Optimizing equine sperm quality: an alternative to single layer centrifugation for sperm isolation.

Abstract: In vitro semen purification techniques have been developed that seek to mimic the in vivo selection process in order to generate the highest possible chance of oocyte fertilization following artificial insemination. Numerous methods have been developed to isolate functional spermatozoa for artificial insemination, yet only one method, single-layer centrifugation using commercial preparations like EquiPure, has been widely employed. In this study, we have introduced a novel approach for isolating spermatozoa and compared their quality to those isolated using EquiPure. The AI port system (Memphasys, Ltd. in Sydney, Australia) features a disposable cartridge with an inoculation chamber for depositing extended semen and a harvest chamber for extracting isolated spermatozoa. These chambers are separated by a 5 µm polyethylene terephthalate (PETE) membrane, allowing highly motile spermatozoa to migrate from the inoculation chamber to the harvest chamber over a 20-minute period. This migration effectively leaves behind seminal plasma and other cell types, such as leukocytes. Comparative analyses between spermatozoa isolated with the AI port and EquiPure demonstrated that, across all measured sperm parameters, including yield, vitality, motility, morphology, DNA fragmentation, and mitochondrial superoxide generation, the AI port-isolated cells exhibited comparable or superior performance, particularly in terms of DNA fragmentation. In summary, the AI port system demonstrates the potential to efficiently isolate high-quality spermatozoa, possibly offering a cost-effective and user-friendly alternative that may enhance the success rates of artificial insemination in breeding programs. Lay summary: This study aimed to create a new method for refining stallion semen to increase the likelihood of a successful pregnancy through artificial insemination. While there are existing techniques for isolating high-quality sperm, the most common involves a complicated process using a centrifuge, which spins the semen to separate it. This research introduces a new approach called the AI port system that uses a disposable cartridge with two separate chambers for putting in semen and getting out isolated sperm. A membrane between the chambers acts like a filter, letting highly motile sperm swim across, leaving behind unwanted substances like bacteria and blood cells. Compared to the centrifugation method, the AI port system effectively produces sperm with comparable or better quality in various aspects, including vitality, movement, shape, DNA integrity, and energy production. In summary, the AI port system is an easy-to-use alternative with the potential to improve the success of artificial insemination in horse breeding programs.