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.
Int J Mol Sci. 2023 Jul 26;24(15):11947. doi: 10.3390/ijms241511947.
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.
精子性别鉴定技术在动物生产领域具有巨大的经济效益。例如，精子分选技术有望达到 90%以上的精子性别鉴定准确率。然而，为了正确规范这项技术，需要一些实验室方法。本文详细描述了一种使用基于绝对 qPCR 的标准化马精子性别鉴定方案。此外，通过使用压电系统（Piezo-ICSI）将可能分选的马精子注入牛卵母细胞中，生成马/牛异种胚胎，实现并验证了绝对 qPCR 的结果。我们的结果表明，分选的马精子中 X 精子和 Y 精子的确定性分别为 97%和 94%，而通过 Piezo-ICSI 生成的假定雌性和雄性马/牛杂种胚胎的准确性为 92%，符合预期性别。因此，可以得出结论，所提出的方法是标准化马精子分选的一种可靠、具有成本效益且相对简单的选择。这得到了使用分选精子生成的 Piezo-ICSI 异种胚胎正确性别鉴定结果的支持，验证了这些配子的正确性别鉴定和活力。
