FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
International journal of molecular sciences2023; 24(15); doi: 10.3390/ijms241511947

Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR.

Abstract: Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed equine spermatozoa into bovine oocytes using a piezoelectric system (Piezo-ICSI). Our results indicated that equine sex-sorting spermatozoa had a 97% and 94% certainty for X and Y sperm, respectively, while presumptive female and male equine/bovine hybrid embryos, generated by Piezo-ICSI, had an accuracy of 92% with respect to the desired sex. Therefore, it is concluded that the presented methodology is a reliable, cost-effective, and relatively simple option for standardizing sex-sorting of equine spermatozoa. This is supported by the results of the correct sexing of Piezo-ICSI heterologous embryos generated with the sexed spermatozoa, validating the correct sexing and viability of these gametes.
Get Full Text
Publication Date: 2023-07-26 PubMed ID: 37569324PubMed Central: PMC10419253DOI: 10.3390/ijms241511947Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research study focuses on standardizing a method of accurately determining the sex of sperm cells from stallions using a process known as ‘sex-sorting’, validated by a technique called ‘absolute RT-qPCR’. The research demonstrated a successful rate of 97% accuracy for X chromosome sperm and 94% accuracy for Y chromosome sperm. The findings suggest this method could be a cost-effective option for the equine industry.

Sperm Sexing and its Benefits

  • The process of sperm sexing is a technique of interest among the animal production sector, as it offers economic benefits by allowing producers to more accurately determine the gender of offspring.
  • Sex-sorting is a method where the X and Y chromosomes in sperm, which determine the sex of the offspring, are distinguished. The promise of this technology is over 90% accuracy. This rate of precision can allow for significant control over the sex outcomes in animal populations, something that can be highly beneficial in many sectors of animal agriculture.

Standardization Protocol and Absolute RT-qPCR

  • The process of standardization ensures that the technique can be relied upon to produce valid and repeatable results when performed under the same conditions.
  • Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) is employed, an advanced laboratory technique that measures the amount of a specific DNA molecule. In this research, it is used as the base methodology to determine the sex of Stallion spermatozoa.
  • The ‘absolute’ quantification by qPCR indicates an exact count of target DNA molecules in the sample, allowing for a precise determination of X and Y sperm.

Results and Validation

  • The accuracy of the sex sorting process utilizing the absolute RT-qPCR technique resulted in a 97% certainty for X (female) sperm and a 94% certainty for Y (male) sperm.
  • To validate these findings, equine/bovine hybrid embryos were created by injecting presumably sex-sorted horse sperm into cow oocytes (a process known as intracytoplasmic sperm injection, or ICSI), using a system called Piezo-ICSI.
  • The resulting embryos showed an accuracy of 92% regarding their sex, demonstrating the validity of the sexing process.

Conclusion

  • The study concludes that this method of sex-sorting equine spermatozoa using the absolute RT-qPCR-based methodology is a reliable, cost-effective, and simpler approach for discriminating the sex of sperm.
  • The high accuracy rate in created embryos supports the validity of this technique, pointing towards its potential for widespread application in the animal production industry.

Cite This Article

APA
Muñoz E, Castro M, Aguila L, Contreras MJ, Fuentes F, Arias ME, Felmer R. (2023). Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR. Int J Mol Sci, 24(15). https://doi.org/10.3390/ijms241511947

Publication

International journal of molecular sciences
ISSN: 1422-0067
NlmUniqueID: 101092791
Country: Switzerland
Language: English
Volume: 24
Issue: 15

Researcher Affiliations

Muñoz, Erwin
  • Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, P.O. Box 54-D, Chile.
  • Doctoral Program in Applied Cellular and Molecular Biology, Universidad de La Frontera, Temuco P.O. Box 54-D, Chile.
Castro, Macarena
  • Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, P.O. Box 54-D, Chile.
  • Master of Science Program with Mention in Biology of Reproduction, Universidad de La Frontera, Temuco P.O. Box 54-D, Chile.
Aguila, Luis
  • Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, P.O. Box 54-D, Chile.
Contreras, María José
  • Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, P.O. Box 54-D, Chile.
  • Doctoral Program in Applied Cellular and Molecular Biology, Universidad de La Frontera, Temuco P.O. Box 54-D, Chile.
Fuentes, Fernanda
  • Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, P.O. Box 54-D, Chile.
  • Doctoral Program in Applied Cellular and Molecular Biology, Universidad de La Frontera, Temuco P.O. Box 54-D, Chile.
Arias, María Elena
  • Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, P.O. Box 54-D, Chile.
  • Department of Agricultural Production, Faculty of Agriculture and Environmental Sciences, Universidad de La Frontera, Temuco P.O. Box 54-D, Chile.
Felmer, Ricardo
  • Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, P.O. Box 54-D, Chile.
  • Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Environmental Sciences, Universidad de La Frontera, Temuco P.O. Box 54-D, Chile.

