By Maria Paz Zuñiga, DVM, WEVA Board Member

Scientists and breeders have been pursuing sex preselection before conception for years. In dairy and meat cattle industries there are financial benefits to producing one calf sex or the other. However, choosing a sex for horses tends to have more subjective reasons or be related to athletic performance.

Until now, the only replicable and efficient way to separate the sperm that contains the X chromosome from those that contain the Y chromosome from a semen sample is by sorting the cells with a high-flow cytometer. However, it has only been used commercially (not in day-to-day equine practice) and on a large scale in the dairy cattle industry. The number of spermatozoa that can be effectively selected per hour in the flow cytometer has been a limitation for its mass application, especially for species that require a high number of sperm to inseminate a female, as is the case with horses.

The main factors that affect gestation rates with flow-cytometer-sexed semen are the low numbers of spermatozoa available for insemination; the sperm damage by high pressure speed flow, DNA stain, and ultraviolet light exposure; and the cryopreservation process of these sex-sorted spermatozoa.

In 2015, to find a solution to these problems, Hernan Ramirez, DVM, from Chile, and Luis Losinno, DVM, PhD, from Argentina, began investigating and testing whether magnetic nanoparticles (NP) could sort spermatozoa based mainly on membrane charge differences. Nanotechnology refers to the technology that treats or uses particles synthesized at the nanometer scale. These nanoparticles can be made in different sizes and compositions, and their biocompatibility with biological fluids makes them an excellent device or tool for interactions with labeled and unlabeled cells for purposes that use fluorescence or magnetism.

Magnetic-activated cell sorting (MACS) is a technique for preparing sperm for use in assisted reproduction techniques (ARTs). This technology has shown that it can separate motile, viable, and morphologically normal spermatozoa that exhibit good cryopreservation tolerance and fertilization potential.

Ramirez and Losinno´s main objective was to determine if magnetic nanoparticles could effectively separate “X” and “Y” stallion spermatozoa and maintain sperm viability in fresh, cooled, and frozen sex-sorted semen. A secondary objective was to test fertility rates in mares inseminated with fresh and cooled sex-sorted semen.

The pair used three technologies to determine the efficacy of sex-sorting spermatozoa with magnetic nanoparticles: fluorescent in situ hybridization, high-flow cytometry, and real-time polymerase chain reaction. Currently, the average value obtained for X sperm separation in their trials is 91%, 92%, and 94%, respectively, with each technology being tested on more than 70 ejaculates.

They’ve also used several other tests to evaluate spermatozoa parameters (such as motility, straight linear velocity, sperm DNA integrity evaluation, and more). Results have shown no significant differences in any parameters evaluated between control (which have not been exposed to nanoparticles) and sex-sorted groups.

At press time, the team had achieved 27 pregnancies aimed at producing fillies with NP sex-sorted semen. The fertility rates for fresh and cooled sex-sorted semen were 81.2% and 75%, respectively. Seventeen of the 27 pregnancies have been confirmed with ultrasonography at 65 and 125 days for filly pregnancies with a 100% efficacy. And, so far, 11 of the pregnant mares have already given birth to fillies—a 100% efficiency.

The present trials have demonstrated that magnetic nanoparticles are an effective means of sorting spermatozoa containing the “X” chromosome. Furthermore, the sex-sorting technique has not affected sperm viability or fresh or cooled semen fertility rates. At the date of this study, to the author’s and researchers’ knowledge there are no other published studies reporting on the ability to sex-sort stallion semen with NP.

Ramirez and Losinno continue to investigate and add more results to their trials. They believe this is a very promising technology that will some day be available for all breeders seeking foals of a specific sex.