Effect of overnight staining on the quality of flow cytometric sorted stallion sperm: comparison with tradtitional protocols.

The research article investigates the possibility of using cooled, overnight stained sperm for flow cytometric sorting in horses, providing an alternative to the requirement of using fresh stallion semen, which has logistical limitations.

Introduction

• The research delves into the field of animal reproduction, specifically equine, and examines the use of flow cytometry for sperm separation based on the X and Y chromosomes.
• Flow cytometry is dubbed the only reliable method for this procedure, and MoFlo SX DP sorter is the device that makes this possible. The process aids in the production of offspring of a desired sex.
• However, the existing protocol demands the use of fresh semen, which is typically obtained near or at the sorting facility, thus limiting its applicability.

Method

• The researchers proposed and tested a new protocol over two breeding seasons. The protocol involved using fresh stallion semen cooled for 20 hours, during which it was stained using Hoechst 33342, a fluorescent DNA stain used to identify and separate cells.
• Post these 20 hours, the semen was sorted via flow cytometry and compared to sperm from the same ejaculate batch that had been sexed the previous day.

Results

• The results demonstrated that all sperm parameters, including velocities, kinetics, viability, membrane permeability, the production of reactive oxygen species, mitochondrial membrane potential, and DNA fragmentation index, remained unchanged when comparing fresh sorted and refrigerated sorted semen.
• The study thus concluded that pre-sorting storage at a low temperature (5°C) did not alter the sorted sample’s key characteristics.

Implications

• The findings point towards a potential shift in methodology for equine sperm sorting that would enable the use of semen from stallions housed far from the sorting facilities.
• The new protocol could allow for processed and stained sperm to be refrigerated and shipped on the previous day, to be sorted and inseminated the following day.
• Such a change in protocol could streamline processes, reduce logistical constraints, and widen opportunities for practical applications in the field of equine reproduction.