MeSH Terms

  • Horses
  • Male
  • Animals
  • Cattle
  • Female
  • Semen
  • Spermatozoa
  • Oocytes
  • Sperm Injections, Intracytoplasmic / veterinary
  • Sperm Injections, Intracytoplasmic / methods
  • Reference Standards

Grant Funding

  • FONDECYT 1201166 / Agencia Nacional de Investigación y Desarrollo
  • National Doctoral Scholarship #21191434 / Agencia Nacional de Investigación y Desarrollo

Conflict of Interest Statement

The authors declare no conflict of interest.

References

This article includes 26 references
  1. Naniwa Y, Sakamoto Y, Toda S, Uchiyama K. Bovine sperm sex-selection technology in Japan.. Reprod. Med. Biol. 2019;18:17–26.
    doi: 10.1002/rmb2.12235pmc: PMC6332832pubmed: 30655718google scholar: lookup
  2. Hirst CE, Major AT, Smith CA. Sex determination and gonadal sex differentiation in the chicken model.. Int. J. Dev. Biol. 2018;62:153–166.
    doi: 10.1387/ijdb.170319cspubmed: 29616724google scholar: lookup
  3. González-Marín C, Góngora CE, Moreno JF, Vishwanath R. Small ruminant SexedULTRA™ sperm sex-sorting: Status report and recent developments.. Theriogenology 2021;162:67–73.
    doi: 10.1016/j.theriogenology.2020.12.028pubmed: 33444918google scholar: lookup
  4. Quelhas J, Santiago J, Matos B, Rocha A, Lopes G, Fardilha M. Bovine semen sexing: Sperm membrane proteomics as candidates for immunological selection of X- and Y-chromosome-bearing sperm.. Vet. Med. Sci. 2021;7:1633–1641.
    doi: 10.1002/vms3.540pmc: PMC8464243pubmed: 34037311google scholar: lookup
  5. Pozdyshev DV, Kombarova NA, Muronetz VI. Biochemical Features of X or Y Chromosome-Bearing Spermatozoa for Sperm Sexing.. Biochemistry 2023;88:655–666.
    doi: 10.1134/S0006297923050085pubmed: 37331711google scholar: lookup
  6. Bhat Y, Sharma M. X-sperm enrichment of bovine semen by Percoll density gradient method and its effect on semen quality, sex ratio and conception rate.. Indian J. Anim. Res. 2020;54:1181–1187.
    doi: 10.18805/ijar.B-3823google scholar: lookup
  7. Ohlweiler LU, Mezzalira JC, Mezzalira A. Porcine IVF embryo development and estrogen receptors are influenced by the concentration of percoll gradients during sperm selection.. Mol. Reprod. Dev. 2020;87:135–141.
    doi: 10.1002/mrd.23290pubmed: 31691431google scholar: lookup
  8. Thomas JM, Locke JWC, Bonacker RC, Knickmeyer ER, Wilson DJ, Vishwanath R, Arnett AM, Smith MF, Patterson DJ. Evaluation of SexedULTRA 4M™ sex-sorted semen in timed artificial insemination programs for mature beef cows.. Theriogenology 2019;123:100–107.
    doi: 10.1016/j.theriogenology.2018.09.039pubmed: 30296650google scholar: lookup
  9. Kalbfleisch TS, Rice E, DePriest MS, Walenz BP, Hestand MS, Vermeesch JR, O’Connell BL, Fiddes IT, Vershinina AO, Petersen JL. Eq쪳, an updated reference genome for the domestic horse.. BioRxiv 2018.
    doi: 10.1101/306928pmc: PMC6240028pubmed: 30456315google scholar: lookup
  10. Raudsepp T, Santani A, Wallner B, Kata SR, Ren C, Zhang HB, Womack JE, Skow LC, Chowdhary BP. A detailed physical map of the horse Y chromosome.. Proc. Natl. Acad. Sci. USA 2004;101:9321–9326.
    doi: 10.1073/pnas.0403011101pmc: PMC438975pubmed: 15197257google scholar: lookup
  11. Hargreaves A. The hunt for dark DNA.. New Sci. 2018;237:29–31.
    doi: 10.1016/S0262-4079(18)30440-8google scholar: lookup
  12. Khamlor T, Pongpiachan P, Sangsritavong S, Chokesajjawatee N. Determination of sperm sex ratio in bovine semen using multiplex real-time polymerase chain reaction.. Asian-Australas. J. Anim. Sci. 2014;27:1411.
    doi: 10.5713/ajas.2014.14223pmc: PMC4150173pubmed: 25178292google scholar: lookup
  13. Park JH, Lee JH, Choi KM, Joung SY, Kim JY, Chung GM, Jin DI, Im KS. Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere.. Theriogenology 2001;55:1843–1853.
    doi: 10.1016/S0093-691X(01)00526-Xpubmed: 11414489google scholar: lookup
