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.

Keywords: PIEZO-ICSI; absolute RT-qPCR; plasmids; sex-sorting; sperm stallion.

Figure 1

Confirmation of the correct design of the pUC19-TNMD and pUC19-SRY vectors by PCR. Lanes L correspond to the DNA Ladder “GeneRuler 50 bp DNA Ladder (ThermoFisher, Waltham, MA, USA)”. Lane pUC-TNMD corresponds to the pUC19-TNMD vector with the qPCR-TNMD primers. Lane pUC-SRY corresponds to the vector pUC19-SRY with the qPCR-SRY primers. The CX lane corresponds to the pUC19-TNMD vector with the qPCR-SRY primers and the CY lane corresponds to the pUC19-SRY vector with the qPCR-TNMD primers.

Figure 2

Standard curves to determine the percentage error of sex-sorting. (a) The curve of standards made with the pUC19-TNMD plasmid and the number of spermatozoa with a copy of the TNMD gene that carries the X chromosome are shown. (b) The curve of standards made with the pUC19-SRY plasmid and the number of spermatozoa with a copy of the SRY gene that carries the Y chromosome are shown. Squares represent the standards made with the plasmids, the triangles represent the sexed spermatozoa, and the circles represent the unsexed spermatozoa, in both groups. In red is the control of unsexed sperm and blue is sexed sperm samples in both graphs.

Figure 3

Equine/bovine hybrid embryos produced by Piezo-ICSI. Representative images of equine/bovine hybrid embryos on day 3 of culture. Both embryos are in the morula stage with 8–16 blastomeres.

Figure 4

PCR sexing of equine/bovine hybrid embryos generated by Piezo-ICSI. Lanes L correspond to the DNA Ladder GeneRuler 50 bp DNA Ladder (ThermoFisher, Waltham, MA, USA). Lanes XX and XY correspond to the sexing of female and male embryos, respectively. Lanes CX and CY correspond to the confirmation that the qPCR-TNMD and qPCR-SRY primers have no amplification sites in the bovine parthenotes genome. (a) Representative electrophoresis of the PCR sexing of 4 female embryos (89 bp) and 4 male embryos (141 bp), produced with the respective sexed spermatozoa, can be observed. (b) Representative electrophoresis shows that primers qPCR-TNMD and qPCR-SRY do not generate an amplicon by PCR, with the DNA of bovine parthenotes as template.

Figure 4

PCR sexing of equine/bovine hybrid embryos generated by Piezo-ICSI. Lanes L correspond to the DNA Ladder GeneRuler 50 bp DNA Ladder (ThermoFisher, Waltham, MA, USA). Lanes XX and XY correspond to the sexing of female and male embryos, respectively. Lanes CX and CY correspond to the confirmation that the qPCR-TNMD and qPCR-SRY primers have no amplification sites in the bovine parthenotes genome. (a) Representative electrophoresis of the PCR sexing of 4 female embryos (89 bp) and 4 male embryos (141 bp), produced with the respective sexed spermatozoa, can be observed. (b) Representative electrophoresis shows that primers qPCR-TNMD and qPCR-SRY do not generate an amplicon by PCR, with the DNA of bovine parthenotes as template.

Figure 5

pUC19-TNMD and pUC19-SRY vectors. The two constructs designed for making the standards for absolute qPCR quantification are shown. (a) pUC19-TNMD vector contains a fragment of the TNMD gene, endogenous to the X chromosome. (b) pUC19-SRY vector containing a fragment of the SRY gene, endogenous to the Y chromosome.

Figure 6

Dot Plot of sperm populations. Representative image showing the two sperm populations identified by the differences in fluorescence of the X and Y chromosomes during sex-sorting by flow cytometry. In blue is the X sperm population, orange is the Y sperm population, green is indetermined sperm population and yellow is debris.