., Merton, J.S., Haring, R.M., Bakker, P.J.M. and Aten, J.A. (1998). Improving the resolution of cryopreserved X- and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm. J. Anim. Sci. 76(7): 1896- 1902.
  19. Vega, M.D., Pena, A.I., Gullon, J., Prieto, C., Barrio, M., Becerra, J.J., Herradon, P.G. and Quintela, L. A. (2008.). Sex ratio in rabbits following modified artificial insemination. Animal Reproduction Science. 103: 385–391.
  20. Windsor, D.P. and Evans, G. (1993). White Sex predetermination by separation of X and Y chromossome- bearing sperm: A review. Reprod. Fert. Develop. 5: 155-171.
  21. Wolf, C.A., Brass, K.E., Rubin, M.I.B. Pozzobon, S.E., Mozzaquatro, F.D. and De La Corte, F.D. (2008). The effect of sperm selection by Percoll or swim-up on the sex ratio of in vitro produced bovine embryos. Anim. Reprod. 5: 110-115.
  22. Zeidan, A.E.B., El-Zaia, A.A., Radwa, H.E.H., Rowida, J., Riad, M. and El-Aasar, T.A. (2008). Viability, Acrosomal Status and Sex Ratio of the Centrifuged Rabbit Spermatozoa. American -Eurasian J. Agric. and Environ. Sci. 4 (3): 318-325.
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Indian Journal of Animal Research
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    Research Article

    volume 54 issue 10 (october 2020) : 1181-1187,   Doi: 10.18805/ijar.B-3823

    X-sperm Enrichment of Bovine Semen by Percoll Density Gradient Method and Its Effect on Semen Quality, Sex Ratio and Conception Rate

    Y
    Y. Bhat
    M
    M. Sharma
    1Department of Veterinary Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, G.B Pant University of Agriculture and Technology, Pantnagar-263 145, Uttarakhand, India.
    Cite article:- Bhat Y., Sharma M. (2020). X-sperm Enrichment of Bovine Semen by Percoll Density Gradient Method and Its Effect on Semen Quality, Sex Ratio and Conception Rate. Indian Journal of Animal Research. 54(10): 1181-1187. doi: 10.18805/ijar.B-3823.

    ABSTRACT

    In cattle, sex selection has a significant economic impact when it improves herd capacity of milk or meat production and is commonly used for the production of calves of the desired gender.  New separation techniques which present both better accuracy and low costs are necessary. Density gradient centrifugation might be an approach to sexing spermatozoa because of the additional DNA content and volume of X-bearing sperm head. The present study was designed to apply the modified percoll discontinuous density gradient for X-sperm enrichment in cross bred bovine semen. We attempted to amplify the level of X-sperm enrichment by using double centrifugation in percoll gradient. In addition, effect of percoll density gradient on fertility of semen was also evaluated at three stages; before centrifugation (stage I), after 1st centrifugation (stage II) and after 2nd centrifugation (stage III) respectively and ultrasonography was carried out to confirm the sex of fetus at 60-90 days post AI. Semen was collected from two cross bred bovine bulls and 34 cows were inseminated with liquid X-sperm enriched semen after double centrifugation in percoll gradient. 22 cows were inseminated with non-sexed fresh liquid semen (control). Results showed that the volume, progressive motility, live sperms, concentration and HOST reactive sperms significantly (P<0.05) decreased at stage II and stage III compared to stage I. However, no significant (P>0.05) change was observed in pH and mass motility of semen at all three stages. There was non-significant effect on per cent spermatozoa with intact acrosomal membrane and fully damaged acrosomal membrane at all three stages however, partially damaged acrosomal membrane sperms were significantly (P<0.05) decreased at Stage II and Stage III compared to Stage I. The conception rate significantly (P<0.05) decreased with X-enriched semen (41.17%) compared to non-sexed liquid semen (59.09%). The female sex ratio of calves significantly (P<0.05) increased to 66.66% with X- sperm enriched semen compared to the male percentage of 33.33% with sexed semen and female percentage of 46.15% with non-sexed semen.