  14. Ren F, Xi H, Ren Y, Li Y, Wen F, Xian M, Zhao M, Zhu D, Wang L, Lei A. TLR7/8 signalling affects X-sperm motility via the GSK3 α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos.. J. Anim. Sci. Biotechnol. 2021;12:89.
    doi: 10.1186/s40104-021-00613-ypmc: PMC8330071pubmed: 34340711google scholar: lookup
  15. Yadav SK, Gangwar DK, Singh J, Tikadar CK, Khanna VV, Saini S, Dholpuria S, Palta P, Manik RS, Singh MK. An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm.. Vet. World. 2017;10:498.
    doi: 10.14202/vetworld.2017.498-504pmc: PMC5465762pubmed: 28620252google scholar: lookup
  16. Kurtz S, Petersen B. Pre-determination of sex in pigs by application of CRISPR/Cas system for genome editing.. Theriogenology 2019;137:67–74.
    doi: 10.1016/j.theriogenology.2019.05.039pubmed: 31208775google scholar: lookup
  17. Azam A, Awan MA, Khalid M, Sami A, Shahzad Q, Ansari MS, Rakha BA, Naqvi SMS, Akhter S. Efficiency of sucrose density gradient method for sex separation of buffalo spermatozoa as validated by SYBR Green Real Time PCR.. Anim. Sci. Pap. Rep. 2020;38:22–23.
  18. Gaddam N, ST VR, Putty K, Sakaram D, Ganji VK. Quantitative and qualitative assessment of sex sorted semen of Bos indicus and Bos taurus breeds.. Res. Sq. 2022:1–13.
    doi: 10.21203/rs.3.rs-1625203/v1google scholar: lookup
  19. Saffari M, Heidari F, Shamsara M, Hashemi E. The Effect of Diffrent Factors on X-and Y Bearing Sperm in Bull Semen.. Iran. J. Vet. Med. 2018;12:219–227.
    doi: 10.22059/ijvm.2018.247199.1004863google scholar: lookup
  20. Laghi L, Randolph AE, Malesci A, Boland CR. Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA.. J. Gen. Virol. 2004;85:3383–3388.
    doi: 10.1099/vir.0.80039-0pubmed: 15483255google scholar: lookup
  21. Hou Y, Zhang H, Miranda L, Lin S. Serious overestimation in quantitative PCR by circular (supercoiled) plasmid standard: Microalgal pcna as the model gene.. PLoS ONE 2010;5:e9545.
    doi: 10.1371/journal.pone.0009545pmc: PMC2832698pubmed: 20221433google scholar: lookup
  22. Barrachina F, Soler-Ventura A, Oliva R, Jodar M. A Clinician’s Guide to Sperm DNA and Chromatin Damage.. Springer; Cham, Switzerland 2018.
    doi: 10.1007/978-3-319-71815-6_2google scholar: lookup
  23. Wang D, Zhu H, Guo J, Lin B, Zhang L, Hao H, Du W, Zhao X. A study of a method to assess the purity of sorted bovine semen using rapid single-sperm sexing PCR.. J. Anim. Vet. Adv. 2011;10:750–756.
    doi: 10.3923/javaa.2011.750.756google scholar: lookup
  24. Vázquez M. Optimización de una Técnica de Extracción de ADN Genómico de Espermatozoides de Toro.. Instituto de Fisiología Celular; Mexico City, Mexico 2014.
  25. Arias ME, Sánchez R, Felmer R. Effect of anisomycin, a protein synthesis inhibitor, on the in vitro developmental potential, ploidy and embryo quality of bovine ICSI embryos.. Zygote 2016;24:724–732.
    doi: 10.1017/S0967199416000034pubmed: 27140503google scholar: lookup
  26. Kurokawa M, Fissore RA. ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphosphate receptor-1 down-regulation, and embryo development.. Mol. Hum. Reprod. 2003;9:523–533.
    doi: 10.1093/molehr/gag072pubmed: 12900511google scholar: lookup

Citations

This article has been cited 2 times.
  1. Álvarez-Rodríguez M, Catalán J. Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization. Int J Mol Sci 2025 Apr 25;26(9).
    doi: 10.3390/ijms26094049pubmed: 40362289google scholar: lookup
  2. Wu Y, Wang C, Fan X, Ma Y, Liu Z, Ye X, Shen C, Wu C. The impact of induced pluripotent stem cells in animal conservation. Vet Res Commun 2024 Apr;48(2):649-663.
    doi: 10.1007/s11259-024-10294-3pubmed: 38228922google scholar: lookup
Subscribe to our newsletter
Join 99,928 horse owners like you who receive our email updates on horse health and nutrition.