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.
Publication Date: 2023-07-26 PubMed ID: 37569324PubMed Central: PMC10419253DOI: 10.3390/ijms241511947Google Scholar: Lookup
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Summary
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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
Researcher Affiliations
- 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.
- 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.
- Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, P.O. Box 54-D, Chile.
- 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.
- 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.
- 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.
- 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.
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Citations
This article has been cited 2 times.- Álvarez-Rodríguez M, Catalán J. Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization. Int J Mol Sci 2025 Apr 25;26(9).
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