    REFERENCES

    1. Blottner, S., Schwerin, M., Bottcher, M. Pitra, C. (1993). Selective enrichment of bovine X- and Y-spermatozoa by Percoll™ density gradient. Arch. Tierz. 36: 153-162.
    2. Buranaamnuay, K., Sangsuwan, P., Changsangfa, C., Faisaikarm, T. and Kaeoket, K. (2015) Influence of Discontinuous PureSperm and OptiPrep Gradient Centrifugations on Bovine Sperm Quality and the Sex Ratio of in vitro Produced Embryos. Chiang Mai J. Sci. 42(3): 637-649.
    3. Garner, D.L. (2006). Flow cytometric sexing of mammalian sperm. Theriogenology, Stoneham, 65: 943-957.
    4. Garner, D. L. (2009): The dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm. Theriogenology. 71: 11-21.
    5. Hossepian de Lima, V.F.M., Ramalho, M.F.P.D-T., Rodrigues, L.H., Malheiros, E.B., Moreira-Filho, C. A. (2000). Separation of X- and Y- bearing bovine spermatozoa by Percoll™ density gradient centrifugation. Theriogenology. 53: 280.
    6. Hossepian De Lima, V.F.M., Ramalho, M.D.T., Alves, B.C.A., Lucio, A.C., Oliveira, L.Z., Filho, A.M., Carneiro, L.C. (2015). Enrichment of Bovine Semen with X-Bearing Sperma- tozoa Using Percoll™ and Optiprep® Discontinuous Gradients. Animal and Veterinary Sciences. 3(1): 1-7.
    7. Lange, W., Pemsel, H., Blottner, S., Roselius, R., Pfeilsticker, J. and Rommel, P. (1995). Uber de einflu des bullen auf die selective anreicherung der X-und Y- spermienmit der Percoll™-dichtegradienten-zentrifugation. Arch. Tierz. 38: 155-161.
    8. Iizuka, R., Kaneko, S., Aoki, R. and Kobayashi, T. (1987). Sexing of human sperm by discontinuous percoll density gradient and its clinical application. Human Reproduction, Oxford. 2: 573-575.
    9. Lucio, A.C., Resende, M.V., Dernowseck, J.A., Perini1, A.P., Oliveira, L.Z., Miguel, M.C.V., et al. (2012). Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production. Arq. Bras. Med. Vet. Zootec. 64(3): 525-532.
    10. Malik, A., Haron, A.W., Yusoff, R., Bukar, M., Kasim, A. and Sabri, M. (2011). Verification of X- and Y-spermatozoa separation by nested polymerase chain reaction (PCR), motility and membrane integrity in bovine. African Journal of Biotech- -nology. 10(85): 19796-19801.
    11. Morton, K.M., Herrmann, D., Sieg, B., Struckmann, C., MaxwellL, W.M.C., Rath, D., et al. (2007). Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm. Molecular Reproduction and Development, New York.74: 931-940. 
    12. Oliveira, L. Z., Arruda, R. P. and Celeghini, E. C C. (2011). Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association. Andrologia. 44: 9-15. 
    13. Promthep, K., Satitmanwiwat, S., Kitiyanant1, N., Tantiwattanakul, P., Jirajaroenrat, K., et al. (2016). Practical use of percoll density gradient centrifugation on sperm sex determination in commercial dairy farm in Thailand. Indian J. Anim. Res. 50(3): 310-313.
    14. Resende, M.V., Bezerra, M.B., Perecin, F., AlMeida, A.O., Lucio, A.C. and De Lima, V.F.M.H. (2009). Separation of X bearing bovine sperm by centrifugation in continuous percoll and optiprep density gradient: Effect in sperm viability and in vitro embryo production. Ciência Animal Brasileira. 10(2): 581-587.\
    15. Resende, M.V., Lucio, A.C., Perini, A.P., Oliveira, L.Z., Almeida, A.O., Alves, B.C.A., et al. (2011). Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient. Arq. Bras. Med. Vet. Zootec. 63(3): 544-551.
    16. Seidel, G.E., Schenk, J.L., Herickhoff, L.A., Doyle, S.P., Brink, Z. and Green, R.D. (1999). Insemination of heifers with sexed sperm. Theriogenology. 52: 1407–20.
    17. Snedecor, G.W. and Cochran, W.G. (1989) Statistical Methods. 8th Edition, Iowa State University Press, Ames
    18. Stap, J., Hoebe, R.A., Merton, J.S., Haring, R.M., Bakker, P.J.M. and Aten, J.A. (1998). Improving the resolution of cryopreserved X- and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm. J. Anim. Sci. 76(7): 1896- 1902.
    19. Vega, M.D., Pena, A.I., Gullon, J., Prieto, C., Barrio, M., Becerra, J.J., Herradon, P.G. and Quintela, L. A. (2008.). Sex ratio in rabbits following modified artificial insemination. Animal Reproduction Science. 103: 385–391.
    20. Windsor, D.P. and Evans, G. (1993). White Sex predetermination by separation of X and Y chromossome- bearing sperm: A review. Reprod. Fert. Develop. 5: 155-171.
    21. Wolf, C.A., Brass, K.E., Rubin, M.I.B. Pozzobon, S.E., Mozzaquatro, F.D. and De La Corte, F.D. (2008). The effect of sperm selection by Percoll or swim-up on the sex ratio of in vitro produced bovine embryos. Anim. Reprod. 5: 110-115.
    22. Zeidan, A.E.B., El-Zaia, A.A., Radwa, H.E.H., Rowida, J., Riad, M. and El-Aasar, T.A. (2008). Viability, Acrosomal Status and Sex Ratio of the Centrifuged Rabbit Spermatozoa. American -Eurasian J. Agric. and Environ. Sci. 4 (3): 318-325.
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